JOHN B. HYNES VETERANS MEMORIAL CONVENTION CENTER April 30

JOHN B. HYNES VETERANS MEMORIAL CONVENTION CENTER April 30 ­– May 2, 2015

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APRIL 30 – ­ MAY 2, 2015

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 3

TABLE OF CONTENTS Welcome 5 Charter of the Weinstein Meeting and International Weinstein Committee

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Weinstein Conference History

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General Announcements

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Floor Plan

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The Traveling Weinstein Heart Carpet

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Sponsors 11 Condensed Conference Schedule

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Detailed Conference Schedule

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Keynote Speakers

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Venue 18 Abstracts: Platform Sessions

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Abstracts: Technology breakout Session

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Abstracts: Poster Sessions

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List of Authors

258

Notes 266

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WELCOME

TO THE 2015 WEINSTEIN CONFERENCE IN BOSTON

On behalf of the organizing committee, it is a great pleasure to welcome you to the 2015 Weinstein Cardiovascular Development Conference, hosted in Boston, MA, USA. Known for its old-world architecture, rich history and premier academic institutions, Boston is well on its way towards becoming a truly cosmopolitan city. The 2015 Weinstein Conference is hosted by Boston Children’s Hospital at the John B. Hynes Veterans Memorial Convention Center. The Hynes Center is located in the heart of Boston’s Back Bay neighborhood, a short distance from many of Boston’s most well-loved attractions such as the Public Garden, Fenway Park and Newbury Street. This year, the Weinstein Conference welcomes around 325 attendees, with 72.3 percent from North America, 25.2 percent from Europe, 7.3 percent from Asia, and .6 percent from Australia. The meeting has been organized to maximize scientific discussion on all aspects of heart development and

congenital heart disease through platform and poster sessions. This year we are all honored to have the participation of two keynote speakers: Mark Krasnow, MD, PhD (Stanford, CA, USA) and Christopher Walsh, MD, PhD (Boston Children’s Hospital, MA, USA). As is traditional for this meeting, the talks for platform sessions were selected from submitted abstracts to feature the most exciting new work in the field. We received a large number of excellent abstracts, which were all subjected to blind evaluation by an internal panel of 25 judges representing the interests of all researchers. This year, we are happy to feature over 200 poster presentations. The conference schedule includes talks, discussion times, poster sessions, meals and free time, all intended to promote interaction and collaboration. We hope you enjoy the Weinstein 2015 Conference!

Boston Children’s Hospital Organizing Committee Co-chairs:

Geoffrey Burns

William T. Pu

Assistant Professor of Medicine, Harvard Medical School, Department of Medicine, Massachusetts General Hospital

Professor of Pediatrics, Harvard Medical School, Department of Cardiology, Boston Children’s Hospital

Da-Zhi Wang Associate Professor of Pediatrics, Harvard Medical School, Department of Cardiology, Boston Children’s Hospital

Members: Jonathan Seidman Professor of Genetics, Harvard Medical School

Calum MacRae Associate Professor of Medicine, Harvard Medical School; Chief of Cardiology, Brigham and Women’s Hospital

Ibrahim Domian Assistant Professor of Medicine, Harvard Medical School, Department of Medicine, Massachusetts General Hospital

Maria Kontaridis Assistant Professor of Medicine, Harvard Medical School, Department of Medicine, Beth Israel Deaconess Medical Center

Boston Children’s Hospital Planning Committee: Michael Anderegg Executive Director, Heart Center

Francisco J. Naya

Janine Santimauro

Associate Professor of Biology, Boston University; Member, Whitaker Cardiovascular Institute

Department Administrator, Heart Center

Sean Wu Assistant Professor of Medicine, Stanford University; Member, Stanford Cardiovascular Institute

Caroline Burns Assistant Professor of Medicine, Harvard Medical School, Department of Medicine Massachusetts General Hospital

Martha Degnan Marketing Director, Heart Center

Erin Jemiola Marketing Communications Manager

Victoria Cunningham Continuing Medical Education

Priscila Paulino Heart Center Marketing

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 5

CHARTER

OF THE WEINSTEIN MEETING AND INTERNATIONAL WEINSTEIN COMMITTEE

Scope of the Conference

Obligations of the Participants

The Weinstein Cardiovascular Development Conference is an annual meeting for scientists investigating normal and abnormal development of the heart and vasculature as it may ultimately relate to human disease. It is a freestanding meeting unaffiliated with any society or parent organization. Interested individuals or groups from host institutions organize it on a rotating basis. The intent of the meeting is to advance the overall field of cardiovascular development through the sharing of information and the facilitation of collaborative investigations. True to the vision of Dr. Constance Weinstein, who first organized this conference, the meeting is intended to include as many perspectives as possible. Investigators in any relevant area who can provide contributions to our understanding of heart and vascular development are welcome to contribute.

One of the most important aspects of the Weinstein Conference has been the willingness of the participants to share new and unpublished information. This has provided opportunities for the participants to devise new experiments and develop new hypotheses in a collaborative manner. It is expected that all participants will participate in a collegial and ethical manner with respect to information obtained at the Weinstein Conference. Permission should be obtained before disclosure of another investigator’s unpublished data.

Organization of the Conference In order to provide a corporate memory and to maintain the quality of the conferences, the participants of the 1998 meeting voted to form an organizing committee called the “Weinstein Committee.” The makeup of the Committee is comprised of representatives from the next two proposed meeting sites and two “at large” members voted upon by the conference participants. “At large” members serve a three-year term. The responsibility of the Committee is to assist the local organizing committee with meeting arrangements, organization and funding. In addition, the Committee is charged with soliciting nominations for future meeting sites and hosts. Such nominations will then be brought to a vote by attendees during the meeting. Meeting sites will be selected by vote such that the local organizing committee will have a two-year lead time. In the event that multi-year funding is sought from the National Institutes of Health or other national sources, the Weinstein Committee will participate in this process. Local Organizing Committees To provide a varied flavor and the opportunity for new approaches, each host institution will form a local organizing committee to select a meeting venue and format. The site should be selected for its potential to optimize informal communication and interaction. As a way to emphasize new and topical information, organizers from the host institution should select speakers from among the submitted abstracts. Scheduling should include opportunities for new voices and encourage the development of students, fellows and younger faculty. Ample time for discussion is to be provided.

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Similarly, investigators pursuing similar experiments should inform a presenter if the divulged information has a bearing on their own work. All participants in the conference should be willing to share their expertise and reagents in the collective advancement of the area of cardiovascular development. Annual Business Meeting Each Weinstein Conference will include time set aside for a business meeting. At this time, participants will vote on future host and meeting site selection and may consider changes in the direction of the conference or its organization. At the 1999 meeting in Tucson, Arizona, this Charter was distributed to the participants and ratified. Its provisions commenced at the business meeting of the 1999 Tucson, Arizona Conference. The Charter will remain in effect until modified by a vote of participants at an annual business meeting. International Weinstein Committee (term expiration indicated in parentheses) Ivan Moskowitz University of Chicago, 2015 Parker Antin University of Arizona, 2016 Jose Luis de La Pompa Centro Nacional de Investigaciones Cardiovasculares, Madrid, 2017 William Pu Harvard, 2018 Frank Conlon Univ. North Carolina, 2019 Vidu Garg Ohio State University, 2020 Joy Lincoln At large member; Ohio State University Robert Kelly At large member; Developmental Biology Institute of Marseille

WEINSTEIN CONFERENCE HISTORY The Weinstein Cardiovascular Development Conference is a continuation of an annual meeting of investigators funded by three separate RFAs on cardiac development in 1986, 1988 and 1990 at the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH). Grants funded under the RFA mechanism resulted in a total of 26 research programs in the area of cardiac development. A separate program of Specialized Centers in Research (SCORs) in congenital heart disease resulted in the addition of investigators from the Universities of Iowa, Pennsylvania and Rochester. Investigators were brought together annually at the National Institutes of Health to discuss their latest research results and potential areas of collaboration. The first meeting was held in a basement meeting room at the NIH and included approximately a dozen participants. These annual meetings at the NIH between 1986 and 1993 continued to grow as

new research programs were funded and by word of mouth with collaborators. These meetings coincided with the emergence of molecular, biological and genetic approaches to the investigation interest in keeping these collegial and informative meetings going. Independent meetings have been held every year since 1994.

Weinstein Conference Host Institutions and Sites

15. Texas A&M University of Houston (Houston, 2008)

1. Medical University of South Carolina (Charleston, 1994)

16. University of California, San Francisco (San Francisco, 2009)

2. University of Rochester (Rochester, 1995) 3. University of Pennsylvania (Philadelphia, 1996) 4. University of Cincinnati (Cincinnati, 1997) 5. Vanderbilt University (Nashville, 1998) 6. University of Arizona (Tucson, 1999) 7. Washington University (St. Louis, 2000) 8. University of Texas, Southwestern Medical School (Dallas, 2001) 9. University of Utah (Salt Lake City, 2002)

Dr. Constance Weinstein of the NHLBI was instrumental in focusing the attention of the NIH on the need for research in this area. She obtained the funds for the RFA and SCOR programs and organized the intramural meetings held at the NIH. To honor Dr. Weinstein, in 1995, the meeting was formally named the Weinstein Cardiovascular Development Conference. Dr. Weinstein is retired from NHLBI, but she tries to attend these meetings to monitor progress in the field that she spent so much

17. University of Amsterdam, Netherlands (Amsterdam, 2010) 18. University of Cincinnati (Cincinnati, 2011) 19. University of Chicago (Chicago, 2012) 20. University of Arizona (Tucson, 2013) 21. Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III Madrid 22. Boston Children’s Hospital (Boston, 2015)

10. Harvard University (Cambridge, 2003) 11. Leiden University (Leiden, Netherlands, 2004) 12. University of Arizona (Tucson, 2005) 13. University of South Florida (Tampa/St. Petersburg, 2006) 14. Indiana University School of Medicine (Indianapolis, 2007)

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 7

GENERAL ANNOUNCEMENTS Oral Presentations

Catering

All platform sessions will be held in Ballroom A. Each session is scheduled to last 90 minutes in total with 5 speakers. This includes 15 minutes for the presentation followed by 3 minutes for discussion. It is important that the participants do not exceed the allocated time.

Throughout the conference, we will be offering breakfast, lunch and snacks. On Thursday and Friday evenings, we will have light receptions with hors d’oeuvres, and on Saturday evening, we will host a dinner for all attendees. If you have any allergies or dietary restrictions, please inform an event staff member.

Posters Poster sessions will be held in Ballroom B. All posters will be displayed for the entirety of the allotted poster session time. Posters are all numbered according to the abstract portion of this program book. The first poster session will be held on Thursday, April 30. If you are planning on showing a poster during the poster session, please be sure to hang your poster between 12:30 p.m. and 1:15 p.m. on Thursday, April 30. You will be able to find your abstract number in this program book (list begins on page 13). Please hang your poster on the poster board assigned to this number. We ask that all presenters with even numbers stand by their posters for discussion during poster session A. All presenters with odd numbers should stand by their posters for discussion during poster session B.

Business Meeting The business meeting will be held on Saturday, May 2 from 8 to 9 a.m. in room 301 (across from registration). Awards The following awards will be presented at the end of the meeting: • Markwald Award for the best oral presentation by predoc/postdoc • Weinstein Award for best poster • Travel Awards Farewell Party All participants are invited to attend a farewell party held at the Hynes Convention Center on Saturday, May. The farewell party will be held room in 302. Evaluations Evaluations will be sent to all participants via e mail after the conference. These evaluations provide important feedback and help the organizers improve future meetings. Acknowledgements We are very grateful to the people who have worked diligently behind the scenes to make this meeting a success. First of all, we thank Boston Children’s Hospital for hosting the 2015 Weinstein Cardiovascular Development Conference. We also very sincerely thank all of the sponsors and the reviewers for selecting the stand-out abstracts for oral presentations. We thank Mark Krasnow and Christopher Walsh for serving as our keynote speakers this year. And finally, we thank Dr. Constance Weinstein for her continued dedication to the mission of this meeting.

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FLOOR PLAN Hynes Convention Center 900 Boylston Street Boston, Massachusetts 02115 LEVEL 3

Drop-Off (Lower Level)

Boylston Street

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 9

THE TRAVELING WEINSTEIN HEART CARPET Commissioned by Dr. Nadia Rosenthal, this is a silk Persian rug from the Kerman Province of Iran. The design was based on a “boteh”, or medallion design from a 19th century Afshar rug. The images in the rug represent stages of heart development in the embryo. The second row shows the lateral heart masses after gastrulation. The third row shows the cardiac crescent. Subsequent rows show stages in the heart tube as looping proceeds. The last row shows a four-chambered heart with lungs on each

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side. Somite structures can be seen to change during the growth of the embryo as well. The reciprocating trefoils around the edge represent the DNA double helix with four colors to represent the four bases. The image of this rug was used for the first edition of “Heart Development,” edited by Richard P. Harvey and Nadia Rosenthal. The rug was donated to the Weinstein Cardiovascular Development Conference in 2001 and is transferred every year to the host institution for display.

SPONSORS Hosted by

Thank you to our 2015 Weinstein Cardiovascular Development Conference Sponsors GOLD LEVEL SPONSORS

SILVER LEVEL SPONSORS

Cardiovascular Institute ADDITIONAL SUPPORT PROVIDED BY American Association of Anatomists American Heart Association’s Councils on Basic CV Sciences, CV Disease in the Young, and Functional Genomics and Translational Biology Biocytagen, LLC Cyagen Biosciences, Inc. Developmental Dynamics Fisher Scientific Life Technologies Mouse Specifics, Inc. Nikon Instruments, Inc. VisualSonics, Inc.

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 11

CONDENSED CONFERENCE SCHEDULE THURSDAY 4/30/2015 13:15 13:30 Welcome and opening remarks: Ballroom A 13:30 15:00

Platform session I, Ballroom A Cardiogenesis, cardiac lineages, and early heart development Moderators: Geoff Burns and Brian Black

15:00 15:20 Break 15:20 16:50

Platform session II, Ballroom A Second heart field, outflow tract, and vascular development Moderators: Caroline Burns and Robert Kelly

16:50 17:15 Break 17:15 18:15

Keynote Lecture I: Mark Krasnow, Stanford and HHMI, Ballroom A Dissecting lung and vascular development at single cell resolution

18:15 22:00 Light reception & Poster Session A, Ballroom B Poster presenters at posters from 18:30-20:00

FRIDAY 5/1/2015 7:00

9:00

Breakfast & Posters, Ballroom B

9:00 10:30 Platform session III, Ballroom A Myocardial development and cardiomyopathies Moderators: Frank Naya and Ibrahim Domian 10:30 11:00 Break 11:00 12:30 Platform session IV, Ballroom A Trends in cardiovascular development Moderators: Joe Yost and Weinian Shou 12:30 12:45 Break 12:45 14:15 Breakout sessions I: 1. Career development I, Ballroom A Moderator: Maria Kontaridis 2. Technology fair, Room 302 Organizer: William Pu 14:15

14:30 Break

14:30 16:00 16:00

Platform session V, Ballroom A Epicardium, coronary vessels, conduction system, and arrhythmias Moderators: William Pu and Bin Zhou

16:30 Break

16:30 18:00

Platform session VI: Ballroom A Endocardium and cardiac valves Moderators: Maria Kontaridis and Joy Lincoln

18:00 22:00 Light reception & Poster Session B, Ballroom B Poster presenters at posters from 18:15-19:45

SATURDAY 5/2/2015 7:00

8:30

Breakfast and posters, Ballroom B

8:30

9:00

Business meeting, Ballroom A

9:00 10:30

Platform session VII, Ballroom A Cardiac stem cells, growth, and regeneration Moderators: Sean Wu and Ken Poss

10:30 11:00 Break 11:00 12:30 Platform session VIII, Ballroom A Cardiovascular genomics and transcriptional and epigenetic regulation Moderators: Da-Zhi Wang and Frank Conlon 12:30 12:45 Break 12:45 14:15 Breakout sessions II: 1. Career development II. Ballroom A Moderator: Maria Kontaridis 2. Trends and controversies in cardiovascular development Room 304 Moderator: Da-Zhi Wang 14:15

14:30 Break

14:30 16:00

Platform session IX: Ballroom A Cardiovascular genetics Moderators: Calum MacRae and Vidu Garg

16:00 16:30 Break 16:30 17:30

Keynote Lecture II: Christopher Walsh, Boston Children’s and HHMI Ballroom A Genes underlying human developmental brain disorders

18:00 23:00 Closing banquet and awards, Room 302 12

DETAILED CONFERENCE SCHEDULE

AND LIST OF POSTERS

THURSDAY 4/30/2015 13:15 13:30 Welcome and opening remarks, Ballroom A 13:30 15:00

Platform session I, Ballroom A Cardiogenesis, cardiac lineages, and early heart development Moderators: Geoff Burns and Brian Black

1-01. Lionel Christiaen: New York University Regulation of cardiopharyngeal fates specification in a simple chordate 1-02. Ian Scott: The Hospital for Sick Children Aplnr and its ligand Elabela have opposite effects on Nodal signaling during cardiac development 1-03. Daniela Panakova: Max Delbrück Center PCP-driven Cardiac Remodeling Couples Changes in Actomyosin Tension with Myocyte Differentiation 1-04. Tao P Zhong: Fudan University Regulation of Vertebrate Ciliogenesis and Heart Development

2-03. Megan Rowton: University of Chicago Hedgehog signaling modulates cardiac progenitor differentiation status 2-04. Kelly Smith: University of Queensland Transmembrane protein 2 (tmem2) is required during cardiovascular development to modulate the ECM 2-05. Sean Li: Boston Children’s Hospital Identification of intrapericardial arterial trunk smooth muscle progenitors 16:50

17:15 Break

17:15 18:15

Keynote Lecture I: Mark Krasnow, Stanford and HHMI, Ballroom A Dissecting lung and vascular development at single cell resolution

18:15 22:00

Light reception & Poster Session A, Ballroom B Poster presenters at posters from 18:30-20:00

1-05. Yuika Morita: IMCB, the University of Tokyo Cardiac cell induction and regeneration by Sall+;Mesp1 derived cells 15:00

15:20 Break

15:20 16:50

Platform session II, Ballroom A Second heart field, outflow tract, and vascular development Moderators: Caroline Burns and Robert Kelly

2-01. Zaffran Stephane: INSERM Expression of HOXB1 in second heart field progenitor cells is essential for normal heart development 2-02. Ariel Rydeen: Cincinnati Children’s Hospital Cyp26 enzymes are required for second heart field addition and ventricular maintenance

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 13

FRIDAY 5/1/2015 7:00 9:00 Breakfast & Posters, Ballroom B 9:00 10:30 Platform session III, Ballroom A Myocardial development and cardiomyopathies Moderators: Frank Naya and Ibrahim Domian 3-01. Silvia Martín Puig: CNIC HIF1 and cardiovascular development: how metabolic regulation influences ventricular chamber formation 3-02. Ibrahim Domian: MGH Atypical Protein Kinase C Dependent Polarized Cell Division Directs Myocardial Trabeculation 3-03. Mingfu Wu: Albany Medical College Lineage tracing reveals that oriented division underlies trabecular morphogenesis and differentiation 3-04. Ethan David Cohen: University of Rochester SMD Daam1 and Daam2 are redundantly required for myocardial maturation 3-05. Zhanpeng Huang: Boston Children’s Hospital CIP/MLIP senses pathophysiological stresses to regulate cardiac homeostasis 10:30 11:00 Break 11:00 12:30 Platform session IV, Ballroom A Trends in cardiovascular development Moderators: Joe Yost and Weinian Shou 7-06. Ayhan Atmanli: Massachusetts General Hospital Multiplex Analysis of Gene Expression in Individual Living Cells 1-06. Laurie Boyer: Massachusetts Institute of Technology Transcriptional Control of Cardiac Cell Fate 3-06. Dan DeLaughter: Harvard Medical School Single Cell Transcriptional Atlas of Cardiac Development 9-06. Xiaoqin Liu: University of Pittsburgh School of Medicine Etiology of hypoplastic left heart syndrome: insights from analysis of mutant mouse models 8-06. Pingzhu Zhou: Boston Children’s Hospital Identifying cell type specific enhancers using Cre-activated, lineage-restricted p300 ChIP-seq 12:30 12:45 Break 14

12:45 14:15 Breakout sessions I: 1. Career development I, Ballroom A Moderator: Maria Kontaridis 2. Technology Fair, Room 302 Organizer: William Pu 14:15 14:30 Break 14:30 16:00 Platform session V, Ballroom A Epicardium, coronary vessels, conduction system, and arrhythmias Moderators: William Pu and Bin Zhou 5-01. Ching-Ling (Ellen) Lien: Children’s Hospital Los Angeles Cxcl12 Chemokine guided angiogenesis directs coronary vasculature formation in zebrafish 5-02. Bin Zhou: Albert Einstein College of Medicine Notch signaling controls coronary angiogenesis by endocardial progenitors 5-03. Jinhu Wang: Duke University Epicardial regeneration is directed by the cardiac outflow tract and Hh signaling 5-04. Wenduo Ye: Tulane University Shox2 and Nkx2-5 antagonistically determine pacemaking cell fate in the pulmonary vein myocardium 5-05. Ozanna Burnicka-Turek: University of Chicago, Departments of Pediatrics T-box Rheostat Patterns Cardiac Conduction System Functional Domains 16:00 16:30 Break 16:30 18:00 Platform session VI, Ballroom A Endocardium and cardiac valves Moderators: Maria Kontaridis and Joy Lincoln 6-01. Diego Franco: University of Jaen miR-23b and miR-199a impairs EMT during atrioventricular endocardial cushion formation 6-02. Katelynn Toomer: MUSC Cilia and their function in Valve Development and Mitral Valve Prolapse 6-03. Fernanda Bosada: University of Oregon Wnt signaling has distinct and dynamic roles in semilunar and atrioventricular canal valve development 6-04. Eva Lana-Elola: National Institute for Medical Research New Down syndrome mice show AVSD with intact vestibular spine and reveal heart defects map to two loci 6-05. Lindsey J. Miller: The Ohio State University Exploring endothelial cell dynamics in aging heart valves 18:00 22:00 Light reception & Poster Session B, Ballroom B Poster presenters at posters from 18:15-19:45

SATURDAY 5/2/2015 7:00

8:30

8:30

9:00

9:00 10:30

Breakfast and posters, Ballroom B Business meeting, Ballroom A Platform session VII, Ballroom A Cardiac stem cells, growth, and regeneration Moderators: Sean Wu and Ken Poss

7-01. Hua Shen: University of Southern California Embryonic heart proliferation and neonatal heart regeneration controlled by IGF2 7-02. Vahid Serpooshan: Stanford University Nkx2.5+ Cardiomyoblast Contribution to Postnatal Cardiogenesis 7-03. Zhiqiang Lin: Boston Children’s Hospital Acetylation of VGLL4 regulates postnatal cardiac growth 7-04. Ge Tao: Baylor College of Medicine Pitx2 Promotes Heart Repair by Regulating Respiratory Chain Components and the Antioxidant Response 7-05. Christopher Antos: DFG-Center for Regenerative Therapies Dresden Calcineurin Inhibition Enhances Regeneration: Fish Appendages Can Lead to Understanding Organ Allometry 10:30

11:00 Break

11:00 12:30 Platform session VIII, Ballroom A Cardiovascular genomics and transcriptional and epigenetic regulation Moderators: Da-Zhi Wang and Frank Conlon 8-01. Brian L. Black: UCSF Cooperative transcriptional activation of paired MEF2 sites by Myocardin and MEF2C 8-02. Lauren Waldron: University of North Carolina An evolutionarily evolved Tbx5/ Chd4 interaction provides mechanistic insight into atrial septation. 8-03. Luis Luna-Zurita: Gladstones Institute of Cardiovascular Disease Genomic and structural basis for regulation of cardiogenesis by heterotypic transcription factors 8-05. Jian Ding: Boston Children’s Hospital Deletion of Trbp reveals a novel linear miR-208a-mediated pathway required for normal cardiac function

8-04. Youngsook Lee: Universtiy of Wisconsin-Madison Transcriptional Mechanisms Critical for Ventricular Wall Maturation 12:30

12:45 Break

12:45 14:15 Breakout sessions II: 1. Career development II, Ballroom A Moderator: Maria Kontaridis 2. Trends and controversies in cardiovascular development, Room 304 Moderator: Da-Zhi Wang 14:15

14:30 Break

14:30 16:00

Platform session IX: Ballroom A Cardiovascular genetics Moderators: Calum MacRae and Vidu Garg

9-01. Janel R. Cabrera: Beth Israel Deaconess Medical Center/Harvard Medical School Aberrant Endothelial-Myocardial Crosstalk Causes Hypertrophy in Noonan Syndrome with Multiple Lentigines 9-02. Anne-Karin Arndt: Department for Congenital Heart Disease and Pediatric Cardiology, University Kiel PRDM16 - a novel key player in personalized medicine 9-03. Silvia E. Racedo: Albert Einstein College of Medicine Increased Tbx1 gene dosage and the 22q11.2 duplication syndrome 9-04. H. Joseph Yost: University of Utah Recessive and compound heterozygous variants in novel gene pathways in congenital heart disease 9-05. Kern: Medical University of South Carolina Lumican Deficiency Results In Cardiomyocyte Hypertrophy With Altered Collagen Assembly 16:00

16:30 Break

16:30 17:30 Keynote Lecture II: Christopher Walsh, Boston Children’s and HHMI, Ballroom A Genes underlying human developmental brain disorders 18:00 23:00 Closing banquet and awards, Room 302

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 15

KEYNOTE SPEAKERS Christopher A. Walsh, MD, PhD [email protected] Christopher Walsh is Bullard Professor of Pediatrics and Neurology at Harvard Medical School, Chief of the Division of Genetics and Genomics at Boston Children’s Hospital, an Investigator of the Howard Hughes Medical Institute, and an Associate Member of the Broad Institute. Dr. Walsh completed his MD and PhD degrees (with Dr. Rainer Guillery) at the University of Chicago. After a neurology residency and chief residency at Massachusetts General Hospital, he completed a postdoctoral fellowship in genetics at Harvard Medical School with Dr. Connie Cepko. He joined the faculty at Beth Israel Deaconess Medical Center and Harvard Medical School in 1993, and has held the Bullard Professorship at Harvard since 1999. He joined Boston Children’s Hospital as Chief of Genetics in 2006. Dr. Walsh’s research has focused on the development, evolution, and function of the human cerebral cortex, the part of our brain responsible for our highest cognitive abilities. He has studied the basic biology of cell division and migration in the developing brain, and has pioneered the analysis of human genetic diseases that disrupt the structure and function of the cerebral cortex. His laboratory has identified genetic causes for more than twenty brain diseases of children, associated with autism, intellectual disability, seizures, and cerebral palsy, by fostering worldwide collaborations with physicians and families. Among his awards are a Jacob Javits Neuroscience Investigator Award from the National Institute of Neurological Disorders and Stroke, the Dreifuss-Penry Award from the American Academy of Neurology, the Derek Denny-Brown Award and the Jacoby Award from the American Neurological Association, the Research Award from the American Epilepsy Society, the Krieg Award from the Cajal Club, and the Wilder Penfield Award from the Middle Eastern Medical Assembly. He is an elected member of the American Neurological Association, the American Association of Physicians, and the Institute of Medicine, and is an elected fellow of the American Association for the Advancement of Sciences.

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Mark Krasnow, M.D., Ph.D. [email protected] Mark Krasnow received his BS summa cum laude from University of Illinois, and his PhD. in biochemistry and MD from the University of Chicago in the Medical Scientist Training Program, where he worked with Dr. Nicholas Cozzarelli elucidating the molecular mechanisms of site-specific recombination enzymes. He was a Helen Hay Whitney postdoctoral fellow with Dr. David Hogness at Stanford University, where he demonstrated the transcriptional regulatory function of Drosophila homeotic proteins. In 1988, he joined the faculty of the Department of Biochemistry at Stanford University School of Medicine, and he is currently Professor of Biochemistry and an Investigator of the Howard Hughes Medical Institute. In his own lab he has pioneered the use of genetic and genomic approaches to identify the cellular and molecular programs of respiratory and vascular system development and maintenance, and the elucidation of respiratory control circuits. He began this work by establishing the Drosophila tracheal (respiratory) system as a tractable model, and his current work focuses on the mammalian lung and vasculature. His long-term goal is to understand how these programs go awry in disease and how this information can be used to develop new treatments, including regenerative strategies. Krasnow is also the Executive Director of the Vera M. Wall Center for Pulmonary Vascular Disease, and he previously served as Chair of the Department of Biochemistry and Director of the Stanford Medical Scientist Training Program. He was a Lucille P. Markey Scholar and an NSF Presidential Young Investigator, and is a Fellow of the American Association for the Advancement of Science and of the American Academy of Arts and Sciences.

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 17

WELCOME TO THE HYNES CONVENTION CENTER

THE BEST-LOCATED MAJOR CONVENTION CENTER IN THE COUNTRY! While you’re here at the Hynes in Boston’s bustling and historic Back Bay neighborhood, you’re just steps away from our beloved Newbury Street, the Shops at the Prudential Center, and Copley Square, offering hundreds of worldclass dining and shopping attractions, public transportation and other amenities. Named for a former Boston mayor, the Hynes Auditorium opened in 1965 and was officially renamed the John B. Hynes Veterans Memorial Convention Center in 1994 after a major expansion made the facility into what it is today. Not only are you surrounded by history, culture, scenery and activities at the Hynes, but our facility offers state-of-the-art technology—including complimentary WiFi— dramatic views of the city, and unparalleled customer service. In Boston, we pride ourselves on unbeatable meetings technology and exceptional customer service. Delivering remarkable events is not just something we aim to do—it is our signature. You’ll find creativity, attention to detail and a highly knowledgeable staff at our convention centers. And this signature level of service runs throughout Boston’s hospitality culture from our restaurants to our hotels to our tour guides and beyond. Our dedication to exceeding visitors’ expectations is what has made Boston a top North American destination for meetings and conventions for the last eight years in a row, and ICCA’s number one U.S. destination in 2014 for international association meetings. Boston is a city that has always been celebrated for its role in American history, particularly as the birthplace of the American Revolution. Today, Boston is still shaping history by playing a major role in our knowledge-based world economy as a hub of ideas, innovation and entrepreneurship. The mix of renowned educational and research institutions, leading medical centers, the growing Innovation District and massive amounts of culture make Boston a vibrant and fun place to live, visit and do business. We’re confident you’re going to enjoy every minute you spend here.

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PLATFORM SESSIONS

ABSTRACTS

2015 WEINSTEIN CARDIOVASCULAR DEVELOPMENT CONFERENCE 19

Platform Session 1 1. Cardiogenesis/cardiac lineages/early heart development 1-01

REGULATION OF CARDIOPHARYNGEAL FATES SPECIFICATION IN A SIMPLE CHORDATE Lionel Christiaen Florian Razy-Krajka, Wei Wang, Nicole Kaplan, Karen Lam, Lionel Christiaen New York University In vertebrates, the pharyngeal subset of head muscles shares a common origin with cardiomyocytes in the cardiopharyngeal mesoderm of early embryos. The ascidian Ciona intestinalis offers an opportunity to study cardiopharyngeal fate specification with cellular resolution in a simple chordate model. In ascidians, bipotent cardiopharyngeal progenitors undergo asymmetric cell divisions to produce first and second heart precursors and pharyngeal muscle precursors following a stereotyped clonal pattern. Using targeted perturbations, Fluorescence Activated Cell Sorting (FACS), transcriptome profiling and fluorescent in situ hybridization, we showed that multipotent cardiopharyngeal progenitors express a mixture of early cardiac and pharyngeal muscle regulators. This multilineage transcriptional priming is resolved through regulatory cross-antagonisms, whereby Tbx1/10 inhibits Gata4/5/6 and heart specification in the pharyngeal muscle precursors, thus promoting Ebf expression and muscle fate specification; while, in the heart precursors, Nk4/Nkx2-5 inhibits Tbx1/10 and Ebf, permitting Gata4/5/6 expression and cardiac specification. We demonstrate how polarized signaling events regulate the initial asymmetries and trigger the transcriptional cascades underlying initial heart vs. pharyngeal muscle fate choices. Current single cell RNA-seq analyses and perturbation studies further characterize the regulatory dynamics underlying fate choices in the early cardiopharyngeal mesoderm.

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Platform Session 1 1. Cardiogenesis/cardiac lineages/early heart development 1-02

APLNR AND ITS LIGAND ELABELA HAVE OPPOSITE EFFECTS ON NODAL SIGNALING DURING CARDIAC DEVELOPMENT Ian C Scott Ashish R Deshwar, Serene C Chng, Lena Ho, Bruno Reversade, Ian C Scott The Hospital for Sick Children The Apelin receptor (Aplnr) and its early ligand Elabela (Ela) are essential for heart development by controlling the migration of cardiac progenitors to the anterior lateral plate mesoderm, but how this happens is unclear. In this study we demonstrate that Aplnr signaling modulates Nodal/TGFβ signaling during gastrulation, a key pathway essential for mesendoderm induction and migration. Loss of Aplnr function leads to a reduction in Nodal target gene expression whereas activation of Aplnr increases the expression of these same targets. Furthermore, loss of Aplnr results in a delay in the expression of the cardiogenic transcription factors mespaa/ ab. By elevating Nodal levels in aplnrb mutant embryos we are able to fully restore cardiac differentiation. We find that loss of Aplnr attenuates the activity of a point source of the Nodal ligands Squint and Cyclops in part by regulating their extracellular processing. Most unexpectedly, we find that in the context of Nodal modulation the loss of Ela results in increased Nodal signaling, pointing to the possibility that Ela may work as an inhibitory ligand to Aplnr. We favour a model in which the antagonism between Ela and its receptor Aplnr is able to fine tune Nodal output to initiate the migration of lateral margin cells to the heart forming region. We propose that the Elabela-Aplnr signaling cascade may therefore act as a specific rheostat for the Nodal/TGFβ pathway during the earliest stages of cardiogenesis.

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Platform Session 1 1. Cardiogenesis/cardiac lineages/early heart development 1-03

PCP-DRIVEN CARDIAC REMODELING COUPLES CHANGES IN ACTOMYOSIN TENSION WITH MYOCYTE DIFFERENTIATION Daniela Panakova Marie Swinarski, Anne M. Merks, Alexander M. Meyer, Stefan Donat, Salim Abdelilah-Seyfried, Daniela Panakova Max Delbrück Center During vertebrate cardiogenesis the two-chambered looped heart is formed from a simple linear tube. To date, this process is incompletely understood. Changes in tissue architecture are controlled by junctional remodeling, cell intercalations, and collective cell migration. Wnt/PCP signaling plays a crucial role in guiding the tissue remodeling. Here, we show that the non-canonical ligands Wnt11 and Wnt5b and the PCP core components Fzd7, Vangl2, Dvl2, and Pk1 are involved in the regulation of cell re-arrangements during cardiac chamber formation. Downstream effectors of the PCP pathway target cell adhesion, cytoskeleton, and migration. We show that deficient PCP signaling affects the cardiac chamber architecture through changes in actomyosin organization. We observe specific changes in the localization of the phosphorylated non-muscle myosin II regulatory light chain that are regulated by the PCP pathway core components. Moreover, this process is accompanied by impaired SRF signaling. Taken together, our data indicate that the Wnt/PCP pathway regulates cardiac chamber remodeling by modulating actomyosin activity that is coupled to mechanosensitive signaling controlling cardiomyocyte differentiation.

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Platform Session 1 1. Cardiogenesis/cardiac lineages/early heart development 1-04

REGULATION OF VERTEBRATE CILIOGENESIS AND HEART DEVELOPMENT Tao P Zhong D Jin, TT Ni, J Sun, JD Amack, G Yu, B Zhou, IA Drummond, JD Schuetz, J Malicki, Chiang Chin, Tao P. Zhong Fudan University Cilia are microtubule-based organelles that mediate signal transduction and regulate heart development. The signaling cascades that regulate cilia formation and heart development remain incompletely understood. Here we report that prostaglandin signaling is crucial to ciliogenesis and cardiac left-right asymmetry. We analyzed the zebrafish leakytail (lkt) mutant that displays ciliogenesis defects and randomized heart looping. Positional Cloning reveals that lkt encodes an ATP-binding cassette transporter expressed in a subset of epithelia. We found that Lkt localizes on the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCCT804M mutant. Consistently, PGE2 synthesis enzymes, Cyclooxygenase-1 and its receptor, EP localized in the cilium, are required for proper cilia formation and elongation. Increased cAMP synthesis rescues ciliogenesis and randomized heart looping in embryos deficient for EP activity. Importantly, PGE2 signaling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt-mediated PGE2 signaling acts through a ciliary G-protein-coupled receptor, EP, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis in regulating left-right asymmetry of the heart and other organs. Our findings thus delineate a novel ciliogenic cascade and mechanism that regulates vertebrate ciliogenesis and heart development.

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Platform Session 1 1. Cardiogenesis/cardiac lineages/early heart development 1-05

CARDIAC CELL INDUCTION AND REGENERATION BY SALL+;MESP1- DERIVED CELLS Yuika Morita Yuika Morita, Peter Anderson, Yuko Tsukahara, Chulan Kwon, Kazuko Koshiba-Takeuchi, Jun K Takeuchi IMCB, the University of Tokyo It has been considered that Mesp1, a key regulator for cardiac lineage commitment, regulate all cardiac cell lineages before e9.5. However, Mesp1-lineage analysis showed that 15~20% cells in heart were formed from non-Mesp1 derived cells at 8.5~9.5, and these cell number was increased up to 30% at postnatal heart. Here we show that non-Mesp1 derived cardiac cell population, mainly marked by Sall1, contributes to cardiac lineage and its regeneration in the mice heart. Sall1 regulating non-Mesp1 derived cardiac progenitor, clearly differentiated into functional cardiomyocytes and conduction cells from Sall-GFP;Mesp1cre;ROSA-RFP mice embryos, but not other organ cells fate (ex; skeletal muscle, vascular, neuron, pancreas gene programs). Loss-of-function analysis using Sall1/Mesp1 DKO mice led to completely no heart morphology, and gain-of-function analysis showed that DOX-inducible overexpression of Sall1-Mesp1 in hiPS cells efficiently differentiate into cardiomyocytes. Interestingly, Sall+ cells were re-induced in heat during early cardiac regeneration, and positively contributed into cardiomyocytes from non-Mesp1 derived cell lineage in mice model, suggesting that Sall+ cells will be an effective source for heart repair in human in the future. In this conference, we will show an origin of Sall+ cells in cardiac regeneration and its potential for cardiomyocyte renewal.

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Platform Session 2 2. Outflow tract/second heart field/cardiac neutral crest/vasculature 2-01

EXPRESSION OF HOXB1 IN SECOND HEART FIELD PROGENITOR CELLS IS ESSENTIAL FOR NORMAL HEART DEVELOPMENT ZAFFRAN Stephane LAFOREST Brigitte, ROUX Marine, BERTRAND Nicolas, PUCEAT Michel, ZAFFRAN Stephane INSERM During development the embryonic heart tube grows by progressive addition of second heart field (SHF) progenitor cells to its arterial and venous poles. Our previous study in the mouse identified the expression of Hoxa1 and Hoxb1 in distinct sub-domains of the SHF that contribute to the atrial and sub-pulmonary myocardium. We recently showed that Hoxb1-/- and compound Hoxa1-/-;Hoxb1+/- embryos displayed premature differentiation of cardiac progenitors in the SHF leading to misalignment of the OFT. To investigate a role of Hoxb1 in cardiac progenitors, we generated a novel CAG-Hoxb1 transgenic allele in mice, allowing activation of Hoxb1 in cells in which it is normally not expressed. Our initial results demonstrated that ectopic expression of Hoxb1 in the anterior SHF, using Mef2c-AHF-Cre, compromises the contribution of this region and give rise to heart with a hypomorphic right ventricle. Interestingly, we observed that markers of the left ventricular myocardial cells are expressed in the right ventricle of transgenic hearts, suggesting that right ventricular identity was affected. To determine how Hoxb1 restricts SHF identity we are evaluating potential target genes by ChiP sequencing and RNA seq using mouse embryonic stem cell and embryonic models. These results strongly indicate that Hox genes need to be restricted both spatially and temporally for normal heart development to occur.

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Platform Session 2 2. Outflow tract/second heart field/cardiac neutral crest/vasculature 2-02

CYP26 ENZYMES ARE REQUIRED FOR SECOND HEART FIELD ADDITION AND VENTRICULAR MAINTENANCE Ariel Rydeen Ariel Rydeen, Joshua Waxman Cincinnati Children’s Hospital Excess retinoic acid (RA) is teratogenic, including a prevalence of outflow tract (OFT) defects. The main factors that limit RA in vertebrates are Cyp26 enzymes. While Cyp26 deficient embryos have cardiac malformations, the mechanisms underlying these defects are not completely understood. We found that Cyp26 deficient embryos have OFT defects, which arise from two phases of ventricular cardiomyocyte (VC) loss. First, there is a failure of VC addition from the second heart field (SHF). Second, there is a progressive loss of first heart field VCs, due to VCs exiting the heart. Interestingly, we found the VC defects in Cyp26 deficient embryos are mediated by inverse effects on FGF signaling and matrix metalloproteinases (MMPs), both of which can affect interactions with the extracellular environment. Importantly, we found restoring FGF signaling in Cyp26 deficient embryos, which have decreased fgf8 expression, can restore SHF addition. Furthermore, attenuating MMP function in Cyp26 deficient embryos, which have increased mmp9 expression, can restore SHF addition and VC maintenance. However, restoring FGF signaling does not attenuate mmp9 expression and attenuating MMP function does not restore fgf8 expression, suggesting that FGF and MMPs act in parallel to disrupt SHF addition. Altogether, our results indicate a novel mechanism by which excess RA signaling, due to Cyp26 deficiency, causes perturbations in the extracellular environment and promotes ventricular OFT defects.

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Platform Session 2 2. Outflow tract/second heart field/cardiac neutral crest/vasculature 2-03

HEDGEHOG SIGNALING MODULATES CARDIAC PROGENITOR DIFFERENTIATION STATUS Megan Rowton Megan Rowton, Andrew Hoffman, Jeff Steimle, Chul Kim, Alex Guzzetta, Shuhan Yu, Erika Hanson, Ivan P. Moskowitz University of Chicago Cardiac septation requires Hedgehog (Hh) signaling in the second heart field (SHF). The Hh signal is transduced by degradation of the Gli3T transcriptional repressor and induction of the Gli1 transcriptional activator. We found that during murine embryonic stem cell (mESC) differentiation, Gli1 expression decreased as cells transitioned from mesoderm to cardiomyocytes, and conversely, that Gli3T expression increased as cardiomyocytes differentiated. In vivo, Gli3 expression is absent from SHF progenitors and nuclear-localized in atrial cardiomyocytes. In mESC lines with inducible expression of Gli1 and Gli3T, Gli1 overexpression at cardiac progenitor stages caused upregulation of SHF Hedgehog targets and reduced cardiomyocyte differentiation. Gene expression and chromatin accessibility studies revealed that the differentiation of Gli1-overexpressing cells was arrested at the cardiac mesoderm stage. We used RNA-seq and ChIP-seq to define direct SHF Hh targets in vivo. We found that Hh signaling acts as a molecular switch on SHF-specific enhancers, activating them in cardiac progenitors and repressing them in cardiomyocytes. Removal of Gli binding sites afforded inappropriate activation of SHF enhancers in cardiomyocytes in vivo. These results reveal the molecular logic of Gli action on SHF enhancers and suggest that Gli1 / Gli3T dynamics modulate the differentiation state of cardiac progenitors and cardiomyocytes.

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Platform Session 2 2. Outflow tract/second heart field/cardiac neutral crest/vasculature 2-04

TRANSMEMBRANE PROTEIN 2 (TMEM2) IS REQUIRED DURING CARDIOVASCULAR DEVELOPMENT TO MODULATE THE ECM Kelly A. Smith Jessica De Angelis, Anne Lagendijk, Huijun Chen, Neil Bower, Mathias Francois, Jeroen Bakkers, Alpha Yap, Benjamin M. Hogan, Kelly A. Smith University of Queensland The endocardium and blood vasculature are comprised of endothelial cells and share common genetic regulators. tmem2 regulates cardiac morphogenesis in the zebrafish and we show that tmem2 is also required for angiogenesis. Tmem2 is a single-pass transmembrane protein, with the C-terminus oriented extracellularly. We show the C-terminus is sufficient to rescue the tmem2 angiogenesis defect but only if secreted. Tmem2’s nearest homologue can degrade hyaluronic acid (HA) so we examined HA in tmem2 mutants. By fusing an HA-binding protein domain to GFP, we made a fluorescent reporter for HA localization in live zebrafish embryos. This tool can label cardiac jelly and the matrix surrounding major vessels and angiogenic sprouts. Quantification shows increased fluorescence in tmem2 mutant embryos compared with siblings, indicating excess matrix associated with tmem2 mutant phenotypes. Surprisingly, injection of hyaluronidase into tmem2 mutants rescues the angiogenesis defect but causes cardiac dysmorphology in both siblings and mutants, suggesting that, whilst the cellular mechanism causing the heart and vascular defects may be similar, the heart is more sensitive to ECM modulation. Finally, we show that whilst expression of vegf components is unchanged, vegf intracellular signaling is reduced in tmem2 mutants. This data suggests that when Tmem2 is absent, excess HA results, preventing ligand-induced signaling and morphogenesis defects of the cardiovascular system.

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Platform Session 2 2. Outflow tract/second heart field/cardiac neutral crest/vasculature 2-05

IDENTIFICATION OF INTRAPERICARDIAL ARTERIAL TRUNK SMOOTH MUSCLE PROGENITORS Sean (Xue) Li Jingying Wang, Zhengfang Zhou, Ping Zhu, Sean (Xue) Li Boston Children’s Hospital Intrapericardial arterial trunks are the direct conduits to supply the systemic and pulmonary circulations from heart. Developmental mechanism, including the smooth muscle origin and potential lineage relationship with cardiomyocytes are largely unknown. Using the Cre/loxP-mediate genetic lineage mapping strategy, we show that the entire pulmonary trunk (PT) but only few of the aortic trunk (AT) smooth muscle cells (SMCs) comes from Six2+ progenitors. On the other hand, a majority of the AT SMCs is derived from the neural crest cells. Selective ablation of Six2+ cells using diphtheria toxin A causes pulmonary atresia, demonstrating that Six2+ progenitors are critical for AT formation. Further comprehensive gene expression analyses as well as a series of prospective and retrospective analyses of Six2+ progenitors demonstrate that the Six2+ PT SMC progenitors are located at the caudal part of the second heart field (SHF). These progenitors are committed to the PT fate between embryonic day 8.0 and 10.0, a few days before the PT is morphologically obvious. Together, these studies demonstrate that Six2 defines a novel population of SHF progenitors, which are critical for pulmonary trunk formation. Results from this further suggest that the intrapericardial arterial trunks are prefigured and have different embryonic origins.

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Platform Session 3 3. Myocardial development/cardiomyopathies 3-01

HIF1 AND CARDIOVASCULAR DEVELOPMENT: HOW METABOLIC REGULATION INFLUENCES VENTRICULAR CHAMBER FORMATION S Martin-Puig I Menendez-Montes, B Escobar-Rodriguez, B Palacios-Argandoña, M Torres, S Martin-Puig CNIC Hypoxia Inducible Factor-1 (HIF1) is a critical regulator of cellular metabolism but the molecular mechanisms linking hypoxia and embryonic cardiomyocyte (CM) bioenergetics remain unexplored. We analyzed HIF1 expression during chamber formation finding that it is dynamically regulated, progressively decreasing from E9.5 to E14.5, and showing higher levels in CM of compact myocardium (cm) versus those of trabecules (tb). To unravel the meaning of this pattern, we generated gain and loss of function (GOF/LOF) models of HIF pathway in progenitors and CM. LOF mutants present hypertrabeculated and thicker myocardium while GOF mutants display a dramatic thinning of cm and interventricular septal defects, dying by E15.5. RNASeq analysis indicates that HIF1 controls the metabolic status of embryonic heart, mostly regulating glycolysis and mitochondrial enzymes. We propose that HIF1 heterogeneity could mediate a metabolic compartmentalization, with higher levels in cm driving glycolysis and reduced HIF1 in tb promoting oxidative metabolism in parallel with CM maturation. In fact, LOF mutants show increased mitochondrial network and activity compared to controls, while significant reduction is found in GOF myocardium, which contains mitophagosomes in agreement with induction of autophagy genes. In summary, compartment-specific regulation of the VHL/HIF pathway in the embryonic myocardium leads to local bioenergetics modulation essential for cardiac chamber regionalization and growth.

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Platform Session 3 3. Myocardial development/cardiomyopathies 3-02

ATYPICAL PROTEIN KINASE C DEPENDENT POLARIZED CELL DIVISION DIRECTS MYOCARDIAL TRABECULATION Ibrahim Domian Derek Passer, Annebel van de Vrugt, Ibrahim Domian MGH A hallmark of cardiac development is theformation of trabeculations exclusively from the luminal surface of the primitive heart tube. Although a number of genetic defects in the endocardium and cardiac jelly disrupt myocardial trabeculation, the role of polarity machinery in driving this process remains unclear. Herein, we demonstrate that atypical protein cinase C iota (Prkci) and its interacting partners of Par polarity complex are localized to the luminal side of luminal myocardial cells. Remarkably, these cells undergo polarized cell division with the mitotic divisional angle oriented perpendicular to the heart’s lumen. Disruption of the polarity complex through cardiac specific deletion of Prkci or its downstream interacting partner NuMA, results in aberrant mitotic spindle alignment, loss of polarized cardiomyocyte division, and loss of normal myocardial trabeculation. Collectively these results layout a new mechanism for cardiac morphogenesis where in response to inductive signals, Prkci and its downstream partners direct polarized cell division of luminal myocardial cells to drive trabeculation in the early developing heart.

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Platform Session 3 3. Myocardial development/cardiomyopathies 3-03

LINEAGE TRACING REVEALS THAT ORIENTED DIVISION UNDERLIES TRABECULAR MORPHOGENESIS AND DIFFERENTIATION Mingfu Wu Jingjing Li, Chen Zhao, Ernest Spiotto, Mingfu Wu Albany Medical College The early embryonic heart does not have a coronary circulatory system to perfuse itself and has to form sheet-like trabecula to increase surface area for exchanges. To understand cellular and molecular mechanisms of trabecular formation in the mouse heart, we use stochastic multicolor clonal analysis to define the contribution of individual cardiomyocyte to the heart morphogenesis. We genetically label a cardiomyocyte prior to trabeculation via the brainbow multicolor system and then trace and analyze the labeled clone during trabeculation by whole-embryo clearing and imaging. The labeled cell undergoes oriented cell division (OCD) and directory migration to form clone. The clones display different patterns including perpendicular, migratory, parallel, and mixed. Different from Zebrafish, in which only migratory clones contribute to trabeculae, perpendicular, migratory, and mixed clones contribute to trabecular formation in the mouse. Further studies show that perpendicular division is an extrinsic asymmetric cell division, as some of mRNAs were asymmetrically distributed in mitotic cells, which might contribute to trabecular differentiation. N-Cadherin deletion in the whole heart or in the single labeled cell/clone disrupts the OCD and clonal patterns, resulting in trabeculation and differentiation defects. In summary, our data demonstrate that N-Cadherin dependent OCD and directional migration contribute to trabecular morphogenesis and cardiomyocyte differentiation.

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Platform Session 3 3. Myocardial development/cardiomyopathies 3-04

DAAM1 AND DAAM2 ARE REDUNDANTLY REQUIRED FOR MYOCARDIAL MATURATION Ethan David Cohen Ethan David Cohen, Edward E. Morrisey, Terry P. Yamaguchi, Vickas V. Patel University of Rochester SMD Wnt ligands regulate heart morphogenesis but the means they use to do so are unclear. Two Formin-related proteins, Daam1&2, were found to bind the Wnt effector Disheveled. Since Daam1&2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function in the myocardium. Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor heart function. The defects in Daam1 CKOs were observed by mid-gestation and associated with a loss of protrusions from myocytes invading the OFT. Daam1 CKO hearts also had Hypertrabeculation/noncompaction (HT/ NC) and deranged cardiomyocyte polarity. To determine if Daam1&2 act redundantly, Daam1 CKOs homozygous for an insertion into Daam2 were generated. These Daam1/2 double knockout (DKO) mice had small hearts with strong HT/NC, severe losses of heart function and sarcomere structure and increased myocyte proliferation. While RhoA was unaffected in Daam1/2 DKOs, Akt activity was lost relative to controls raising the question of whether or not Daam1 was truly involved in Wnt signaling. Daam1floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO; Wnt5anull/+ mice were small, had HT/NC like Daam1/2 DKOs and more disrupted heart function than Daam1 CKOs suggesting that Daam1 and Wnt5a act in a common pathway. Yet deleting Daam1 further disrupted Wnt5a null hearts suggesting it also has Wnt-independent roles in heart development.

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Platform Session 3 3. Myocardial development/cardiomyopathies 3-05

CIP/MLIP SENSES PATHOPHYSIOLOGICAL STRESSES TO REGULATE CARDIAC HOMEOSTASIS Zhanpeng Huang Zhanpeng Huang, Masaharu Kataoka, Jinghai Chen, Bin Zhou, Hiroko Wakimoto, Jan Kyselovic, Christine E. Seidman, Jonathan G. Seidman, William T. Pu, Da-Zhi Wang Boston Children’s Hospital Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins have been associated with cardiomyopathy. However, how cardiomyocytes sense pathophysiological stresses to modulate cardiac remodeling remains poorly understood. Here we describe a novel regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. The expression of CIP, a myocyte-enriched protein highly conserved between human and mouse, is reduced in dilated cardiomyopathy patients. Genetic deletion of CIP accelerates the progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated overexpression of this protein prevents pathologic remodeling and preserves cardiac function. CIP interacts with lamin A/C and dystrophin genetically to modulate dilated cardiomyopathy. In addition, CIP and dystrophin proteins are located at the sarcolemma of cardiomyocytes and interact. Transcriptome analysis indicates that loss of CIP has little impact on gene expression under normal physiological conditions. However, the p53 and Foxo1-mediated gene network related to homeostasis is disturbed in CIP mutant hearts upon pressure-overload stress. Our studies define a novel key regulator of cardiomyopathy that may be an appropriate therapeutic target to attenuate progression to heart failure.

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Platform Session 4 4. Trends in cardiovascular development 7-06

MULTIPLEX ANALYSIS OF GENE EXPRESSION IN INDIVIDUAL LIVING CELLS Ayhan Atmanli Ayhan Atmanli, Dongjian Hu, Annebel Marjolein van de Vrugt, Ibrahim John Domian Massachusetts General Hospital The capacity of pluripotent stem cells (PSC) to recapitulate many of the in vivo developmental programs provides an attractive in vitro model system for studying lineage commitment and cellular differentiation. Advances in the generation of patient-specific PSC have now also allowed for the development of novel and evolving approaches for the study of human disease. Despite this progress and the promise of stem cell biology in clinical medicine, a significant obstacle for their use is the lack of robust assays to simultaneously examine the expression of multiple genes in living cells undergoing normal differentiation or pathogenesis. This consideration is particularly relevant in view of the fact that PSC differentiation results in a diverse population of heterogeneous cells with complex and interacting regulatory networks. To overcome these limitations, we have developed a novel FRET-based technology for the multiplex analysis of gene expression in individual living cells (MAGIC) to perform concurrent gene expression and functional analyses of single cardiac myocytes (CMs). We apply our live-cell mRNA imaging technology to demonstrate distinct physiological profiles of human PSC-CMs expressing MLC2v and MHCx as markers of the ventricular cell fate and myocardial maturity, respectively. By tailoring our live-cell gene expression imaging with functional assays, we open new avenues for the study of myocardial lineage commitment and maturation.

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Platform Session 4 4. Trends in cardiovascular development 1-06

TRANSCRIPTIONAL CONTROL OF CARDIAC CELL FATE Laurie Boyer Zhihong Xue, Gizem Rizki, Laurie Boyer Massachusetts Institute of Technology Heart development depends critically on tight coordination of gene expression patterns, and disruption of transcriptional networks during embryogenesis underlies congenital heart disease. Long non-coding RNAs (lncRNAs) have emerged as a new regulatory layer of developmental gene expression patterns. We identified Braveheart (Bvht), a novel lncRNA necessary for activation of a core network of cardiovascular transcription factors during cardiac lineage commitment. Using biochemical and genetic approaches, we find that Bvht comprises distinct structural motifs that mediate its function, opening the door for understanding the broader developmental roles of lncRNAs. In addition to embryonic development, postnatal cardiomyocyte (CM) maturation involves the dramatic rewiring of the transcriptional landscape during a narrow developmental window, concomitant with a switch from a proliferative to hypertrophic mode of growth and cell cycle exit, the latter of which is a major roadblock to adult cardiac regeneration. Using Bvht as an example, we identified candidate pro-maturation lncRNAs whose disruption leads to a significant increase in CM proliferation and cytokinesis. We are currently taking a multi-disciplinary approach to precisely define the mechanism of each candidate. Together, our work demonstrates that lncRNAs are integral components of the transcriptional regulatory landscape that drives cardiac cell fate and provides a basis for improving methods for regenerative therapy.

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Platform Session 4 4. Trends in cardiovascular development 3-06

SINGLE CELL TRANSCRIPTIONAL ATLAS OF CARDIAC DEVELOPMENT Dan DeLaughter Dan DeLaughter, Alex Bick, Hiroko Wakimoto, Irfan Kathiriya, Benoit Bruneau, J. G. Seidman, Christine E. Seidman Harvard Medical School Cardiac development from linear heart tube to four-chamber heart is incompletely understood in part because of the inability of conventional transcriptome analysis to deconvolute cell populations. Single-cell RNA sequencing was performed on 1229 murine cells isolated from three cardiac chambers and seven time points (E9.5, E11.5, E14.5, E18.5, P0, P7, P21). Cells were classified using unbiased transcriptome-wide analysis without a priori knowledge of constituent cell types or markers. We confirmed the spatial and temporal expression patterns of many classical markers of cardiac development and identified previously unknown cell specific markers. Cardiomyocytes and endocardial cells transition through multiple developmental states. In particular, left ventricular myocytes do not mature as a homogenous, synchronized cell population, but exist as a transcriptional continuum of more or less mature cells that overlap in a stepwise fashion with the neighboring time points until P21. Left and right ventricular myocytes were quite distinct at E11.5, but converged into remarkably similar expression patterns by P0. By contrast endothelial cells from each chamber are quite similar at each stage of development. Analyses of these data enabled a robust delineation and transcriptional description of cardiac cell lineages, identifying how these lineages uniquely mature during cardiogenesis while identifying novel markers previously undescribed using conventional transcriptome analysis.

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Platform Session 4 4. Trends in cardiovascular development 9-06

ETIOLOGY OF HYPOPLASTIC LEFT HEART SYNDROME: INSIGHTS FROM ANALYSIS OF MUTANT MOUSE MODELS Xiaoqin Liu Xiaoqin Liu, Shazina Zaed, Zhaohan Chen, George Gabriel, Kevin Peterson, Abha Bais, Dennis Kostka, Stephen Murray, George A. Porter, Jr., Cecilia W. Lo University of Pittsburgh School of Medicine Hypoplastic left heart syndrome (HLHS) is a congenital heart disease comprising severe hypoplasia of the left ventricle (LV), aorta and mitral valve. Previous work suggests a genetic etiology, but the genes and mechanism of HLHS pathogenesis remain unknown. Here, we report findings from the first HLHS mouse models. Ultrasound scanning >100,000 fetal mice in a ENU mutagenesis screen identified 9 HLHS fetuses in 8 mutant lines. Exome sequencing showed no mutations are shared among the 8 HLHS lines. In the Ohia line, two genes were identified to cause HLHS: Sap130, a chromatin remodeling protein and Pcdha9, protocadherin mediating cell adhesion. Mouse embryos generated by CRISPR/Cas9 editing of Sap130/Pcdha9 showed the same constellation of HLHS cardiac lesions. Ohia HLHS mutants had defects in cell cycle progression and increased apoptosis in the LV but not RV. EM analysis and functional assays revealed defects in mitochondrial maturation and cardiomyocte differentiation. RNAseq profiling showed gene expression changes in the LV associated with metabolic pathways, energy metabolism, cell proliferation, cancer, chromatin remodeling, and Tgfβ signaling. Together, these findings show HLHS is genetically heterogeneous with an oligogenic etiology. In the Ohia HLHS mutants, LV hypoplasia is associated with cell intrinsic defects involving abnormalities in cell cycle regulation, mitochondrial maturation and cardiomyocyte differentiation. Supported by NIH grant HL098180.

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Platform Session 4 4. Trends in cardiovascular development 8-06

IDENTIFYING CELL TYPE SPECIFIC ENHANCERS USING CRE-ACTIVATED, LINEAGERESTRICTED P300 CHIP-SEQ Pingzhu Zhou Pingzhu Zhou, Fei Gu, Bin Zhou, Aibin He, Sean Stevens, Qing Ma, William Pu Boston Children’s Hospital Transcriptional enhancers are fundamental components of gene regulatory networks that govern cell type specific gene expression and function. Because currently used techniques to determine genome-wide chromatin occupancy by ChIP-seq lack cell type specificity, we are particularly uninformed about enhancers in cell types that compose numerically small fractions of tissues, such as endothelial cells (ECs) of the developing vascular system. To overcome this limitation, we developed a method to selectively biotinylate in Cre-labeled cells the transcriptional coactivator p300, which decorates many tissue specific enhancers. Subsequence streptavidin pulldown of p300-associated chromatin followed by massively parallel sequencing thus delineated EC-specific p300 bound regions. A subset of the EC-specific p300 bound regions were functional angiogenic enhancers, based on several criteria. First, the genes linked to p300-bound regions were enriched in ECs, as defined by translating ribosome affinity purification (TRAP) of EC vs whole embryo transcripts. Second, these EC p300-associated genes were overrepresented for genes involved in angiogenesis. Third, these EC bound regions include several previously validated EC-enhancers. A subset of these EC p300-associated regions are being validated and further dissected in transient transgenic assay. Our work establishes a system to perform high affinity, cell type specif transcriptional networks in vivo.

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Platform Session 5 5. Epicardium, coronaries, conduction system, and arrhythmias 5-01

CXCL12 CHEMOKINE GUIDED ANGIOGENESIS DIRECTS CORONARY VASCULATURE FORMATION IN ZEBRAFISH Ching-Ling (Ellen) Lien Michael R.M. Harrison, Jeroen Bussmann, Ying Huang, Arthela Osorio, Long Zhao, C. Geoffrey Burns, Caroline E. Burns, Henry M. Sucov, Arndt F. Siekmann, Ching-Ling (Ellen) Lien Children’s Hospital Los Angeles Coronary vasculature plays critical roles in heart functions by maintaining a continuous supply of oxygen and nutrients. Coronary disease is a major cause of myocardial infarction and heart failure, which continues to be the leading cause of mortality worldwide. Malformation of coronary vasculature can also cause severe congenital defects. Despite this critical importance, the processes and factors required for coronary vessel development remain to be elucidated. Here we report the first detailed analysis of coronary vessel formation in zebrafish. We observe that coronary vessels form in zebrafish by angiogenic sprouting of cells emerging at the atrioventricular canal. Lineage tracing and clonal analysis suggest that coronary endothelial cells in developing zebrafish hearts are derived from endocardium. Endothelial cells express the CXC-motif chemokine receptor Cxcr4a and migrate to vascularize the ventricle under the guidance of the myocardium-expressed ligand Cxcl12b. cxcr4a mutant zebrafish fail to form a vascular network, whereas ectopic expression of Cxcl12b ligand induces coronary vessel formation. Importantly, cxcr4a mutant zebrafish fail to undergo heart regeneration following injury. Our results suggest that Cxcl12/Cxcr4 chemokine signaling has an essential role in coronary vessel formation by directing migration of endothelial cells. Poorly developed vasculature in cxcr4a mutants likely underlies decreased regenerative potential in adults.

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Platform Session 5 5. Epicardium, coronaries, conduction system, and arrhythmias 5-02

NOTCH SIGNALING CONTROLS CORONARY ANGIOGENESIS BY ENDOCARDIAL PROGENITORS Bin Zhou Yidong Wang, Bingruo Wu, Bin Zhou Department of Genetics, Albert Einstein College of Medicine Understanding of the developmental origin of coronary vessels would inspire novel regenerative treatment for coronary artery diseases. However, the developmental origins of coronary arteries still remain controversial with multiple sources, sinus venosus (SV), ventricular endocardium (VE) and proepicardium. Recent study using Nfatc1-Cre lineage tracing interpreted that VE contributes to the majority of coronary arteries in the embryonic ventricle wall. Here we generated Nfatc1-Cre and Nfatc1-Dre recombinase mouse lines, and found Nfatc1-based constitutive recombinase actually labeled both SV and VE. Therefore, restricted VE lineage tracing requires a specific marker that could distinguish VE from SV. Here we identified a new VE marker natriuretic peptide receptor 3 (Npr3). By in situ hybridization and immunostaining on tissues of Npr3-GFP mice, we found that Npr3 was specifically expressed in VE but not SV in the developing heart. By Npr3-CreER line, we labeled VE at E9.5 before coronary vessels form. At E13.5 and E15.5, these early labeled VE contribute to only about 10% coronary vessels in the ventricular wall. As a control, VE contributes significantly to the coronary vessels in the inter-ventricular septum. Taken together, our lineage tracing data based on new tools demonstrated that VE contributes to a minority of coronary vessels in the embryonic ventricle wall.

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Platform Session 5 5. Epicardium, coronaries, conduction system, and arrhythmias 5-03

EPICARDIAL REGENERATION IS DIRECTED BY THE CARDIAC OUTFLOW TRACT AND HH SIGNALING Jinhu Wang Jinhu Wang, Jingli Cao, Amy L. Dickson, Kenneth D. Poss Duke University In response to cardiac damage, a mesothelial tissue layer enveloping the heart called the epicardium is activated to undergo cell division and accumulate at the injury site. The epicardium is a new target for enhancing heart regeneration, yet its adult biology and dynamism is poorly understood. Here, we find that genetic depletion of the epicardium after partial ventricular resection inhibits cardiomyocyte proliferation and delays muscle regeneration. The epicardium vigorously regenerates after its ablation, through proliferation and migration of spared epicardial cells as a sheet to cover the exposed ventricular surface in a wave from the chamber base toward its apex. By reconstituting epicardial regeneration ex vivo, we show that extirpation of the bulbous arteriosus (BA), a distinct, smooth muscle-rich tissue structure that distributes outflow from the ventricle, prevents epicardial regeneration. Conversely, experimental repositioning of the BA by tissue recombination initiates epicardial regeneration and can govern its direction. Hedgehog (Hh) ligand is expressed in the BA, and treatment with Hh signaling antagonist arrests epicardial regeneration. Transplantation of Shhsoaked beads at the ventricular base stimulates epicardial regeneration after BA removal, indicating that Hh signaling can substitute for the BA influence. Thus, the ventricular epicardium has pronounced regenerative capacity, regulated by the neighboring OFT and Hh signaling.

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Platform Session 5 5. Epicardium, coronaries, conduction system, and arrhythmias 5-04

SHOX2 AND NKX2-5 ANTAGONISTICALLY DETERMINE PACEMAKING CELL FATE IN THE PULMONARY VEIN MYOCARDIUM Wenduo Ye Wenduo Ye, Jun Wang, Yingnan Song, Diankun Yu, Yanding Zhang, Fen Wang, Richard Harvey, Laura Schrader, James Martin, Yiping Chen Tulane University In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. However, the genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. Here we present evidence that Shox2 antagonizes the transcription output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5+ domain within the SAN to determine the cell fate. Shox2 deletion in the Nkx2-5+ domain of the SAN caused sick sinus syndrome, associated with the loss of pacemaker program. Explanted Shox2+ cells from the PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2- cells, and Shox2 deletion led to Hcn4 obliteration in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. Such mechanism is also adopted in differentiating embryoid-bodies. Moreover, we discovered a substantial genome wide co-occupancy of Shox2, Nkx2-5, and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development.

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Platform Session 5 5. Epicardium, coronaries, conduction system, and arrhythmias 5-05

T-BOX RHEOSTAT PATTERNS CARDIAC CONDUCTION SYSTEM FUNCTIONAL DOMAINS Ozanna Burnicka-Turek Ozanna Burnicka-Turek, Nataliya B. Peterenko, Vincenzo Macri, Sebastain Clauss, Vicas V. Patel, Patrick Ellinor, Ivan P. Moskowitz University of Chicago, Departments of Pediatrics Despite the significant clinical consequences of cardiac conduction system (CCS) disorders, the molecular mechanisms and transcriptional networks that establish and maintain regional function of CCS domains are unknown. For example, the nodal conduction system conducts slowly and autonomously depolarizes, where the ventricular conduction system (VCS) conducts quickly without spontaneous depolarization. Using a VCS-specific inducible Cre recombinase, we removed Tbx5 from the adult VCS. In the absence of Tbx5, the fast conducting VCS became functionally slow. Expression analysis disclosed an efficient switch from VCS to nodal-like myocardial gene expression. This involved suppression of genes required for fast ventricular conduction, including gap junction subunits (e.g. Cx40), cardiac Na+ channels (e.g.Nav1.5/INa) and inwardly rectifying K+ ion channels (e.g. Kir2.1/IK1). In contrast, genes required for slow conducting nodal phenotype, including Tbx3, were maintained. In myocytes isolated from the Tbx5-deficient VCS, we observed a significant increase in action potential (AP) duration and reductions in INa and IK1 currents. Furthermore, in the absence of functional Tbx5, VCS myocytes generated spontaneous diastolic depolarizations and spontaneous APs, normally observed only in the nodal cells. Our findings suggest that Tbx5 determines the functional and molecular identity of the adult VCS and that in the absence of Tbx5, the underlying nodal potential of VCS is uncovered.

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Platform Session 6 6. Endocardium and cardiac valves 6-01

MIR-23B AND MIR-199A IMPAIRS EMT DURING ATRIOVENTRICULAR ENDOCARDIAL CUSHION FORMATION Diego Franco Fernando Bonet, Angel Dueñas, Amelia Aranega, Diego Franco University of Jaen Valve development is a multistep process involving the activation of the cardiac endothelium, an epithelial-mesenchymal transition (EMT) and the progressive lining and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tbf-beta and/or Vegf signaling have been involved in crucial steps of valvulogenesis. We have previously demonstrated discrete changes on microRNAs expression during cardiogenesis, which are predicted to target Bmp- and Tgf-beta signaling. We now analyzed the expression profile of candidate microRNAs in atrial, ventricular and atrioventricular canal regions at four different developmental stages. qRT-PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profile within the atrial, ventricular and atrioventricular canal regions of the developing chicken heart. miR-23b, miR-199a and miR-15a displayed increased expression at early AVC developmental whereas others such as miR-130a and miR-200a display decreased expression levels. Functional analyses of miR-23b, miR199a and miR-15a overexpression, respectively, led to EMT blockage in vitro. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT-related genes that are predicted to be targeted by these microRNAs. Thus, our data demonstrate that miR-23b and miR-199a over-expression can impair atrioventricular EMT.

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Platform Session 6 6. Endocardium and cardiac valves 6-02

CILIA AND THEIR FUNCTION IN VALVE DEVELOPMENT AND MITRAL VALVE PROLAPSE Katelynn Toomer Katelynn Toomer, Kimberly Sauls, Amanda Johnson, Russell Norris MUSC Mitral valve prolapse affects 1 in 40 people worldwide and is the leading indication for surgical repair for mitral regurgitation in the United States. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. Previous work has shown that mutations in DCHS1 gene cause MVP in humans, a phenotype that is recapitulated in dcsh1 deficient mice. Interestingly, global knockout of Dchs1 resulted in development of polycystic kidney disease, a well-established ciliopathy. Additionally, patients with PKD and/or various other ciliopathies have increased incidence of aortic and mitral valve diseases. Thus, we hypothesized that cilia may play a key role in upstream regulatory pathways important for valve morphogenesis and that disruption of cilia may result in valve diseases. We see cilia expression primarily in mitral and aortic valve interstitial cells during development, which are restricted to the spongiosa postnatally. Genetic removal of cilia resulted in both mitral valve defects and bicuspid aortic valve disease, highlighting the importance for cilia in valve morphogenesis. Additionally, profound disturbance in ciliogenesis were observed in the Dchs1 model of MVP. Signaling pathway analyses have led to potential unifying mechanisms for valve disease pathogenesis and we present drugable pathways that could be beneficial in abrogating valve disease progression. These findings as well as mechanistic ciliary function will be discussed.

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Platform Session 6 6. Endocardium and cardiac valves 6-03

WNT SIGNALING HAS DISTINCT AND DYNAMIC ROLES IN SEMILUNAR AND ATRIOVENTRICULAR CANAL VALVE DEVELOPMENT Fernanda Bosada Fernanda Bosada, Vidusha Devasthali, Kimberly Jones, Yujung Choi, Brynn Akerberg, Andrew McKay, ChingPin Chang, Calvin Kuo, Bin Zhou, Kryn Stankunas University of Oregon Heart valve development proceeds through coordinated steps by which endocardial cushions (EC) form mature, elongated and stratified valves. Previous studies suggest that Wnt signaling and its canonical effector β-catenin have roles from endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define direct roles of the Wnt pathway at different stages of valve formation. We have developed a transgenic mouse system that provides spatiotemporal inhibition of LRP-dependent Wnt signaling by chemically-inducible expression of Dkk1. Unexpectedly, this approach indicates Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Further, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt signaling is activated in cushion mesenchymal cells where it has a permissive role supporting FGF-driven expansion of the ECs and the elongation and remodeling of AVC valves. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that broad Axin2 expression in maturing valves prevents tissue overgrowth rather than simply reporting Wnt activity. Our data suggests that while canonical Wnt signaling is active and dynamically regulated at various stages of valve development, its roles may be less discrete than currently understood.

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Platform Session 6 6. Endocardium and cardiac valves 6-04

NEW DOWN SYNDROME MICE SHOW AVSD WITH INTACT VESTIBULAR SPINE AND REVEAL HEART DEFECTS MAP TO TWO LOCI Eva Lana-Elola Eva Lana-Elola, Sheona Watson-Scales, Amy Slender, Dorota Abucewicz, Alexandrine Martineau, Charlotte Douglas, Timothy Mohun, Elizabeth M. C. Fisher, Victor L. J. Tybulewicz National Institute for Medical Research Down syndrome (DS), caused by trisomy of human chromosome 21 (Hsa21) is the most common genetic cause of congenital heart defects (CHD), particularly atrio-ventricular septal defects (AVSD). However, the precise genetic or mechanistic causes of these CHD remain unclear. Using high-resolution episcopic microscopy (HREM) we found that a new mouse with a duplication of 135 genes orthologous to Hsa21, results in CHD that closely resemble those observed in individuals with DS. Interestingly, these mice present a specific subtype of incomplete AVSD with exclusive ventricular shunting. We combined genetic lineage markers and HREM to model in 3D the development of the vestibular spine or dorsal mesenchymal protrusion (DMP), a tissue derived from the second heart field shown to be involved in the etiology of AVSDs. The development of the DMP appears to be unaffected in the DS models and we propose that this specific subtype of AVSD is not caused by a failure in the formation of the DMP. Moreover, in order to identify dosage-sensitive genes that when present in 3 copies cause CHD in DS, we generated a high-resolution mapping panel of 16 new mouse strains with partial duplications and deletions for regions of mouse chromosome 16, orthologous to Hsa21. Analysis of embryonic hearts from strains with short duplications allowed us to narrow down the critical genomic region for DS-CHD and demonstrate that DS-associated AVSDs are caused by an additional copy of at least 2 different loci/genes.

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Platform Session 6 6. Endocardium and cardiac valves 6-05

EXPLORING ENDOTHELIAL CELL DYNAMICS IN AGING HEART VALVES Lindsey J. Miller Lindsey J. Miller, Blair F. Austin, Joy Lincoln The Ohio State University Heart valve insufficiency affects ~2.5% of the population and aging is a significant risk factor, however its not clear why. In the elderly, composition of the valve extracellular matrix (ECM) is altered, and studies have shown that valve interstitial cells (VICs) mediate ECM homeostasis, while overlying valve endothelial cells (VECs) regulate VIC function. Based on this, we hypothesize that age-dependent changes in endothelial cell dynamics underlie valve disease in the aging population. Using electron microscopy, we observe changes in VEC morphology and distribution in aging valves. In addition using RNA-seq, we have generated age-dependent molecular profiles of VECs isolated from mice at embryonic (E14.5), post-natal (PN), adult (4 months) and aged (12-15 months) stages. Additional analysis identified pluripotency as a significantly affected pathway between post-natal and adult time points and using reporter mice, we show telomerase activity in VECs after birth. As cell cycle genes were markedly reduced in VECs following post-natal growth, we examined if circulating cells play a role in maintaining the adult valve endothelium. To do this, we performed bone marrow transplants and observe donor cells co-expressing endothelial markers on the surface of the valve cusps. Together this study highlights age-dependent differences in plasticity and phenotype of the valve endothelium and provides insight into the therapeutic potential of VECs for heart valve regeneration and repair.

49

Platform Session 7 7. Cardiac stem cells, growth, and regeneration 7-01

EMBRYONIC HEART PROLIFERATION AND NEONATAL HEART REGENERATION CONTROLLED BY IGF2 Hua Shen Hua Shen, Henry Sucov University of Southern California The ventricular wall of the embryonic heart increases in thickness by cardiomyocyte (CM) proliferation. We previously found that insulin like growth factor 2 (IGF2) is an essential factor to regulate cardiomyocyte proliferation and ventricular wall formation. Igf2 is expressed in endocardium and epicardium, but tissue-specific mutation demonstrated that epicardial IGF2 is the primary inducer of ventricular wall expansion. Igf2 continues to be expressed in the epicardium and endocardium through late gestation. As previously reported, the neonatal heart can regenerate after injury whereas this capacity is lost by postnatal day 7. Interestingly, we found that Igf2 expression correlates with this regenerative capacity: Igf2 expression persists as in the embryonic heart at P1-P3, but decreased dramatically by P7. To test the functional role of Igf2 expression in neonatal heart regeneration, we resected hearts of control and conditional Igf2 mutant pups. Unlike the complete restoration that occurred in control hearts by 3 weeks, Igf2 deficient hearts healed by scar formation rather than by cardiomyocyte regeneration. These results clarify the tissue interactions that underlie embryonic heart growth, and demonstrate that neonatal heart regeneration is accomplished by the redeployment of signaling pathways that support embryonic cardiomyocyte proliferation.

50

Platform Session 7 7. Cardiac stem cells, growth, and regeneration 7-02

NKX2.5+ CARDIOMYOBLAST CONTRIBUTION TO POSTNATAL CARDIOGENESIS Vahid Serpooshan Yuan-Hung Liu, Vahid Serpooshan, David Rawnsley, Anusha Kumar, Xiaojing Huang , Sean M. Wu Stanford University Two mechanisms have been proposed for adult mammalian heart limited renewal of cardiomyocytes: differentiated myocyte replication and progenitor/immature cell differentiation. This study aims to identify and characterize a population of postnatal cardiomyocyte precursors and determine their impact on neonatal development and cardiogenesis. We tracked the expression of a cardiac-specific enhancer of Nkx2.5 from embryonic to postnatal development and identified a pool of Nkx2.5+ cardiomyoblasts in the early neonatal heart. Genome-wide expression profiling of neonatal Nkx2.5 enh-eGFP+ cells demonstrated their similarity to early embryonic cardiac progenitor cells. We developed new transgenic mouse lines that express Cre recombinase under the Nkx2.5 cardiac enhancer to track the progeny of these Nkx2.5+ cardiomyoblasts. Neonatal Nkx2.5 enh-eGFP+ cells differentiated into cardiomyocytes in coculture assays in vitro and following CreLoxP based lineage tracing in vivo. Genetic ablation of Nkx2.5 enh-eGFP+ cardiomyoblasts in ROSA-2LoxDTA mice yielded cardiac hypertrophy and subsequent dilation, indicating a functional requirement of Nkx2.5 enh-eGFP+ cells in normal heart development. This study provides direct lineage tracing evidence that a neonatal cardiomyoblast population contributes to cardiogenesis in the postnatal heart. The cell population identified here may serve as the target of regenerative therapies if they can be expanded in situ using pharmacological agents.

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Platform Session 7 7. Cardiac stem cells, growth, and regeneration 7-03

ACETYLATION OF VGLL4 REGULATES POSTNATAL CARDIAC GROWTH Zhiqiang Lin Zhiqiang Lin, Haidong Guo, Pingzhu Zhou, Qing Ma, William T. Pu Boston Children’s Hospital Cardiomyocyte (CM) loss is a major cause of heart failure. Mammalian CMs retain proliferation capacity shortly after birth, but most CMs exit the cell cycle in the adult heart. Understanding the mechanisms that lead to neonatal CM cell cycle exit will lay the foundation for reversing adult CM cell cycle quiescence. The Hippo-Yap pathway is crucial for regulation of cardiomyocyte proliferation, and the YAP-TEAD complex is the terminal transcription effector controlling the signal output of this pathway. In this study, we found that VGLL4, a negative regulator of YAP, was mainly expressed in the CM, and its expression increased with age. During postnatal cardiac growth, the major interaction partner of TEAD1 switched from YAP in the neonatal heart to VGLL4 in the adult heart. Mass spectrometric analysis identified several VGLL4 acetylation sites including a major acetylation site within the VGLL4-TEAD interaction interface, and VGLL4 acetylation blocked VGLL4 binding to TEAD. Mutation of the acetylated lysine to arginine (VGLL4-KR) reduced VGLL4 acetylation and increased VGLL4-TEAD1 interaction. Overexpression of VGLL4-KR but not wild type VGLL4 inhibited neonatal cardiac growth, resulting in cardiac hypoplasia and heart failure. Together, our study defines VGLL4 as a novel inhibitor of cardiac growth and CM proliferation that competitively modulates YAP interaction with TEAD through an acetylation-dependent switch.

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Platform Session 7 7. Cardiac stem cells, growth, and regeneration 7-04

PITX2 PROMOTES HEART REPAIR BY REGULATING RESPIRATORY CHAIN COMPONENTS AND THE ANTIOXIDANT RESPONSE Ge Tao Ge Tao, Peter C. Kahr, Yuka Morikawa, Min Zhang, Lele Li, Zhao Sun, Brad A. Amendt, James F. Martin Baylor College of Medicine The lack of self-renewal capacity in mature mammalian hearts is a major reason for heart failure after myocardial infarction (MI). To repopulate the heart with de novo cardiomyocytes, one promising strategy is to reintroduce mature cardiomyocytes into mitotic cycle. We have previously reported that the mouse Hippo signaling constrains heart size during development, and knocking-down of Hippo (Hippo-deficient) promotes juvenile and adult myocardial regeneration after MI. Here we dissected the signaling downstream of Hippo and identified the paired-like homeodomain transcription factor 2 (pitx2) as a potential cofactor of Yap, the target of Hippo-kinase-cascade. Co-IP assays indicated a direct interaction between Pitx2 and Yap; knocking-down of pitx2 in Hippo-deficient heart resulted in severe scarring after MI, implicating a requirement of pitx2 for the regeneration of Hippo-deficient heart. In addition, Pitx2 expression is induced in cardiomyocytes of injured myocardium, and is required in neonatal cardiac regeneration. Immuno-staining, ChIP-seq and RNA-seq showed that pitx2 regulates cell proliferation and the expression of antioxidant scavenger genes. Further studies revealed that over-expression of pitx2 in adult cardiomyocytes is sufficient to promote the restoration of myocardial structure and function after MI. Together, these evidences revealed a novel role of pitx2 during injury response and restoration of myocardium.

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Platform Session 7 7. Cardiac stem cells, growth, and regeneration 7-05

CALCINEURIN INHIBITION ENHANCES REGENERATION: FISH APPENDAGES CAN LEAD TO UNDERSTANDING ORGAN ALLOMETRY Christopher L. Antos Satu Kujawski, Weilin Lin, Michael Wagner, Ali El-Armouch, Yixin Zhang, Christopher L. Antos DFG-Center for Regenerative Therapies Dresden Heart and appendage regeneration involves growth control of progenitor cells to reform the missing tissues to the original dimensions. Restoration to the correct dimensions involves coordinating rapid allometric regenerative outgrowth with the reinstatement of isometric cell proliferation once the correct tissue dimensions are reached. It is unknown what executes this control. We show that calcineurin regulates the coordinated growth of multi-tissue fish appendages. Its inhibition continues allometric regeneration beyond the original fin dimensions. Calcineurin activity is low when the rate of progenitor cell proliferation is highest, and its activity increases as the regeneration rate decreases. Despite continued rapid cell proliferation, this growth is coordinated: tumors never form. Previous results show that the rate of regeneration is positionally controlled, but it is unknown what this positional control is. Our data suggest that calcineurin inhibition shifts regeneration from a distal growth program to a proximal program. This shift is associated with retinoic acid signaling, which can change positional information. We provide evidence that calcineurin acts through a potassium channel also expressed in the fish heart that controls allometric growth. Thus, we identified a calcineurin-mediated mechanism that operates as a molecular switch that controls regenerative growth. The targets of this switch can lead to the mechanisms governing regenerative growth of the heart.

54

Platform Session 8 8. Cardiovascular genomics/transcriptional regulation/epigenetics 8-01

COOPERATIVE TRANSCRIPTIONAL ACTIVATION OF PAIRED MEF2 SITES BY MYOCARDIN AND MEF2C Brian L. Black Jianxin Hu, Courtney M. Anderson, Diane E. Dickel, Len A. Pennacchio, Brian L. Black UCSF AMP-activated protein kinase (AMPK) is a master regulator of energy balance and homeostasis and plays a central role in the switch from fatty acids to glycolysis during cardiac hypertrophy and heart failure. AMPKα2 is the predominant isoform of the catalytic subunit expressed in the heart and is encoded by the prkaa2 gene. Although AMPK and its role in metabolism and heart failure have been extensively studied, the transcriptional control of prkaa2 is completely unknown. We identified a myocardial-specific enhancer of prkaa2 that is active throughout heart development. Deletion of the enhancer using CRISPR/Cas9 reduces prkaa2 expression in vivo by ~60%. We also found that the prkaa2 cardiac enhancer requires the MADS box transcription factor MEF2C and its cofactor Myocardin for activity in vivo. Moreover, the prkaa2 enhancer contains two MEF2 binding sites that function together in a synergistic fashion in response to MEF2C binding to each site and bridging of the two MEF2-bound sites by Myocardin dimerization. This work establishes a molecular basis for the cooperative activity of two proximally located MEF2 sites by Myocardin-MEF2. Additionally, this work may provide additional strategies for manipulating AMPK expression during heart failure.

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Platform Session 8 8. Cardiovascular genomics/transcriptional regulation/epigenetics 8-02

AN EVOLUTIONARILY EVOLVED TBX5/CHD4 INTERACTION PROVIDES MECHANISTIC INSIGHT INTO ATRIAL SEPTATION Lauren Waldron Lauren Waldron (University of North Carolina), Junghun Kweon (University of Chicago), Kerry M. Dorr (University of North Carolina), Brenda Temple (University of North Carolina), Todd Greco (Princeton University), Ileana Cristea (Princeton University), Ivan Moskowitz (University of Chicago), Frank Conlon (University of North Carolina) University of North Carolina Mutations in Tbx5 are causative to the congenital cardiac disease Holt-Oram Syndome (HOS). However, little is known about the mechanisms of Tbx5 function. We have knocked in the Avi epitope into the Tbx5 locus and by coupling Tbx5AVI with a directed proteomics approach we determined the composition of the endogenous Tbx5 interactome, including the Nucleosome Remodeling and Deacetylase (NuRD) complex. We confirmed the Tbx5-NuRD interaction biochemically and genetically and show the interaction is required for atrio-ventricular septation. We have gone on to identify 11 new targets repressed by Tbx5. Molecular modeling of the Tbx5 interaction domain shows it forms a Coil-α-Helix and that HOS missense mutations mapping to this region align to a single interaction surface. Consistently, these HOS mutations reduce or abolish interaction with Chd4, the catalytic core component of the NuRD complex. Moreover, mutations fail to repress targets of Tbx5. Analysis of the interaction domain across Tbx5 orthologues reveals a complete conservation of those amino acid mutated in HOS patients amongst animals with a septated heart. However, orthologues of Tbx5 in vertebrates lacking a septated heart have amino acid substitutions that ablate the Tbx5-Chd4 interaction. Thus, these data imply the Tbx5-Chd4 interaction arose coincident with cardiac atrial septation and demonstrate the Tbx5-Chd4 interaction is essential for human cardiac development and function.

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Platform Session 8 8. Cardiovascular genomics/transcriptional regulation/epigenetics 8-03

GENOMIC AND STRUCTURAL BASIS FOR REGULATION OF CARDIOGENESIS BY HETEROTYPIC TRANSCRIPTION FACTORS Luis Luna-Zurita Luis Luna Zurita, Sebastian Glatt, Sean Thomas, Bogaç L. Kaynak, Daniel He, Christian Stirnimann, Alisha K. Holloway, Katherine S. Pollard, Christoph W. Müller, Benoit Bruneau Gladstones Institute Of Cardiovascular Disease Transcriptional regulation events leading cardiomyocyte differentiation are also crucial for proper establishment of the different structures in the developing heart. These events are carried out by heterotypic transcription factor interactions, such as those between the T-box transcription factor TBX5 and the homeodomain transcription factor NKX2-5, which have been proposed as a mechanism for human congenital heart defects. Using an embryonic stem (ES) cell -based directed differentiation system we are able to recapitulate the different stages of cardiomyocyte differentiation. Through the use of WT and KO ES cells for Tbx5 and/or Nkx2-5, here we show that Tbx5 and Nkx2-5 coordinately control cardiac morphogenesis and gene expression via distinct modes, classified as individual, cooperative, or redundant. High-resolution genomic localization of Tbx5, Nkx2-5 and the zinc finger transcription factor Gata4, provided by ChIP-exo experiments, reveals highly interdependent binding that correlates with their modes of regulation and is specific of each differentiation stage. The co-crystal structure of the Tbx5 and Nkx2-5 DNA-binding domains on target DNA shows direct interaction between the two factors that stabilizes a ternary complex, and provides a biophysical basis for motif grammar dictating interdependent binding. Our work reveals the molecular basis of a combinatorial logic for heterotypic transcription factors in mammalian heart development.

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Platform Session 8 8. Cardiovascular genomics/transcriptional regulation/epigenetics 8-04

TRANSCRIPTIONAL MECHANISMS CRITICAL FOR VENTRICULAR WALL MATURATION Youngsook Lee Eunjin Cho, Matthew R Mysliwiec, Robert J Schwartz, Youngsook Lee Universtiy of Wisconsin-Madison Noncompaction cardiomyopathy (thin myocardium) results from the failure of myocardial development during embryogenesis. Jarid2, the founding member of Jumonji histone demethylases, critically regulates cardiac development and ES cell differentiation. Jarid2 is enzymatically inactive but functions as a transcriptional regulator. To identify cardiac-specific roles of Jarid2, Jarid2 was deleted in early cardiac progenitors using Nkx2.5-Cre Knock-in mice (Jarid2Nkx-KI). Jarid2Nkx-KI exhibit hyper-trabeculation, thin myocardium, ventricular septal defects and increased cardiac jelly, partially recapitulating defects in Jarid2 KO. By overlapping ChIP-chip and microarray analyses on Jarid2 KO embryonic hearts, potential transcriptional targets of Jarid2 were identified including Isl1, Bmp10 and FN1, which are occupied by Jarid2, SETDB1, and H3K9me3 or H3K27me3, as well as upregulated in mutants. Jarid2 occupancy was observed at the Isl1 promoter region by ChIP assays. Isl1 transcriptional activity was repressed by Jarid2 using luciferase assays. Bmp10, an essential factor for trabecular formation, and pSMAD1/5/8 were increased in Jarid2Nkx-KI hearts, correlating well with hyper-trabeculation. FN1 was upregulated in Jarid2Nkx-KI hearts, likely contributing to increased cardiac jelly. Thus, the failure to repress targets such as Isl1, Bmp10 and FN1 results in defective ventricular maturation in mutants. We are investigating the histone methyl code on genomic loci of Jarid2 targets.

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Platform Session 8 8. Cardiovascular genomics/transcriptional regulation/epigenetics 8-05

DELETION OF TRBP REVEALS A NOVEL LINEAR MIR-208A-MEDIATED PATHWAY REQUIRED FOR NORMAL CARDIAC FUNCTION Jian Ding Jian Ding, Jinghai Chen, Yanqun Wang, Masaharu Kataoka, Lixin Ma, Pingzhu Zhou, Xiaoyun Hu, William T Pu, Da-Zhi Wang Boston Children’s Hospital Cardiomyopathy, a disease of the heart that causes weak muscle contraction, is associated with altered expression of genes encoding contractile proteins. Here we show that Trbp (Tarbp2), an RNA binding protein, is required for normal heart function. Cardiac-specific inactivation of Trbp (Trbp-cKO) caused progressive cardiomyopathy and lethal heart failure. Genome-wide transcriptome profiling revealed that Trbp loss of function resulted in upregulation of Sox6, repression of genes encoding normal cardiac slow-twitch myofiber proteins, and pathologically increased expression of skeletal fast-twitch myofiber genes. Remarkably, knockdown of Sox6 fully rescued the Trbp mutant phenotype, whereas AAV9-mediated Sox6 overexpression in the heart phenocopied the Trbp-cKO phenotype. Trbp inactivation was mechanistically linked to Sox6 upregulation through altered processing of selected microRNAs. miR-208a, a direct inhibitor of Sox6 expression, was one of the most strongly depleted microRNAs in Trbp-cKO hearts. Transgenic overexpression of miR-208a was sufficient to repress Sox6, restore the balance of fast- and slow- twitch myofiber gene expression, and rescue cardiac function in Trbp-cKO mice. Our studies reveal the critical role of Trbp-mediated microRNA processing in regulating a linear genetic cascade essential for normal heart function.

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Platform Session 9 9. Cardiovascular genetics 9-01

ABERRANT ENDOTHELIAL-MYOCARDIAL CROSSTALK CAUSES HYPERTROPHY IN NOONAN SYNDROME WITH MULTIPLE LENTIGINES Janel R. Cabrera Jessica Lauriol, Janel R. Cabrera, Kimberly Keith, Gabriel C. Segarra, Meaghan E. Flessa, Lauren E. Miller, Roderick Bronson, Kyu-Ho Lee, Maria I. Kontaridis Beth Israel Deaconess Medical Center/Harvard Medical School Congenital heart disease (CHD) is the most common birth defect worldwide; however, underlying mechanisms remain unknown. Loss-of-function mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP2, are implicated in CHD and cause Noonan Syndrome with Multiple Lentigines (NSML). NSML presents with multiple cardiac defects, including hypertrophy. Here, we found that the NSML-associated adult-onset cardiac hypertrophy stems from aberrant signaling originating from developing endocardium. Embryonic NSML hearts showed diminished trabeculation and valvular hyperplasia, defects recapitulated in endocardial-, but not myocardial- or neural crest-, specific NSML mice. NSML hearts also developed ventricular septal defects, a phenotype reproduced only in myocardial-specific NSML hearts, suggesting NSML mutations have both cell autonomous and non-autonomous functions in cardiac development. Importantly, endocardial-specific expression of NSML was sufficient to induce adult-onset cardiac hypertrophy. Mechanistically, we observed aberrant AKT activity in NSML embryos, with decreased downstream FOXP1/FGF and NOTCH1/EPHB2 signaling, two pathways necessary for reciprocal crosstalk between developing endocardium and myocardium. Taken together, our data provide the first functional and disease-based evidence to suggest that critical mechanisms exist to control endocardial-myocardial crosstalk, the aberrant regulation of which may lead to CHD and adult-onset cardiac disease.

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Platform Session 9 9. Cardiovascular genetics 9-02

PRDM16 - A NOVEL KEY PLAYER IN PERSONALIZED MEDICINE Anne-Karin Arndt Anne-Karin Arndt, Manu Beerens, Eva R. Buys, Gabe Musso, Sabine Klaassen, Hans-Heiner Kramer, Calum A. MacRae Department for Congenital Heart Disease and Pediatric Cardiology, University Kiel We identified a nonsense mutation in PRDM16 resulting in left ventricular non-compaction (LVNC) and dilated cardiomyopathy in human patients. To establish a personalized disease model, we recapitulated the LVNC by cardiomyocyte-specific overexpression of both mutant and wildtype (WT) PRDM16 in zebrafish. We observed an impaired cardiomyocyte proliferation with associated physiologic defects in cardiac contractility and cell-cell coupling during development in mutant, but not WT PRDM16 zebrafish and these defects persisted throughout adulthood. Moreover, mutant PRDM16 zebrafish displayed increased mitochondrial membrane potential and elevated levels of oxidative stress in the heart. In a next step, using a phenotype-driven screening approach, we identified a melanocortin 4 receptor (MC4R) antagonist that rescued the physiologic defects associated with mutant PRDM16 during development. Of note, MC4R antagonists could also rescue the cardiac defects seen in zebrafish models for arrhythmogenic right ventricular cardiomyopathy (ARVC), raising the intriguing question whether Prdm16 is also dysregulated and/or mutated in ARVC patients. While future studies to further elucidate the exact mechanism of mutant PRDM16-related cardiomyopathy are warranted, our current findings underline the importance of personalized disease models, especially for the exploration of disease biology and the development of innovative tailor-made therapies.

61

Platform Session 9 9. Cardiovascular genetics 9-03

INCREASED TBX1 GENE DOSAGE AND THE 22Q11.2 DUPLICATION SYNDROME Silvia E. Racedo Silvia E. Racedo, Donna M. McDonald-McGinn, Erica Hasten, Laura DiCairano, Elaine Zackai, Beverly Emanuel, Bernice Morrow Albert Eistein College of Medicine Abnormal meiotic rearrangements on chromosome 22q11.2 can result in either a deletion (22q11.2DS) or duplication (22q11.2Dup) causing congenital heart disease (CHD). Combining our unpublished data and those in the literature, we found a total of 15.6% of 115 individuals with 22q11.2Dup have CHD, primarily of the conotruncal type (CTD). Previous studies have shown that global loss or gain of function (LOF; GOF) of mouse Tbx1, encoding a T-box transcription factor, on 22q11.2, results in similar CTDs. We asked whether LOF or GOF of Tbx1 in the anterior second heart field (aSHF) could result in similar heart defects. We found that Tbx1 LOF in the aSHF results in a persistent truncus arteriosus (PTA) while constitutive aSHF GOF results in a PTA with a hypoplastic right ventricle (RV). We asked whether there is a molecular basis for why there are similar defects in both mutants. Gene expression profiling was performed on the distal pharyngeal apparatus of Tbx1 aSHF LOF and GOF embryos to understand the basis of the defects. Several heart development genes were changed in expression similarly in both GOF and LOF embryos versus WT (Hand1, Foxa2, Aldh1a2, Tbx20, Rxrg, Six2), possibly explaining why both sets of mutants have a PTA. In contrast, other genes (Tbx5, Wnt2, Gata4, Myh6) were changed in the opposite manner in both mutants versus WT. This could explain why GOF but not LOF mutants have a hypoplastic RV. Some of these genes may modify the human 22q11Dup phenotype.

62

Platform Session 9 9. Cardiovascular genetics 9-04

RECESSIVE AND COMPOUND-HETEROZYGOUS VARIANTS IN NOVEL GENE PATHWAYS IN CONGENITAL HEART DISEASE H. Joseph Yost Scott W. Watkins, Brett Kennedy, Brent W. Bisgrove, Martin Tristani-Firouzi, Mark Yandell, H. Joseph Yost University of Utah Protein-altering de novo variants in cardiac genes were found in ~10% of congenital heart disease (CHD) cases identified by whole-exome sequencing (WES) of 362 CHD parent-offspring trios by the Pediatric Cardiac Genomics Consortium (PCGC). Using the FastQForward bioinformatic pipeline designed at the University of Utah, we re-examined 243 PCGC trios of European ancestry for de novo, recessive, hemi-zygous, and compound-heterozygous inheritance. Prioritized variants were intersected with a list of 491 candidate CHD genes. FastQForward exhibited a 96% concordance rate for de novo variants, underscoring the ability to precisely identify the same alleles as the PCGC. Additionally, FastQForward identified rare and highly damaging recessive or compound-heterozygous alleles in CHD candidate-genes in 57 probands, accounting for ~23% of CHD cases. Nine novel, 73 rare (30% of CHD cases.

63

Main Posters 9 9. Cardiovascular genetics 9-05

LUMICAN DEFICIENCY RESULTS IN CARDIOMYOCYTE HYPERTROPHY WITH ALTERED COLLAGEN ASSEMBLY Loren E. Dupuis, Matthew Berger, Samuel Feldman, Lorna Doucette, Vennece Fowlkes, Shukti Chakravarti, Sarah Thibaudeau, Amy D. Bradshaw, Christine B. Kern Medical University of South Carolina Previous studies suggest mice deficient in the small leucine-rich proteoglycan, lumican (LUM) (lum-/-) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, however lum-/- hearts have not been evaluated. These studies show LUM was immunolocalized to non-cardiomyocytes and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and an increase in myocardial tissue without an increase in cell proliferation. Cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum-/- hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum-/- hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in lum-/- as well as the β and γ forms of collagenα1(I). While the total insoluble collagen was significantly reduced, the fibril size was increased in lum-/- hearts, indicating LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart.

64

TECHNOLOGY BREAKOUT

ABSTRACTS

65

Technology breakout T1-1

MULTIPLEX ANALYSIS OF GENE EXPRESSION IN INDIVIDUAL LIVING CELLS Ayhan Atmanli, Dongjian Hu, Annebel Marjolein van de Vrugt, Ibrahim John Domian Massachusetts General Hospital The capacity of pluripotent stem cells (PSC) to recapitulate many of the in vivo developmental programs provides an attractive in vitro model system for studying lineage commitment and cellular differentiation. Advances in the generation of patient-specific PSC have now also allowed for the development of novel and evolving approaches for the study of human disease. Despite this progress and the promise of stem cell biology in clinical medicine, a significant obstacle for their use is the lack of robust assays to simultaneously examine the expression of multiple genes in living cells undergoing normal differentiation or pathogenesis. This consideration is particularly relevant in view of the fact that PSC differentiation results in a diverse population of heterogeneous cells with complex and interacting regulatory networks. To overcome these limitations, we have developed a novel FRET-based technology for the multiplex analysis of gene expression in individual living cells (MAGIC) to perform concurrent gene expression and functional analyses of single cardiac myocytes (CMs). We apply our live-cell mRNA imaging technology to demonstrate distinct physiological profiles of human PSC-CMs expressing MLC2v and MHCalpha as markers of the ventricular cell fate and myocardial maturity, respectively. By tailoring our live-cell gene expression imaging with functional assays, we open new avenues for the study of myocardial lineage commitment and maturation.

66

Technology breakout T1-2

VISUALIZATION OF PRIMARY CILIA INSIDE INTACT ORGANS USING SCALE/CUBIC TISSUE CLEARING Markus Delling, Shu-Hsien Sheu, David Clapham Boston Children’s Hospital Almost all mammalian cells have a single primary cilium, even in adulthood. Primary cilia loss or disruption of function results in severe human developmental malformations and diseases termed ciliopathies. Primary cilia are solitary, non-motile tubular structures less than 40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

85

Technology breakout T6-3

CRISPR/CAS9-MEDIATED GENOME EDITING TO STUDY CARDIAC TRANSCRIPTIONAL CONTROL Jianxin Hu, Shanmei Xu, Brian Black Cardiovascular Research Institute, University of California, San Francisco Next generation genome editing technologies, including CRISPR, provide powerful tools to study genome function. In the present work, we used CRISPR/Cas9 genomic engineering to delete putative cis-regulatory elements and to conduct a mutational screen for novel transcription factors and transmembrane protein-encoding genes expressed in the heart. Deletion of a a cardiac-specific enhancer from the prkaa2 locus dramatically impaired the expression level of prkaa2 in the developing heart. Deletion of the Mef2c-F1 neural crest enhancer abolished the ability of Mef2c to response to Endothelin signaling. In our small scale CRISPR-based mutational screen, we identified two evolutionarily conserved genes that are critical for early embryo development. The results of these experiments will be presented. In addition, our methods and a discussion of successes and caveats with CRISPR-based mutations, deletions, and insertions using mouse oocyte injection will be presented.

86

Technology breakout T6-4

ADENO-ASSOCIATED VIRUS (AAV)-MEDIATED FUNCTIONAL SCREENING OF LONG NONCODING RNAS (LNCRNAS) IN CARDIAC HYPERTROPHY Zhan-Peng Huang, Gengze Wu, Jinghai Chen, Zhiqiang Lin, William T. Pu, Da-Zhi Wang Department of Cardiology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA Long noncoding RNAs (LncRNAs) are RNA transcripts longer than 200 nucleotides that lack protein-coding potential. Although thousands of lncRNAs have been identified, only a few have been linked to cardiogenesis and cardiac function. To better understand how lncRNAs regulate cardiac hypertrophy, we developed an adeno-associated virus serotype 9 (AAV9)-based functional screening in postnatal Mice. An AAV9:cTNT vector, in which the cardiac troponin T (cTNT) promoter was used to direct cardiac-specific expression of target genes, was utilized to overexpress or knockdown candidate lncRNAs in mouse hearts. We showed, in control experiments, that the AAV9 directed GFP transgene expression in the heart in a cardiac-specific manner with high efficacy after being injected into neonatal mice. Postnatal day 1 wild type or CnA transgenic pups were injected with AAV9 viruses for the screening of hypertrophy-related lncRNAs. We tested 15 candidate lncRNAs and confirmed that AAV9-based gain-of-function and loss-of-function constructs were able to overexpress or knock down these lncRNAs efficiently in the heart. Currently, the phenotype of lncRNA-manipulated animals is under investigation. Our results indicated that AAV is a powerful tool to perform in vivo functional screening and analysis of interesting genes in the heart.

87

Technology breakout T6-5

PERMANENT OR TRANSIENT GENE INACTIVATION BY RNA-GUIDED CAS9 NUCLEASE Gang Wang, Luhan Yang, Dennis Grishin, George Church, William Pu Department of Cardiology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA The RNA-guided Cas9 nuclease has emerged as a powerful genome editing tool. Combining Cas9 nuclease with Piggybac transposase technology, we developed a highly efficient platform for permanent genome editing in induced pluripotent stem cells. Using this platform, we permanently knocked out the Tafazzin gene (TAZ), the Barth syndrome disease gene, in hPSC at genome DNA level with over 70% efficiency, with an off-target mutagenesis rate comparable to background mutation rates observed in control cultured cells, as determined by whole genome sequence and targeted deep sequence. Next, we show that the Cas9 nuclease can be used to deplete targeted RNAs to transiently deplete gene transcripts. We were able to knock down TAZ mRNA expression at the RNA level by over 60-80% in hPSC and hPSC derived cardiomyocytes. This mRNA depletion was transient, and cells recovered normal TAZ expression with further culture. Our study provide a proof of concept that CRISPR/Cas9 can achieve highly efficient and specific gene edition both at genomic DNA level and at mRNA levels in hPSCs.

88

Technology breakout T6-6

IN VIVO AND IN VITRO PROTEIN DETECTION WITH LABEL-FREE NANOSENSORS Qimin Quan, Feng Liang Rowland Institute at Harvard University The stochastic expression of genes, proteins and metabolites result in cellular heterogeneity. Minimally-invasive, in situ and in vivo analytical technology for single cells will be especially useful in the area of stem cell, cardiovascular disease and neuron research, and will provide fundamental improvements of understanding in the field of cell biology. We will present two platforms of label-free biosensors towards single cell analysis and single protein detection, both based on optical nanocavities. The first platform is a nanoplasmonic bioprobe. The bioprobe comprises of a tapered optical fiber with 50 nm tip dimension. A single gold nanosensor is attached to the end of the tip, the surface of which is functionalized with antibodies specific to the target analyte. The collective oscillation of electrons at the surface of the gold nanosensor generates a surface plasmon resonance, which can be used to detect small quantity of proteins. This bioprobe can be inserted into a living single cell and detect intracellular proteins without affecting the cell viability. The second platform is an on-chip photonic nanocavity biosensor. Photons are strongly confined in the nanocavity with ultrahigh quality factor. This generates a sharp resonance, the resonance of which can be used to detect protein affinity at high sensitivity.

89

Technology breakout T7-1

EX VIVO APPROACHES TO STUDY EPICARDIAL REGENERATION IN ZEBRAFISH Jingli Cao, Jinhu Wang, Amy Dickson, Kenneth Poss Duke University The epicardium is a multifunctional cell layer covering the heart that is critical for cardiac development and repair. Despite its importance in cardiac repair and its potential as a therapeutic target for heart disease, there is virtually nothing known about the regenerative capacity of the epicardium itself. In this study, we developed an ex vivo explant culture system, in which the dissected heart contracted for weeks. Together with a transgenic line to ablate epicardial cells in adult zebrafish, we could monitor epicardial regeneration in real-time through live imaging, and found a directed regeneration of the epicardial cell sheet from ventricle base to apex. We also manipulated epicardial regeneration ex vivo by extirpating or graft a chamber or tissue in heart explant, or transplantation of epicardial cells from another heart. These manipulations confirmed that ventricular epicardial regeneration was regulated by the neighboring tissue-bulbous arteriosus (BA). The ex vivo approach enables high-resolution phenotyping of epicardial regeneration in molecular lever. Through chemical screen, we found several signling pathways that could possibly regulate epicardial regeneration and Hedgehog (Hh) signaling pathway is one of the key players. The ex vivo system deepened our understanding of epicardial regeneration in both cellular and molecular level, will lead to more findings.

90

Technology breakout T7-2

EPIGENETIC ANALYSIS OF HEART DEVELOPMENT BY BIOTIN MEDIATED CHIP-SEQ Yong Hu, Fei Gu, Aibin He, Pingzhu Zhou, Bing Zhang, William Pu Boston Children’s Hospital A major question in decoding human heart development is the identification of the enhancers that control the spatial and temporal expression of cardiac genes. Traditional chromatin immunoprecipitation (ChIP) has led to novel insights into understanding of the transcription factors and identification of heart enhancer; however, it is limited by availability of suitable antibodies. Biotinylation is an attractive approach for protein purification due to the high and specific affinity of streptavidin. Here, we generated transgenic mice, which have a short bio epitope on transcription factors Gata4, Nkx2-5, Tead1, Mef2c, and Srf respectively. The Rosa26-BriA allele expresess the biotinylation enzyme BirA, resulting in in vivo biotinylation of the tagged transcription factor. The tagged factor can then be pulled down on immobilized streptavidin to permit analysis of bound DNA by ChIP-seq, or interacting proteins. Using this strategy, we have defined cardiac transcription factor occupancy in the fetal and adult heart. These mice are freely available through Jackson Labs and the Mutant Mouse Regional Resource Centers.

91

Technology breakout T7-3

BENCH TO BASSINET MOUSE MODELS OF CONGENITAL HEART DISEASE AND MOUSE GENETIC RESOURCE Nikolai Klena, George Gabriel, You Li, Cecilia Lo University of Pittsburgh We conducted a large-scale mouse ENU mutagenesis screen to recover mutations causing congenital heart disease (CHD) as part of the Bench to Bassinet (B2B) consortium. This entailed using noninvasive fetal echocardiography for CHD diagnosis, followed by necropsy, micro-CT, and/or episcopic confocal microscopy (ECM). This tiered approach yielded over 250 CHD mutant lines. The phenotypes of each line are curated in the Mouse Genome Informatics database. In this curation effort, we added much new vocabulary to the Mouse Phenotype Ontology for structural heart defects. In addition, we also included the clinical Fyler codes to allow cross-referencing to clinical patient phenotypes. Using exome sequencing, we recovered the pathogenic mutations in 135 mutant lines encompassing 91 genes. Statistical modeling indicated 259 CHD genes in the mouse genome, suggesting our screen is at 35% saturation. We also recovered 11,994 incidental mutations in 7,430 genes. The G1 sperm for each line is cryopreserved as part of the B2B collection in the JAX Induced Mutant Strain Resource. This provides public access not only to the pathogenic mutations, but also the many incidental mutations, including knockout alleles of genes not represented in KOMP. These mutations are searchable via the B2B Mouse Mutation Database and via MGI. In summary, we generated a rich genetic resource invaluable to the cardiovascular community, and the biomedical research community at large. Supported by NIH HL1098180.

92

POSTER SESSION

ABSTRACTS

93

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-07

TRANSCRIPTION FACTOR SMAD1 INTEGRATES BMP2 AND WNT3A SIGNALS DURING CARDIAC PROGENITOR CELL MIGRATION James McColl, J F Song, E Camp, N Kennerley, G F Mok, D McCormick, T Grocott, G N Wheeler, A Munsterberg University of East Anglia In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak. They migrate laterally and then towards the ventral midline where they form the heart. Developmental signals involved in the specification of cardiogenic mesoderm have been studied extensively, however, signals controlling CPC migration are poorly understood. Several pathways are involved in the epithelial to mesenchymal transition, ingression and migration of mesoderm cells through the primitive streak, including fibroblast growth factors (FGFs) and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a novel role for BMP/Smad signalling. We identify an interaction of BMP and Wnt/GSK3β pathways via the differential phosphorylation of Smad1. Overall we reveal molecular mechanisms that contribute to the emerging paradigm of signalling pathway integration in embryo development.

94

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-08

RTF1 DRIVES CARDIAC PROGENITOR FORMATION THROUGH TBX20 Fei Lu, Adam D. Langenbacher, Jie Huang, Qingyu Li, Jau-Nian Chen UCLA While the transcriptional programs governing cardiac differentiation have been delineated, genes driving cardiac cell fate decision are less understood. Here we present genetic evidence in zebrafish and mice indicating that Rtf1, a component of the evolutionarily conserved RNA polymerase II-associated factor 1 complex (PAF1C), acts at a very early step in the hierarchy of cardiac transcription program. We found that overexpression of rtf1 in zebrafish embryos promotes cardiac progenitor formation, whereas rtf1 deficiency eliminates the entire cardiac progenitor population. Similarly, knockdown of rtf1 prevents cardiac differentiation in mouse embryonic stem cells, and rtf1 ablation abolishes the formation of cardiac progenitors in mice. Mechanistic studies indicate that Rtf1 regulates cardiac transcription program via its DNA binding Plus3 domain. Mutations in Plus3 domain abolish Rtf1’s ability to drive cardiac progenitor formation. Among Rtf1 targets is Tbx20, a transcription factor expressed early in the heart field. In zebrafish, tbx20 deficiency drastically reduces cardiac tissues and overexpression of tbx20 results in an enlarged heart, attesting to an essential role for Tbx20 in heart formation. Interestingly, tbx20 expression is drastically suppressed in rtf1 deficient embryos and overexpression of tbx20 effectively restores cardiac progenitor population. Together, these findings demonstrate a critical role for the Rtf1-Tbx20 pathway in cardiac progenitor formation.

95

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-09

CHIRAL CELL ALIGNMENT IN DORSAL MYOCARDIUM REGULATES ASYMMETRIC LOOPING OF THE EMBRYONIC HEART Poulomi Ray, Michael J. Raymond, Gurleen Kaur, Leo Q. Wan Rensselaer Polytechnic Institute During development, the embryonic heart starts as a straight tube along the midline of the embryo, which then bends and rotates towards the right, forming a c-shaped loop. The exact mechanism determining the directionality of the rotation of the heart tube is yet to be understood. In this study, we investigated the role of chirality of cell shapes regulating the rightward rotation of the heart tube using chick embryo as a model system. The chick embryonic heart begins as a straight tube at HH9, consisting of an outer myocardium and an inner endocardium, which undergoes an asymmetric 90° rightward rotation around HH10. We mapped the cell alignment of different regions of the heart tube, along the anterior-posterior and dorsoventral axes, before and during rotation using confocal imaging and quantitative image analysis. We measured the angle between apical cell boundaries and the anterior-posterior axis of the embryo to determine right or left bias. Our studies demonstrate that dorsal myocardial cells have biased planar cell shape during rotation. Additionally, there is an asymmetry of cell alignment between right and left side of the dorsal myocardium, the cells on the right side being significantly more biased towards the right. Currently, we are investigating the interplay between left-right asymmetric molecular signals and planar cell chirality of the dorsal myocardium contributing towards right-handed rotation of the heart tube.

96

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-10

MIR-200B MODULATES CARDIAC LINEAGE CELL FATE IN MOUSE EMBRYONIC STEM CELLS José Manuel Vilches, Francisco Hernández-Torres, Estefanía Lozano-Velasco, Diego Franco, Amelia Aranega University of Jaen ESCs represent a potentially attractive source of cardiac cells for the treatment of cardiovascular diseases. The use of pluripotent stem cells may enable the generation of large quantities of specialised cells that can be used as in vitro tools for drug development as well as for future applications in regenerative medicine. However, most of the currently used differentiation protocols yield inefficient quantities of differentiated cells and low purity of the final cell preparations. The discovery of miRNAs and their roles as important post-transcriptional regulators may provide a new means of manipulating stem cell fate. Here we shown that miR-200b, which has been previously showed to be significantly up-regulated during cardiomycyte differentiation, modulates the contractile phenotype during in vitro cardiac differentiation from ESCs. Interestingly, miR-200b regulates the expression of cardiac genes that define the electrophysiological properties of cardiomyocytes, such as the ion channels genes Scn5a and Scn3b and Cx45. Moreover, miR-200b also modulates the expression levels of the transcription factor Tbx5,a cardiac determinant gene, recently linked to atrial arrhythmias. These data suggest relevant impact in regenerative medicine since we showed that miR-200b can modify the cell fate of the cardiac lineage in ESCs. Additional experiments and further analyses could leads us to purpose this miRNA as excellent tools to obtain specific cardiac cell types from ESCs.

97

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-11

CELL CHIRALITY AND ITS IMPLICATION IN CARDIAC C-LOOPING Kathryn Worley , Leo Q. Wan Rensselaer Polytechnic Institute The development of the vertebrate body plan with left-right (LR) asymmetry (also known as handedness and chirality) requires the emerging chiral morphogenesis. Cardiac C-looping was considered as the first evidence of LR asymmetry at an organ level. Changes in orientation of the LR axis can lead to malformations, such as dextrocardia. We previously demonstrated that the cultivation of homogenous cell populations on micropatterns reveals an intrinsic cell left-right asymmetry can be readily determined by image analysis of cell alignment and directional motion. Recently, we found that disrupting adherens junctions resulted in a decrease in velocity difference in opposing boundaries as well as the associated biased cell alignment, along with an increase in the overall random motion. The further examination of cell polarity indicated that disruption of adherens junctions did not affect cell polarization on the boundaries, but decreased the transmission of chiral bias into the interior region. Overall, our results demonstrated the dependence of the scale of collective cell migration on the strength of cell-cell adhesion, and its effects on the chirality of a multicellular structure through mediating cell polarity. These data have strong implications in cardiac C-looping. We propose that micropatterning could be used as an effective in vitro tool to study the initiation of LR asymmetry, to diagnose disease, and to study factors involved with birth defects in laterality.

98

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-12

IDENTIFICATION OF CARDIOVASCULAR LINEAGE DESCENDANTS AT SINGLE-CELL RESOLUTION Guang Li, Karolina plonowska, Rajarajan Kuppusamy, Anthony Sturzu, Sean M. Wu Stanford University The transcriptional profiles of cardiac cells have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single cell level may demonstrate unique variations. In this study, we aimed to establish a single cell qPCR platform and perform side-by-side comparison between cardiac progenitors cells (CPCs) and cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) and embryos. We first generated a reference map for cardiac cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts, and mESCs. This panel was then applied against single day 10.5 embryonic heart cells to demonstrate its ability to identify each cardiac cell types. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single cell expression assays can resolve the lineage relationships between progenies of single CPCs. With this approach, we found that mESC-derived Nkx2-5+ CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5+ CPCs represent two distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex qPCR analysis in single cells is a powerful tool in examining the unique behaviors of individual cardiac cells.

99

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-13

THE CAPZ INTERACTING PROTEIN RCSD1 IS REQUIRED FOR VERTEBRATE CARDIOGENESIS DOWNSTREAM OF WNT11 Annemarie Hempel, Susanne J. Kühl, Purushotama Rao Tata, Barbara Kracher, Ioan Ovidiu Sirbu, Michael Kühl University of Ulm Wnt proteins have been shown to have essential functions during cardiogenesis and cardiomyogenesis, although the molecular details remain elusive. Disruption of non-canonical Wnt11 in mice causes profound disturbances in cardiac morphogenesis and differentiation, deficits in trabeculation as well as proper sarcomer organization. In this study we demonstrated that loss of Wnt11a the Wnt11 homolog in Xenopus, resulted in a similar cardiac phenotype as in mouse wnt11-/- mutants. Furthermore, we characterized the molecular signalling pathways underpinning the role of Wnt11a in heart development. We show for the first time that depletion of the recently identified non-canonical Wnt mediator Rcsd1 in Xenopus resulted in heart defects resembling Wnt11a morphants. Strikingly, impaired cardiomyocytes differentiation of Wnt11a morphants could be rescued by ectopic expression of rcsd1, implicating a requirement of rcsd1 downstream of wnt11a. Moreover, we showed that this activity of Rcsd1 requires its interaction with the capping protein CapZ and that the capping protein interaction domain of Rcsd1 is of functional relevance. In summary, we provide evidence for a novel mechanism for the regulation of cardiogenesis by non-canonical Wnt signalling, hence extending our understanding of heart development during embryogenesis.

100

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-14

PDGF SIGNALING DIRECTS MEDIAL CARDIOMYOCYTE MOVEMENTS DURING THE INITIATION OF HEART TUBE ASSEMBLY Joshua Bloomekatz, Ariel C. Dunn, Megan Vaughan, Deborah Yelon UCSD Heart tube assembly begins with bilateral epithelial sheets of cardiomyocytes moving toward the midline, where they merge together in a process called cardiac fusion. The signals that direct these cardiomyocyte movements remain unknown. Using a combination of forward genetics and high-resolution cell tracking in zebrafish, we have revealed a novel role for platelet-derived growth factor (PDGF) signaling in cardiomyocyte movement. We identified an ENU-induced mutation named refuse-to-fuse (ref) that inhibits cardiac fusion, resulting in a bifurcated ventricle. Positional cloning revealed that the ref mutation disrupts the splicing of pdgfra, causing premature truncation of the receptor PDGFRα. Anterior endoderm formation is known to facilitate medial cardiomyocyte movement; however, there are no evident anterior endoderm defects in ref mutants. Cell tracking revealed that cardiomyocytes in ref mutants are not directed medially, despite other normal aspects of their motility. pdgfra is expressed in cardiomyocytes prior to their movement to the midline, and the PDGF ligands pdgfaa and pdgfab are expressed in the adjacent cranial mesenchyme. Global overexpression of pdgfaa causes cardiac fusion defects, indicating an instructive role for PDGF signaling in cardiomyocyte movement. Together, our data suggest that PDGF signaling is one of the previously unknown pathways that acts within developing cardiomyocytes to direct their medial movement during heart tube assembly.

101

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-15

INVESTIGATION OF THE MECHANISMS REGULATING PROGENITOR CELL SEGREGATION TO THE CARDIAC POLES Christopher De Bono , Magali Théveniau-Ruissy , Alexandre Francou, Stephane Zaffran, Robert G. Kelly Aix Marseille University Cardiac progenitor cells in pharyngeal mesoderm contribute to the poles of the heart tube during looping morphogenesis. These progenitor cells, termed the second heart field (SHF), give rise to right ventricular and outflow tract (OFT) myocardium at the arterial pole of the mouse heart and atrial myocardium at the venous pole. Perturbation of SHF development results in a spectrum of common congenital heart defects including OFT alignment as well as atrial and atrioventricular septation defects (AVSDs). The transcription factor TBX1, encoded by the major DiGeorge or 22q11.2 deletion syndrome gene, regulates key properties of SHF cells, including proliferation, differentiation delay and epithelial cell shape. Tbx1 is required for addition of future subpulmonary myocardium to the inferior wall of the developing OFT. Subpulmonary myocardium originates in Hox-expressing cells in the posterior SHF and is clonally related to venous pole myocardium. In addition to OFT defects, loss of Tbx1 results in partially penetrant AVSDs due to hypoplastic development of the SHF-derived dorsal mesenchymal protrusion. Here we show that during SHF deployment Tbx1 is maintained in arterial pole progenitor cells, progressively downregulated in Tbx5-positive venous pole progenitor cells and required to establish the boundary between these cell populations, identifying a nodal role for Tbx1 in coordinating SHF addition to the cardiac poles.

102

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-16

ALPK2 IS A NOVEL REGULATORY KINASE REQUIRED FOR HEART DEVELOPMENT Peter Hofsteen, Aaron Robitaille, Nathan Palpant, Lil Pabon, Randall T Moon, Charles E Murry University of Washington Understanding the molecular mechanisms of heart development provides clues toward novel therapies for heart disease. Alpha-kinase 2 (ALPK2) is a recently discovered atypical eukaryotic protein kinase with no known function during heart development. Here, we sought to determine the role of ALPK2 by using two models of cardiogenesis: human pluripotent stem cell (hPSC) differentiation towards definitive cardiomyocytes in vitro and zebrafish in vivo. Chip-Seq, quantitative proteomics and RNA analysis over time course cardiomyocyte differentiation revealed ALPK2 is expressed and epigenetically regulated in cardiac progenitor cells (CPCs) and definitive cardiomyocytes. ALPK2 knockdown (KD) results in failure to form CPCs and subsequently contractile cardiomyocytes. Furthermore, cells lacking ALPK2 fail to undergo EMT and show differential expression of proteins involved in cell polarity and microtubule formation. In zebrafish, KD of Alpk2 supports our findings in vitro. Alpk2 KD fish show severe cardiac defects with decreased function and failure to form epicardium. Collectively, ALPK2 is a novel protein kinase required for vertebrate heart development by playing a role in specification and cellular morphogenesis of CPCs.

103

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-17

UNRAVELLING THE ONTOGENY AND FUNCTIONS OF MESENCHYMAL PROGENITORS DURING MURINE CARDIAC DEVELOPMENT Sara González, Simón Méndez-Ferrer, Joan Isern Centro Nacional de Investigaciones Cardiovasculares (CNIC) The complexity of stromal populations within each tissue is an emerging concept. In developing heart, mesenchymal progenitor cells (MPCs) arise from multiple sources, although their contributions to heart morphogenesis are poorly characterized. Moreover, the relationship of these stromal derivatives with the underlying process of vascularization remains unclear. Nestin (Nes) is a marker of neural stem cells, but is also transiently detected in non-neural tissues, including heart. The early onset of Nes-Gfp transgene expression allows the study of dynamics and function of resident MPCs and selected endothelial subsets, during cardiac development. Interestingly, Nes-GFP+ cells are predominantly associated with developing coronary vessels, and encompass supporting pericytes. We also performed genetic fate mapping by tamoxifen-inducible Cre recombinase driven by Nes 2nd-intron enhancer, and we found that Nes-lineage distinguishes a subgroup of coronary microvasculature, reminiscent of the distinct coronary vascular populations recently revealed by means of Apelin lineage tracing. Deletion of the chemokine Cxcl12 in mice results in embryonic lethality due to multiple defects, including cardiac malformations. Our preliminary data indicate that global Cxcl12 null embryos exhibit also alterations in the patterning and formation of main coronary arteries. We speculate whether Cxcl12 produced by nestin-related cardiac lineages is contributing to this phenomenon.

104

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-18

LNCCPR, A LONG NONCODING RNA EXPOSED AS A NOVEL REGULATOR OF CARDIOPOEISIS Ashley Benham, Benjamin Soibam, Matthew Robertson, Kuo-Chan Weng, Linet George, Yu Liu, Robert Schwartz Texas Heart Institute Heart related diseases are a leading cause of death in the United States. Damaged hearts can lead to loss of cardiomyocytes, fibrosis, and an overall loss of heart function. Cardiac development and the processes defining the cardiac lineage need to be further explored in order to develop and evolve therapeutic options. Using a Mesp1 lineage tracing stem cell line, we present the first early mesoderm and early cardiac progenitor transcriptome using RNA-sequencing. From this transcriptome profiling, we have identified 2649 lncRNAs with enriched expression in the mesoderm-derived population(s). Further expression analysis of a selected set of lncRNAs in vivo yielded 4 lncRNAs with consistent expression patterns in mesoderm-derived tissues with 2 of the 4 present in adult heart tissue. Functional characterization of AK015665, lncCPR, revealed to be a novel regulator of cardiac differentiation via regulation of key genes in the core cardiac transcription factor network.

105

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-19

A MOLECULAR SWITCH REGULATING HEART AND PANCREATIC DEVELOPMENT Weijia Luo, Zengbin Wu, Kuo-chan Weng, Wei Yu, Xueping Xu, Yu Liu, Austin Cooney, Robert J. Schwartz, Li Chen University of Houston A population of the intermediate common progenitors of the mesoderm (Me) and the endoderm (En) was identified in vitro as the mesendoderm (Mes), which existence in vivo is still unclear, nor is the molecular regulation of its differentiation. Here we showed in mouse that bHLH transcription factor Mesp1 is transiently expressed in a subset of the epiblast. Lineage tracing revealed the contribution of the Mesp1+ progenitors to both the Me and En, suggesting that Mesp1 marked the bio-potent Mes progenitors. Mesp1-fated En exclusively gave rise to the pancreatic progenitors, and ultimately to the endocrine cells in the pancreas. RNA-seq and ChIP showed that Mesp1 bound directly to the endogenous regulatory elements of Mes and endodermal regulators. Conditional ablation of Foxa2 in the Mesp1-fated cells led to postnatal death due to hyperinsulinemia. Ablation of Mesp1 in vivo abolished the majority of Mes modulators while led to ectopic expression of En transcription factors, indicating that Mesp1 maintained bi-potency of the Mes by inhibiting the En transcription program. We generated chimeric embryos by injecting into WT blastocysts the control or the Mesp1 homozygous (mutant) ES cell lines. Mutant ESCs failed to contribute to the cardiac Me, but adopted the En fate in vivo, indicating that Mesp1 inhibited the En transcription program in a cell-autonomous manner. Our results demonstrated that Mesp1 functioned as a critical molecular switch between the Me and the En formation.

106

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-20

ETV2-MIR-130A NETWORK REGULATES MESODERMAL PRECURSORS THROUGH INHIBITION OF PDGFRA SIGNALING Bhairab N. Singh, Xiaozhong Shi, Ryutaro Akiyama, Tara L. Rasmussen, Mary G. Garry, Wuming Gong, Yasuhiko Kawakami, Naoko Koyano-Nakagawa, Daniel J. Garry University of Minnesota MicroRNAs (small non-coding RNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined a novel Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality by E12.5. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors towards the endothelial lineage. Loss- and gain-of-function experiments demonstrated that miR-130a is an important regulator of the endothelial program at the expense of cardiac program without impacting the hematopoietic lineages. Mechanistically, miR-130a directly suppresses expression level of Pdgfra and promotes the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a over-expression and knockdown, respectively. This is the first report of a miRNA that specifically promotes the divergence of the common hemato-endothelial progenitor to the endothelial lineage.

107

Main Posters 1 1. Cardiogenesis/cardiac lineages/early heart development 1-21

THE AP-1 COMPONENT FOSL2 POTENTIATES MYOCARDIAL DIFFERENTIATION FROM THE ZEBRAFISH SECOND HEART FIELD Leila Jahangiri, Meghan Adams, Noelle Paffett-Lugassy, Long Zhao, Kathleen Nevis, Burcu Guner-Ataman, Caroline E. Burns, C. Geoffrey Burns CVRC/MGH/Harvard Medical School During embryogenesis, second heart field (SHF) progenitors elongate the heart tube through accretion of myocardial cells to the poles. Although defects in this process cause congenital heart defects, factors regulating the initiation, rate, and duration of myocardial accretion remain incompletely defined. Here, we demonstrate that a component of AP-1, Fos-related antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. We isolated two null alleles of fosl2 that cause homozygous embryos to exhibit ventricular myocardial deficits. Specifically, the linear heart tube in fosl2 mutants was indistinguishable from siblings reflecting unperturbed first heart field development. Although Fosl2 null embryos initiated myocardial accretion without delay, a ventricular cardiomyocyte deficit emerged attributable to sluggish production of SHF-derived cardiomyocytes. Concomitantly, we witnessed accumulation of SHF progenitors, demonstrating that Fosl2 facilitates the progenitor to cardiomyocyte transition. Mutant embryos eventually overcame the cardiomyocyte deficit by extending the accretion window for an additional 12 hours. Fosl2 overexpression also perturbed SHF development through precocious myocardial differentiation. Taken together, our data implicate the AP-1 transcription factor in potentiating the rate of SHF-derived cardiomyocyte production and reveal that cessation of myocardial accretion is regulated by variables independent of developmental stage.

108

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-06

ENDOTHELIN REGULATES NEURAL CREST DEPLOYMENT TO FORM GREAT VESSELS THROUGH DLX-INDEPENDENT MECHANISMS Ki-Sung Kim, Yuichiro Arima, Taro Kitazawa, Koichi Nishiyama, Rieko Asai, Yasunobu Uchijima, Takahiro Sato, Giovanni Levi, Yukiko Kurihara, Hiroki Kurihara Department of Cardiology, Kanagawa Children’s Medical Center Endothelin-1 (Edn1), is involved in the development of neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx-independent mechanism. Edn1/Ednra-KO mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra-KO embryos. By contrast, Dlx5/Dlx6-KO little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches by Dlx5/Dlx6.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-07

ENDOCARDIAL AND VENTRICULAR MUSCLE DEFECTS EXPLAIN MID-GESTATIONAL LETHALITY IN CRKL NULL MOUSE EMBRYOS Erica Hasten, Silvia E. Racedo, Bin Zhou, Bernice Morrow Albert Eistein College of Medicine The present study focuses on newly recognized functions of CRKL (Crk-like), a human congenital heart disease gene mapping to chromosome 22q11.2. CRKL encodes a cytoplasmic adaptor protein that links upstream tyrosine kinase and integrin-dependent signals to downstream effectors. Inactivation of Crkl results in conotruncal heart defects (CTDs) in mice, however mice die at E16.5, earlier than expected for having CTDs. Using our Crkl allelic series in mice, we now examined the embryonic hearts for the presence of other malformations to explain the cause of mid-gestational lethality. The percentage of ventricular wall muscle defects increased from 40% (n=25) in hypomorphs to 67% (n=24) in null embryos (KO). Endocardial cushion and valve defects increased from 44% in hypomorphs to 54% in Crkl KO mutants, indicating that these two defects are sensitive to altered gene dosage. Histological analysis of early embryonic stages (E10.5-E11.5) of Crkl KO embryos showed that these defects are seen before CTDs appear, suggesting that they are not secondary phenotypes. Additionally, our endocardial cell lineage tracing studies in Crkl WT and Crkl KO embryos using Nfatc1-Cre showed that the endocardial derivatives; i.e., valves and coronary vessels, are abnormal in Crkl KO embryos. These results implicate further functions of Crkl as a key regulator of multiple cardiac lineages during embryonic development and may explain the unexpected mid-gestation lethality observed in mouse mutants.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-08

ROR2 AND THE NONCANONICAL WNT SIGNALING PATHWAY MODIFY CONOTRUNCAL DEFECTS IN 22Q11.2 DELETION SYNDROME Jonathan H. Chung, Tingwei Guo, Donna M. McDonald-McGinn, Candice K. Silversides, David J. Cutler, Michael E. Zwick, Eva W. Chow, Anne S. Bassett, Beverly Emanuel, Bernice Morrow Albert Eistein College of Medicine Sixty-five percent of individuals with 22q11.2 deletion syndrome (22q11DS; DiGeorge syndrome/VCFS) have congenital heart disease (CHD). Mouse and human genetic studies indicate that haploinsufficiency of TBX1 on 22q11.2, is in part responsible for CHD. Mouse studies have identified Wnt5a, a noncanonical Wnt ligand, as a downstream target of TBX1. However, it is unknown whether other genes in this pathway could act as genetic modifiers in humans. We performed whole exome sequencing on 89 cases with CHD and 96 deleted controls with a normal heart (Cohort 1) as well as whole-genome sequencing on 50 additional cases and 46 controls (Cohort 2); all subjects have 22q11DS. We evaluated 32 genes in the Pathway Interaction Database noncanonical Wnt pathway. ROR2, a receptor for WNT5a, was the most significant gene from gene-based SKAT and Burden association analysis for each cohort and combined meta-analysis using Stouffer’s method (Stouffer’s p-value = 0.0034). We discovered five novel or rare (MAF < 0.01) predicted deleterious nonsynonymous mutations in ROR2 affecting seven CHD cases and no controls, distributed between the two cohorts. We used ARTP (Adaptive Rank Truncation Product) to perform a gene-set association analysis of damaging variants and found the overall non-canonical WNT pathway was significant (ARTP p-value = 0.021; 1,000 permutations). These results indicate that rare variants in ROR2, and possibly the noncanonical Wnt pathway, may act as modifiers of 22q11DS.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-09

TGFBETA2 FUNCTIONS UPSTREAM OF THE SOXC IN CONGENITAL HEART DEFECTS AND BICUSPID AORTIC VALVE Shalom Hre, Abdulaziz Mohamed, Qinbo Lu, Rajneesh Srivastava, Sarath C. Janga, Kai Jiao, Robert J. Schwartz, Veronique Lefebvre, Mohamad Azhar Indiana University Congenital heart defects including the bicuspid aortic valve are found in many patients that have transforming growth factor-beta2 (TGFB2) heterozygous loss-of-function mutations. Here, we generated mice harboring Tgfb2(βgeo) (knockout-first lacZ-tagged) allele and Tgfb2(flox) (conditional-ready) allele to determine cell-specific expression and function of Tgfb2 in the heart. β-galactosidase staining successfully detected Tgfb2 expression in Tgfb2(βgeo) fetal hearts. Systemic Tgfb2 deletion (via EIIaCre+) resulted in heart defects (DORV, VSD, PTA, valve malformations). Endocardial cell Tgfb2 deletion (via Tie2Cre+) recapitulated the cardiac defects seen in the Tgfb2 null hearts. Since PTA is reportedly caused by neural crest-specific deletion of TGFβ receptors (Tgfbr1 or Tgfbr2), the results indicate that the loss of endocardial cell TGFβ2 via TGFβR1/2, acting in a paracrine fashion, significantly contributes to outflow tract septation and alignment defects. Finally, in the Tgfb2 heterozygous fetuses, the endocardial cell-specific heterozygous Sox4 deletion was sufficient to induce bicuspid aortic valve, whereas the simultaneous endocardial heterozygous SoxC (Sox4/Sox11/ Sox12) deletion caused the outflow and atrioventricular septal defects. These genetic findings along with in vitro biochemical Sox4-luciferase reporter studies collectively indicate that the endocardial cell-specific loss of TGFβ2 via repressed SoxC causes congenital heart defects and/or bicuspid aortic valve.

112

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-10

AKT1 COORDINATES BMP SIGNALING AND BETA-CATENIN ACTIVITY TO REGULATE SECOND HEART FIELD DEVELOPMENT Wen Luo, Xia Zhao, Lichan Tao, Hengwei Jin, Zhongzhou Yang Nanjing University Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/beta-catenin signaling. PTEN-Akt signaling regulates the stem/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract (OFT). Cell proliferation of cardiac progenitors was enhanced while cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of beta-catenin, thus enhancing its activity. Reducing beta-catenin level could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and beta-catenin activity.

113

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-11

NEURAL CREST-SPECIFIC FUNCTIONS OF FIBRONECTIN IN NOTCH SIGNALING AND CARDIOVASCULAR MORPHOGENESIS Xia Wang, Sophie Astrof Thomas Jefferson University Molecular mechanisms of signaling by the extracellular matrix (ECM) in vivo are not well understood. A major ECM glycoprotein fibronectin (Fn1) is essential for vertebrate morphogenesis, and we discovered that Fn1 is expressed in strikingly non-uniform patterns during mouse development, suggesting that ECM plays cell-specific, regulatory roles during embryogenesis. To investigate these roles, we ablated Fn1 in the neural crest (NC). We show that Fn1 synthesized by the NC is essential for Notch signaling and vascular morphogenesis by regulating differentiation of NC cells into vascular smooth muscle cells (VSMCs). Our data demonstrate that the enrichment in Fn1 in the NC surrounding pharyngeal arch arteries facilitates Notch activation in the NC and imparts the spatial specificity to Notch signaling. In this process, NC-synthesized Fn1 signals through integrin α5β1 to activate Notch and VSMCs differentiation. Taken together, our data demonstrate the significance of the regionalized synthesis of ECM in vascular morphogenesis.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-12

HEY2 REGULATES SHF-DERIVED PROGENITOR CONTRIBUTION TO THE ZEBRAFISH HEART Natalie Gibb, Savo Lazic, Natasha Choubey The Hospital for Sick Children The Second Heart Field (SHF) is a population of cardiac progenitor cells that contributes to the development and maturation of the vertebrate heart. As defects in SHF development have been implicated in Congenital Heart Disease, the leading cause of birth defects in newborns, it remains imperative to grasp a fuller understanding of how these progenitors help govern the formation and growth of the embryonic heart. In particular, how cardiac progenitors become assigned to a First versus SHF fate, how SHF progenitors are maintained, and how SHF contribution to various structures of the heart is poorly understood. We have identified hey2 as a gene that acts to restrict the development of the SHF. Knockdown of hey2 results in an expanded SHF progenitor pool, with a greater late (SHF-derived) addition of myocardium to the heart at the expense of early (FHF) contribution. Hey2 is expressed early in SHF progenitors, and use of a conditional overexpression system indicates that hey2 can influence heart development at early stages of cardiac development. Using CRISPR/Cas9 genome editing, we have generated a zebrafish hey2 null mutant line, in which embryos demonstrate a bias towards a SHF progenitor cell fate, showing distinctive up-regulation of SHF-specific gene expression patterns. Collectively, our results highlight a new player in SHF development that appears to be required for maintaining the balance between FHF and SHF progenitor contributions to the developing vertebrate heart.

115

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-13

SPATIAL REGULATION OF CELL COHESION BY WNT5A DURING SECOND HEART FIELD PROGENITOR DEPLOYMENT Ding Li, Tanvi Sinha, Rieko Ajima, Hwa-Seon Seo, Terry Yamaguchi, Jianbo Wang University of Alabama at Birmingham Wnt5a, a non-canonical Wnt ligand important for outflow tract (OFT) morphogenesis, is expressed specifically in second heart field (SHF) progenitors in the caudal splanchnic mesoderm (SpM) near the inflow tract. Using a conditional Wnt5a gain of function (GOF) allele and Islet1-Cre, we ectopically expressed Wnt5a throughout the SHF, including the entire SpM between the IFT and OFT. In Wnt5a null background, ectopic Wnt5a expression can rescue the cell polarity and actin polymerization defects in SHF cells in the caudal SpM, but not OFT shortening. In wild-type background, ectopic Wnt5a expression is sufficient to cause OFT shortening and conotruncal defects. Ectopic Wnt5a expression does not perturb SHF cell proliferation, apoptosis or differentiation, but affects the deployment of SHF cells by causing them to accumulate into a large bulge and fail to enter the OFT. Immunostaining reveals an inverse correlation between cell cohesion and Wnt5a level in the wild-type SpM (caudal SpM: low cohesion/high Wnt5a; rostral SpM: high cohesion/no Wnt5a). Ectopic Wnt5a expression in Wn5a-GOF mutants diminishes the upregulation of cell cohesion in the rostral SpM; whereas loss of Wnt5a in Wnt5a null mutants causes premature increase in cell cohesion in the caudal SpM. Our data indicate that confined expression of Wnt5a in the caudal SpM is essential for normal OFT morphogenesis, and suggest a new function of Wnt5a in controlling cell cohesion to promote SHF deployment.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-14

TELEOST-SPECIFIC ECM “ELASTIN2” DEMARCATES TELEOST HEART OFT BY SWITCHING CELL FATE VIA YAP Yuuta Moriyama, Jun K Takeuchi, Kazuko Koshiba-Takeuchi University of Tokyo In teleost, outflow tract (OFT) is called as bulbus arteriosus (BA). BA is an unique organ characterized as elastic and pear-shaped structure and functions as a “windkessel” organ, absorbing the energy of the bolus of blood ejected from the ventricle and smoothing the blood pressure. While OFTs in other vertebrates including non-teleost fish are composed of cardiac muscle, BA is composed of smooth muscle. Through analyzing teleost BA development, we addressed the mechanism of cell fate determination into smooth muscle cells (SMCs) or cardiomyocytes (CMs). Elastin is an extracellular matrix protein (ECM) imparting the physiologically essential properties of extensibility and elastic recoil, and the major component of BA. Interestingly, there are two elastin genes in teleost, elastin1 and elastin2, compared with a single gene in other vertebrates. We found that the expression patterns of elastin2 in teleost embryos were restricted in BA, while these of elastin1 were expressed in various tissues. Surprisingly, in elastin2 knockdown zebrafish embryos ectopic CMs were differentiated in BA. However, migration patterns of cardiac precursor cells were not changed in these embryos. Further we found that knockdown of YAP, which is known as a mechanotransducer, caused ectopic CMs formation in BA. These results imply that the teleost-specific ECM Elastin2 positively regulates cell fate into SMC via YAP and plays a central role in formation of BA in fish development and evolution.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-15

TIE1 IS REQUIRED FOR LYMPHATIC VALVE DEVELOPMENT Xianghu Qu, Bin Zhou, H. Scott Baldwin Vanderbilt University Medical Center Tie1 is a receptor tyrosine kinase with broad expression in endothelium. We have previously shown that reduction of Tie1 levels in mouse embryos by a hypomorphic Tie1 allele results in abnormal lymphatic patterning and architecture, decreased lymphatic draining efficiency, and ultimately, embryonic demise. Here we report that Tie1 expression becomes restricted to lymphatic valve regions in the early postnatal period of development. To investigate later events of lymphatic development, we employed Cre-loxP recombination utilizing a floxed Tie1 allele and an Nfatc1Cre line, to provide loxP excision in lymphatic endothelium. Interestingly, unlike the early prenatal defects previously described by ubiquitous endothelial deletion or hypomorhic attenuation, excision of Tie1 with Nfatc1Cre resulted in abnormal lymphatic defects in postnatal mice and was characterized by agenesis of lymphatic valves and a deficiency of collecting lymphatic vessels. In addition, excision of Tie1 with lymphatic specific inducible Prox1Cre resulted in the same lymphatic valve defects. Lymphatic valve development is initiated by the onset of turbulent flow that results in accentuation of Prox1 in a subset of lymphatic endothelial cells. Attenuation of Tie1 inhibited Prox1 expression in this cell population. Our findings reveal a fundamental role for Tie signaling during lymphatic valve morphogenesis and implicate Tie1 as a primary mediator in hemodynamic mechanotransduction during vascular development.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-16

MEF2C IS REQUIRED FOR PROPER OUTFLOW TRACT ALIGNMENT AND IS A DIRECT TRANSCRIPTIONAL ACTIVATOR OF TDGF1 Ralston M. Barnes, Ian S. Harris, Brian L. Black UCSF Outflow tract (OFT) alignment and ventricular septal defects (VSD) are among the most severe and prevalent forms of cyanotic congenital heart disease, and these structural defects require surgical intervention and are a frequent cause of mortality in infants. To address transcriptional pathways involved in OFT morphogenesis, we performed a conditional deletion of the gene encoding the transcription factor MEF2C in the anterior heart field (AHF). Mef2c AHF knockout mice die at birth with outflow tract alignment and membranous VSD. We identified the Teratoma-derived growth factor 1 (Tdgf1) gene as being completely dependent on MEF2C for expression in the developing outflow tract. Tdgf1 encodes the Nodal co-receptor Cripto, a molecule involved in establishing symmetry in the developing embryo. Moreover, the TDGF1 gene was recently associated with VSD and conotruncal alignment anomalies in humans. To determine if Tdgf1 is a direct target of MEF2C, we identified a Tdgf1 regulatory element that is active within the outflow tract, and we demonstrate that activity of the Tdgf1 regulatory element is dependent on MEF2C. Our data raise the intriguing possibility that Nodal signaling may serve a critical role during heart development to establish proper alignment of the cardiac outflow vessels.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-17

MICROARRAYS USING DEVELOPING LUNGS SUGGEST A PUTATIVE ROLE OF TBX4 IN THE LUNG VASCULATURE Keiko Uchida, Reina Ishizaki, Akimichi Shibata, Takatoshi Tsuchihashi, Hiroyuki Yamagishi Keio University School of Medicine It was previously reported that the vasculature of lungs developed in both proximal and distal sites. The connection between the proximal and distal vessels occurs only after embryonic day (E) 13 in mouse, and later, the vascular structures extended more peripherally. Around birth, the vascular plexus becomes tightly associated with the epithelium of alveoli. The molecular mechanisms of the lung vascular development, however, still remain to be elucidated. In order to explore the molecular regulation of the embryonic lung vessels, we performed microarray analysis using endothelial marker (CD31) positive cells sorted from the lung tissues at E14 and postnatal day (P) 2 to be compared. As a result, a T-box transcription factor, Tbx4, showed significantly higher expression levels at E14 than at P2. In situ hybridization analysis showed that Tbx4 was expressed in the E14 lung mesenchymal cells (LMC) which is a source of lung vessels. Quantitative RT-PCR revealed that the Tbx4 expression level reached to the peak at E14-15 and later downregulated in the LMC. Although several endothelial markers, CD31 and Tie2, were also expressed in LMC at the highest levels at E14-15, the knockdown of Tbx4 in LMC using RNAi promoted tube length in vitro tube formation assay and attenuated Tie2 expression. These results suggest that Tbx4 in the LMC may regulate maturation of lung vessels at the developmental stage just after the connection between proximal and distal vessels is established.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-18

VISUALIZING CARDIOVASCULAR DEVELOPMENT AND REGENERATION USING TARGETED EFNB2AEGFP KNOCK-IN ZEBRAFISH Yanchao Han, Kenneth Poss Duke University Medical Center Several molecular markers including efnb2a have been shown to expressionare known to be specifically expressed in arteries as opposed to veins. To monitor the arterial development in real time, we knocked an EGFP reporter into the zebrafish endogenous efnb2a locus using TALEN and PhiC31 mediated genomic editing and recombination. The EGFP reporter recapitulates efnb2a expression in arteries, neural tube, muscles and retina during early development. The Aarterial efnb2aEGFP expression can be abolished by administration of Shh inhibitor CyA without affecting its expression in spinal cord. In developing hearts, efnb2aEGFP reporter is expressed in the coronary vessels and in epicardial cells surrounding the coronary vessels. The Eepicardial efnb2aEGFP expression gradually disappears in the adult hearts but is elevated in the wound areas after heart injury, indicating a possible endothelial-epicardial interaction during development and regeneration. Further Ongoing characterization and functional studies will uncover thedissect mechanism of efnb2a expression and function during cardiovascular development and regeneration.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-19

CHARACTERIZATION OF THE CARDIAC LYMPHATIC SYSTEM IN ZEBRAFISH Jonathan Semo, Karina Yaniv Weizmann Institute of Science The lymphatic system is a network of blind ended capillaries, which regulates fluid homeostasis, immune cell trafficking and lipid absorption. The heart was shown to possess a well-developed lymphatic vasculature, and defects in lymphatic drainage have been highlighted as an adverse factor affecting cardiac function in several cardiac pathologies. Nevertheless, the embryonic origins of cardiac lymphatic vessels, as well as their exact function in adult physiological conditions, are largely unknown. In this work, we aimed to characterize the anatomical structure, as well as the initial steps of development of cardiac lymphatic vessels in zebrafish. Analysis of adult hearts isolated from transgenic zebrafish, revealed the presence of lymphatic vessels mainly in the ventricle and bulbus arteriosus. Live-imaging of transgenic embryos revealed that the first lymphatic vessels are detected in the heart between 56-74 hours post-fetilization and by 7 days post-fertilization an elaborate network of Lyve1 positive vessels is found within the myocardium. Moreover, our time lapse sequences demonstrate that the development of cardiac lymphatics involves two waves of lyve1+ cells migrating into the heart from the sinus-venosus, the early wave at 32hpf, and the second wave at 50hpf. Altogether this work provides the first anatomical and developmental characterization of cardiac lymphatic vessels in zebrafish.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-20

CASPASES INHIBIT CANONICAL WNT SIGNALING DURING CARDIAC PROGENITOR DEVELOPMENT Joseph A. Bisson, Bradley Mills, Jay-Christian P. Helt, Thomas P. Zwaka, Ethan David Cohen University of Rochester, School of Medicine and Dentistry We recently reported that two non-canonical Wnt ligands, Wnt5a and Wnt11, act cooperatively to attenuate canonical Wnt/β-catenin (β-cat) signaling that would otherwise disrupt second heart field (SHF) progenitor cells, which ultimately give-rise to the majority of cardiomyocytes. Wnt11 was previously shown to inhibit β-cat and promote cardiac differentiation by activating a novel apoptosis-independent function of Caspases (Casp). Consistent with these data, we now show that Wnt5a/11 are capable of inducing Casp activity in differentiating embryonic stem (dES) cells and that hearts from Wnt5a-/-;Wnt11-/- embryos have diminished Casp3 activity. Furthermore, SHF markers are reduced in Casp3-/- ES cells and hearts from Casp inhibitor (C.I.) treated embryos, while the treatment of wild type ES cells with C.I.s blocked the ability of Wnt5a/11 to inhibit Wnt/β-cat signaling and promote SHF gene expression. Interestingly, Wnt5a/11 treatment of dES cells reduced Akt activity through a Casp-dependent mechanism and Akt levels were increased in hearts from C.I. treated embryos. Surprisingly, inhibition of either Akt or PI3K in dES cells was an equally effective means of increasing SHF markers compared to treatment with Wnt5a/11, while Akt inhibition restored SHF gene expression in Casp3-/- ES cells. Taken together, these findings suggest that Wnt5a/11 inhibit β-cat to promote SHF development through Casp-dependent Akt degradation.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-21

WILMS’ TUMOR-1 EXPRESSION IN CARDIAC ENDOTHELIAL CELLS Sjoerd N. Duim, Kondababu Kurakula, Marie-José Goumans, Boudewijn P.T. Kruithof LUMC Wilms’ tumor-1 (Wt1) is expressed in the embryonic and reactivated adult epicardium and is therefore used as a lineage marker for the epicardium. Recently, it is suggested that also other cells within the heart express Wt1. Our goal is to determine the Wt1-expressing cell types during development and after cardiac injury in the murine and human heart, and to study the role of Wt1 in these cells. Immunohistochemical analysis revealed that Wt1 is expressed by cardiac endothelial cells (ECs) of the mouse heart from E12.5 onwards. In the adult heart, the expression of Wt1 is reduced, although a subset of coronary ECs remains positive for Wt1. Interestingly, after myocardial infarction a temporal upregulation of Wt1 in ECs is observed in the infarcted area and the border zone of the heart. In the human fetal heart, Wt1 expression is similar though broader then observed in the embryonic mouse heart. In addition to the ECs, Wt1 expression is also observed in the endocardial cells of the ventricles. The expression of Wt1 in the ECs suggests a role in angiogenesis. To test this hypothesis, we performed in vitro experiments and show that Wt1 can be induced in ECs by hypoxia, an angiogenic inducer, and that Wt1 enhances cell proliferation of ECs through increased mRNA expression of CyclinD1. In addition, ECs lacking Wt1 were not capable to establish a proper vascular network. Together, these results suggest a role for Wt1 in cardiac vessel formation in development and disease.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-22

MEASURING AORTIC ARCH BLOOD FLOW AND SHEAR STRESS WITH DOPPLER OPTICAL COHERENCE TOMOGRAPHY Lindsy M Peterson, Shi Gu, Ganga Karunamuni, Michiko Watanabe, Michael W Jenkins, Andrew M Rollins Case Western Reserve University During development six symmetric pairs of aortic arches sequentially emerge and are selectively remodeled to form the asymmetric great vessels. These dynamic events are vulnerable to perturbations that may result in congenital heart defects. Hemodynamic forces are known to play a significant role in the dynamic process of aortic arch remodeling. Doppler optical coherence tomography (OCT) is a non-contact imaging modality well suited for embryonic imaging due to its high spatial and temporal resolution. We previously demonstrated that Doppler OCT is capable of measuring absolute blood flow from individual cross sectional images using two-beam scanning. This technique allows for rapid flow measurements over the duration of a heartbeat. Absolute blood flow can then be used to calculate shear stress along the lumen wall. We used a model of prenatal ethanol exposure that results in great vessel abnormalities to investigate the role hemodynamics may play in contributing to these abnormalities. We measured blood flow and shear stress in the 3rd aortic arch of control and ethanol exposed embryos prior to their remodeling. Pulsed Doppler traces were also acquired to measure the percentage of retrograde blood flow. Blood flow, shear stress and percentage of retrograde blood flow all increased in the ethanol exposed embryos compared to the control embryos. These increased hemodynamic forces may have deleterious effects for later developmental events.

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Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-23

A REGULATORY LOOP BETWEEN HDAC3 AND A TGF SIGNALING ORCHESTRATES THE SECOND HEART FIELD DEVELOPMENT Sara L Lewandowski, Harish P. Janardhan, Chinmay M. Trivedi University of Massachusetts Medical School About two-third of human congenital cardiac defects involve the second heart field derived structures. Epigenetic regulation of the second heart field progenitor cells remains an area of intense research. Histone-modifying genes play critical roles in human heart development. Here we show that histone-modifying enzyme histone deacetylase 3 (Hdac3) functions in the second heart field progenitor cells to orchestrate various stages of cardiac development. Murine embryos lacking Hdac3 in the second or anterior heart field progenitor cells (Hdac3-SHF/AHF-null) exhibit complete embryonic lethality, outflow tract defects, ventricular septal defects, overriding aorta, aberrant Endothelial to Mesenchymal transition, and multiple valvular defects. Genome-wide transcriptional profiling, histological analysis, biochemical assays, and genetic or pharmacological rescue studies revealed a critical negative feedback loop between Hdac3 and a transforming growth factor signaling pathway that accounts for cardiovascular anomalies observed in Hdac3-SHF/AHF-null embryos. Further, regulatory interplay between Hdac3 and transforming growth factor signaling genes at the chromatin level is required for extracellular matrix homeostasis within the second heart field derived structures. Elucidation of the role of Hdac3 in the second heart field is relevant to human congenital heart defects observed in connective tissue disorders.

126

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-24

VERSICAN SECRETION IS REQUIRED FOR VASCULOGENESIS DURING EMBRYONIC DEVELOPMENT Nishant Mittal, Sung Han Yoon, Hirokazu Enomoto, Keiichi Fukuda, Atsusi Kawakami, Akira Kudo, Misato Fujita, Keiichi Fukuda, Shinji Makino Keio University School of Medicine Extracellular matrix (ECM) provides the dynamic environment for various cellular processes during cardiovascular development. Versican, a glycoprotein, regulates migration and proliferation of vascular smooth muscle cells by affecting cell adhesion and shape. lht (linear heart tube) medaka mutants having point mutation in 3’ UTR of versican were generated using ENU mutagenesis. lht-/- mutants showed absence of functional vascular system, termination of heart development at linear heart tube stage and absence of organised endocardium. RNA expression for versican was similar in WT and lht-/-, however protein expression was very low in lht-/-, compared to WT. To understand the vascular development in lht-/-, we used Tg (fli:EGFP) lht+/-transgenic medaka. Time lapse imaging showed similar fli+ cells migration across yolk sac at initial stage in WT and lht. However, lht -/- embryos did not show lumen development and lacked blood circulation. Interestingly, mutant embryos showed normal haematopoiesis. Further, embryonic transglutaminase (etgase) is expressed in smooth muscle cells and remodelled exclusively in yolk sac blood vessels. The in-situ hybridization for etgase showed prominent expression exclusively in yolk sac blood vessels in WT. However, etgase+ cells were found scattered and unable to organize to form yolk sac veins in lht-/-. These results indicate that versican drives migration of vascular smooth muscle cells required for blood vessel maturation.

127

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-25

ETHANOL-INDUCED CHDS ARISE THROUGH IMPACT ON CARDIAC NEURAL CREST THAT ALTERS STRUCTURE AND FUNCTIONS Pei MA, Lindsy M. Peterson, Ganga Karunamuni, Shi Gu, Yves T. Wang, Michael W. Jenkins, Michiko Watanabe, Andrew M. Rollins Case Western Reserve University 54% of live-born children with fetal alcohol syndrome (FAS) possess congenital heart defects (CHDs). Alcohol exposure has been shown to impact cell death and migration of neural crest cells, which are involved in the development of many organs including the heart. We hypothesized that cardiac neural crest cells (CNCCs) play a role in ethanol-induced CHDs and perturbing quail embryos with alcohol exposure and CNCC ablation should lead to similar cardiac abnormalities. Cohorts of control, ethanol-exposed, and CNCC-ablated embryos were cultured and imaged under physiological conditions with Optical coherence tomography (OCT) at a cardiac looping stage (HH stage 19). Other embryos developed until a four-chambered stage (HH stage 34) and their hearts were fixed, cleared and imaged with OCT. At HH stage 19, treated groups displayed abnormally twisted body flexure phenotypes, increased percentage of retrograde blood flow, abnormal pulsed Doppler trace waveforms and abnormal cardiac cushion formation, including defective atrioventricular cushion closure and abnormal endocardial-mesenchyme transition. At HH stage 34, perturbed groups displayed reduced volume (45% and 40% respectively) and abnormal morphology of atrioventricular valve leaflets as well as reduced size and abnormal patterning of the great vessels. The similarities in structural and functional abnormalities in both perturbation models support that CNCCs may play a substantial role in our observed ethanol-induced CHDs.

128

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-26

TBX5 DRIVES LUNG DEVELOPMENT FOR ATRIAL SEPTATION Jeffrey D. Steimle, Junghun Kweon, Xinan Holly Yang, Ivan P. Moskowitz University of Chicago Although cardiogenic transcription factors have long been implicated in atrial septation, the molecular mechanism underlying this requirement remains unknown. A recent paradigm describes molecular events in second heart field (SHF) mesoderm that drive inflow septation. We have previously implicated Tbx5- and Sonic hedgehog (Shh)-pathways in the SHF and sought to integrate these pathways into a unified molecular description of inflow septation. We used RNAseq to define the Tbx5- and Shh-dependent SHF transcriptomes at E9.5 and E10.5, respectively. Remarkably, the overlap between these datasets was minimal and consisted primarily of Shh-dependent transcripts. Furthermore, although Tbx5 expression is mesoderm specific, transcripts demonstrating the most significant Tbx5-dependent expression revealed endoderm-specific expression. We hypothesized that Tbx5 was required for lung development and found that early lung specification fails in Tbx5 mutant embryos. Furthermore, we found that Tbx5 is required for SHF canonical Wnt signaling, itself required for pulmonary development. Combining ChIP-seq and ATAC-seq data, we have identified Tbx5-dependent mesodermal enhancers in Wnt ligands and are currently undertaking their functional analysis in-vivo. We propose that Tbx5 directly drives mesoderm to endoderm Wnt signaling to coordinate endoderm development and promote subsequent endoderm to mesoderm Shh signaling for SHF progenitor specification and inflow septation.

129

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-27

BMPS REGULATE A CRITICAL HAND FACTOR THRESHOLD NECESSARY FOR OUTFLOW TRACT DEVELOPMENT Joshua W. Vincentz, Anthony B. Firulli Indiana University School of Medicine Congenital heart defects (CHDs) are often caused by abnormal development of the cardiac outflow tract (OFT). During OFT development, cardiac neural crest cells (cNCCs) invade the extracellular matrix adjacent to the myocardial component of the OFT, known as the myocardial cuff (MC). These tissues then remodel the OFT into the aorta and pulmonary arteries. The mechanisms coordinately controlling cNCC and MC morphogenesis are poorly understood. In humans, mutation or overexpression of the bHLH transcription factors HAND1 and HAND2, respectively, are associated with OFT CHDs. In mice, Hand2 ablation results in OFT defects, whereas Hand1 NCC-specific knockouts are phenotypically normal. Hand1 and Hand2 expression overlaps in both NCCs and the MC, and these two factors genetically interact in the developing jaw. The transcriptional programs that regulate Hand factor expression in OFT tissues are not known. Our promoter deletion analyses have uncovered a 444bp fragment 5’ to the Hand1 transcription start site sufficient to drive reporter gene expression in cNCCs and the MC. Trans-activation assays indicate that Bmp signaling directly regulates this enhancer. Hand2 expression is also downstream of Bmp signaling in the OFT, suggesting that Hand factors transcriptionally implement OFT developmental programs via a conserved Bmp-dependent mechanism. Indeed, cumulative reduction of Hand gene dosage within cNCCs produces OFT defects, revealing a critical Hand factor dosage threshold crucial for OFT morphogenesis. These findings provide fundamental insight into the mechanisms by which Hand factors affect cellular interpretation of extracellular signaling cues during OFT development, thereby deepening our understanding of CHDs.

130

Main Posters 2 2. Outflow tract/second heart field/cardiac neural crest/vasculature 2-28

DELETION OF ETS-1 CAUSES CONOTRUNCAL HEART DEFECTS THROUGH A NEURAL CREST CELL AUTONOMOUS MECHANISM Lizhu Lin, Kazumi Fukatsu, Ya Yin, Maoqing Ye, Simon Bamforth, Robert Anderson, Le Anne Sinha-Garrett, Michael Ostrowski, Marianne Bronner, Paul Grossfeld UCSD

Conotruncal defects (CTs) are among the most common congenital heart defects (CHDs) in liveborn infants. The ETS-1 gene is deleted in every patient of Jacobsen syndrome (11q-) with CHDs. ETS-1 knockout mice in a pure C57/B6 background have double outlet right ventricle (DORV). We performed whole mount in situ hybridization using SOX10, a gene that is expressed in early murine cardiac development that is a direct downstream target of ETS-1. Loss of ETS-1 causes decreased SOX10 expression in the dorsal root ganglia and the branchial arches. To further define the neural crest cell(NCC) defect, we employed an explanted NCC culture system, which demonstrated that loss of ETS-1 causes a cardiac NCC migration defect AND decreased NCC numbers. Conditional deletion of ETS-1 in the neural crest using a Pax3Cre promoter also causes DORV, indicating that loss of ETS-1 causes DORV through a NCC-autonomous mechanism. In vivo studies demonstrate a selective decrease in cardiac NCCs in the left ventricular outflow tract, preceded by a decreased number of NCCs in the developing outflow tract in E10.5 embryos. Conditional deletion in the neural crest at a slightly later stage does not cause heart defects, but does result in defects in other, non-cardiac NCC-derived structures. Taken together, these findings indicate that ETS-1 is required for specification and migration of cardiac NCC, and for regulating a NCC-specific gene regulatory network required for normal cardiac development.

131

Main Posters 3 3. Myocardial development/cardiomyopathies 3-07

THE DEGREE OF HEMODYNAMIC PERTURBATION PREDICTS CARDIAC REMODELING IN CHICKEN EMBRYOS Madeline Midgett, Sandra Rugonyi Oregon Health & Science University Normal hemodynamic conditions are essential for proper cardiac development, with altered hemodynamic loads (pressure and shear stresses) on cardiac tissues known to induce heart defects seen in congenital heart disease. This study determines the effects of altered blood flow on cellular structures of the embryonic heart, and characterizes how varied hemodynamics during development affect mature cardiac formation. Our goal is to elucidate the processes that relate altered blood flow to the resulting heart defects. Blood flow was manipulated using outflow tract (OFT) banding in the chicken embryo at HH18. OFT banding increases the hemodynamic load on cardiac tissues, and this increase is highly dependent on the degree of band constriction. 3D reconstruction and segmentation of OFT structures from focused ion beam scanning electron microscopy images demonstrated a disrupted endocardium and increased alignment of myofibrils in banded tissue compared to control. This suggests that the altered hemodynamic load after banding modifies the progression of the endothelial mesenchymal transition and myofibril maturity in the OFT. Ventricular septal defect (VSD) was identified using color Doppler ultrasound biomicroscopy and X-ray micro-computed tomography in 33% of banded embryos (n=9) at HH36. VSD only occurred in banded embryos with greater than 36% constriction, indicating that the altered hemodynamics created by tight bands trigger detrimental processes that result in VSD.

132

Main Posters 3 3. Myocardial development/cardiomyopathies 3-08

LONG NONCODING RNAS REGULATE POSTNATAL CARDIAC MATURATION Gizem Rizki, Simon Gordonov, Eric Bartell, Aprotim Mazumder, Xinchen Wang, Mark Bathe, Laurie Boyer MIT Shortly after birth, cardiac myocytes (CMs) mature, mostly cease to proliferate and lose the capacity to regenerate. Loss of this regenerative potential results in irreversible weakening and dysfunction of the heart during heart diseases. Postnatal CM maturation involves transcriptional changes leading to a switch from a proliferative to hypertrophic mode of growth and cell cycle exit. Given their roles in proliferation and transcription as well as our work identifying the lncRNA Braveheart, we hypothesize that lncRNAs are important transcriptional regulators of postnatal CM maturation. To identify lncRNAs with functions in CM maturation, we transcriptionally profiled mouse CMs isolated at key points during maturation and identified 60 differentially expressed lncRNAs. We chose 5 upregulated lncRNAs and developed a loss of function screen to measure the proliferative index and cytokinesis in explanted CMs as markers of terminal differentiation status. We identified several lncRNAs whose disruption results in an increase in CM proliferation. Furthermore, we found that cell cycle and cardiac genes are misregulated upon lncRNA depletion, suggesting these lncRNAs modulate gene expression networks. We are currently taking a multi-disciplinary approach to define the mechanisms of action of each candidate. Overall, our findings suggest that lncRNAs have critical roles during postnatal cardiac development and our work may lead to new strategies for stimulating cardiac regeneration.

133

Main Posters 3 3. Myocardial development/cardiomyopathies 3-09

SEQUENTIAL NOTCH ACTIVATION UNDERLIES VENTRICULAR CHAMBER DEVELOPMENT Gaetano D’Amato, Guillermo Luxán, Gonzalo del Monte, Beatriz Martínez-Poveda, Carlos Torroja, Fernando Martínez, Rui Benedito, Anna-Katerina Hadjantonakis, Akiyoshi Uemura, Luis J Jiménez-Borreguero, José Luis de la Pompa CNIC The mechanisms underlying the formation of a mature and functional ventricular wall are poorly understood and alterations in this developmental process cause severe congenital heart defects. Endocardial Notch signaling is fundamental for trabeculation and compaction but the spatio-temporal regulation of signaling specificity is not known. We have found that the glycosyltransferase Manic Fringe favors Dll4 activation of Notch1 in the endocardium, essential for trabeculation. As development proceeds, Dll4 is only required in the developing coronary vasculature, and endocardial Fringe expression is down-regulated, allowing myocardial Jag1 and Jag2 signaling to Notch1 to promote chamber maturation, trabecular compaction and normal ventricular function. Forced expression of Manic Fringe in the endocardium disrupts chamber maturation and compaction, presumably by blocking Jag1 and Jag2 signaling to Notch1. Expression profiling and marker analysis of various mutant combinations reveals that during chamber development Notch promotes myocardial proliferation and the generation of signals connecting chamber endocardium and myocardium, to sustain trabeculae formation, ventricular patterning and compaction. This study demonstrates that three Notch ligands are required to sequentially activate Notch1 in ventricular endocardium and give rise to the functional ventricular wall. These results open a new research avenue into the pathogenesis of cardiomyopathies.

134

Main Posters 3 3. Myocardial development/cardiomyopathies 3-10

CLONAL DYNAMICS OF ATRIAL-SPECIFIED CARDIOMYOCYTES IN THE DEVELOPING ZEBRAFISH ATRIUM Matthew J. Foglia, Kenneth D. Poss Duke University During cardiac morphogenesis, the zebrafish heart changes in structure from a linear tube to a multi-chambered pump composed of one atrium and one ventricle. Embryonic cardiomyocytes whose progeny will contribute to the either chamber are specified early in development. How these populations change over time to produce chambers of distinct size and function is not well understood. To define the patterns of cardiomyocyte division during atrial morphogenesis, we have traced the clonal progeny of embryonic atrial cardiomyocytes from embryogenesis to maturity using multicolor genetic labeling. Clones in the atrium expand unequally and, like the ventricle, dominant clones frequently emerge that comprise a disproportionate share of the wall surface. Unlike those of the ventricular myocardium, however, atrial cardiomyocytes undergo a morphologic change during juvenile development that generates a discontinuous structure that persists through maturity. Concomitantly, interior pectinate muscle fibers form that, in contrast to the ventricle, share a clonal relationship with the adjacent wall cardiomyocytes. Our experiments indicate that patterns of atrial cardiomyocyte division differ markedly from those of ventricular cardiomyocytes, explaining at least in part the different chamber morphologies. These experiments stand to shed light on a poorly understood aspect of chamber morphogenesis and provide a foundation for interpreting molecular mechanisms of heart development.

135

Main Posters 3 3. Myocardial development/cardiomyopathies 3-11

PHENOTYPIC AND TRANSCRIPTIONAL ANALYSES OF ZEBRAFISH MYH6-/- ADULT VENTRICLES Panagiotis Kefalos, Claudia Roedel, Stamatia Kalogirou, Dimitris Beis Biomedical Research Foundation Academy of Athens Zebrafish has emerged as a powerful model to study cardiac development and regeneration. However, there are only few adult zebrafish models of Cardiovascular Disease. We identified two alleles of the atrial specific myosin heavy chain 6 (myh6) that show no atrial myocardial contractility. We are able to raise homozygous mutants with only a single functioning chamber: the ventricle. Using RNASeq, we identified changes in the expression profile of mutant ventricles when compared to age and size matched wild types. In addition, we studied the myocardial morphology at the cellular level due to the prolonged ventricular volume overload, which leads to ventricular remodeling. A long list of genes emerged from our analyses and verified, at the molecular level, that adult myh6-/- ventricles resemble mouse models of cardiac hypertrophy. The signaling pathways we identified are involved in activation of the ER Stress response, Remodeling of Epithelial Adherens Junctions, RAR activation and Erythropoietin regulation. In addition, we identified a number of novel genes that have not been previously reported to be involved in cardiac hypertrophy and will be used to better understand the mechanisms underlying this pathological condition. Disturbed intracardiac flow dynamics are observed throughout development of myh6-/- due to the non-contracting atrium. However, myh6-/- are viable and fertile with an enlarged, spherical ventricle and a bicuspid atrioventricular valve.

136

Main Posters 3 3. Myocardial development/cardiomyopathies 3-12

BLOOD FLOW ACTIVATES ENDOCARDIAL NOTCH SIGNALING TO MODULATE CARDIAC CHAMBER MATURATION Leigh Ann Samsa, Li Qian, Jiandong Liu University of North Carolina at Chapel Hill Congenital heart disease often features structural abnormalities that arise in development. Cardiac contraction plays a critical role in shaping the heart, yet the molecular basis of this function is largely unknown. Using zebrafish, we investigated the role of cardiac contraction in chamber maturation, focusing on the formation of muscular protrusions called trabeculae. By genetic and pharmacological ablation of cardiac contraction in Notch reporter fish, we show that cardiac contraction is required for trabeculation through its role in initiating notch1b transcription in the ventricle endocardium. Notch1 signaling is detectable throughout the ventricular endocardium within four hours of initiation of contraction, but becomes restricted to the endocardial cushions during trabeculation. Notch1 activation induces the expression of its downstream effectors ephrinb2a (efnb2a) and neuregulin1 (nrg1) in the endocardium to promote trabeculation. Interestingly, primary cilia-deficient embryos had reduced Notch reporter, notch1b and nrg1 expression, suggesting a potential role for primary cilia flow detection in cardiac contraction-mediated notch1b expression. Using a heat-shock inducible system to overexpress active Notch, overexpression in the absence of cardiac contraction rescues cardiac efnb2a and nrg1 expression. Together, our findings indicate that heartbeat-responsive transcriptional changes are important for cardiac chamber maturation through epistasis of notch1b-efnb2-nrg1.

137

Main Posters 3 3. Myocardial development/cardiomyopathies 3-13

SPARC MEDIATES CARDIAC AGING Paul S. Hartley Bernadine H. E. Chua University of Edinburgh Collagen deposition is crucial for normal development but also contributes to tissue fibrosis and promotes pathological organ dysfunction in ageing and disease. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a collagen binding matricellular protein and attractive anti-fibrotic target. Studying SPARC in mammalian models is challenging and limited by ethical, technical and financial constraints. Here we examined whether SPARC may play a role in cardiac ageing using the fruit fly Drosophila. Collagen deposition was assessed using Picrosirius red and fluorescently tagged collagen IV. Collagen was most pronounced around the heart, especially at the valves and distal heart region. Collagen deposition increased significantly as flies aged, despite reduced expression of two collagen-IV genes and SPARC. These changes correlated with the well-documented age-related decline of cardiac function, characterised by a slower heart rate, longer diastolic and systolic intervals and increased arrhythmia. Importantly, flies heterozygous for a loss-of-function SPARC allele, exhibited normal cardiac development but reduced collagen deposition in ageing and no age-related decline in cardiac function. These findings parallel those from mammalian models and highlight Drosophila as a novel system with which to study the impact of SPARC on age-related collagen deposition and cardiac dysfunction.

138

Main Posters 3 3. Myocardial development/cardiomyopathies 3-14

USING OCT TO INVESTIGATE ALCOHOL-INDUCED CHANGES IN CARDIAC FUNCTION AND STRUCTURE Ganga Karunamuni, Shi Gu, Yong Qiu Doughman, Amanda I. Noonan, Lindsy M. Peterson, Kersti K. Linask, Michael W. Jenkins, Andrew M. Rollins, Michiko Watanabe Case Western Reserve University Over 500,000 American women per year report drinking alcohol during pregnancy, with 1 in 5 who binge drink. As high as 54% of live-born children with Fetal Alcohol Syndrome (FAS) can present with congenital heart defects (CHDs). Currently, most studies fail to consider the role of altered cardiac function in producing CHDs, although changes in hemodynamics can profoundly affect cardiac development. We hypothesized that acute ethanol exposure creates early hemodynamic anomalies that contribute significantly to cardiac structural and functional defects. Previously, we employed optical coherence tomography (OCT), a non-destructive imaging modality capable of real-time, micrometer-scale resolution imaging, to analyze early-stage ethanol-exposed embryos that exhibited abnormalities in body flexure, blood flow, and cardiac cushions during heart looping stages. OCT also allowed for the rapid identification and quantification of CHDs in late-stage ethanol-exposed embryos, including ventriculoseptal and outflow defects, reduced vessel sizes, and smaller cardiac valve volumes. Furthermore, in preliminary rescue trials, the administration of nutritional supplements (choline, betaine) improved survival rates after ethanol exposure and reduced gross structural defects. We thus demonstrated that functional analyses using our novel technologies can act as early, accurate gauges of cardiac normalcy and abnormalities, and may provide a platform for the testing of new therapeutic strategies.

139

Main Posters 3 3. Myocardial development/cardiomyopathies 3-15

PROFILIN MODULATES SARCOMERIC ORGANIZATION AND MEDIATES HYPERTROPHIC REMODELING OF CARDIOMYOCYTES Viola Kooij, Meera C. Viswanathan, Dong I. Lee, Peter P. Rainer, William Schmidt, William A. Kronert, David A. Kass, Sanford I. Bernstein, Jennifer E. Van Eyk, Anthony Cammarato Johns Hopkins University Heart failure is often preceded by cardiac hypertrophy, which is characterized by increased cell size, altered protein abundance and actin-sarcomeric reorganization. Profilin is a well-conserved, ubiquitously expressed, multi-functional actin-binding protein. Given its role in vascular hypertrophy, we hypothesized that profilin is a key mediator of hypertrophic cardiomyocyte remodelling. In multiple animal models, we found that the quantity of profilin was elevated during cardiac hypertrophy. Cardiomyocyte-specific overexpression of profilin in Drosophila resulted in a significant increase in cardiac heart tube dimensions. Furthermore, profilin silencing in cellular models depressed the hypertrophic response, as measured by preserved myocyte size and attenuated fetal gene re-expression. Mechanistically, we found that profilin regulates hypertrophy through, at least, activation of the ERK1/2 signaling cascade. Moreover, the role of profilin is likely not confined to signaling. Confocal microscopy showed profilin localized to the Z-line of Drosophila myofibrils, as well as to the M-line when overexpressed. Elevated profilin levels led to elongated sarcomeres and thin filaments, myofibrillar disorganization and sarcomeric disarray, which correlated with impaired muscle function. In conclusion, our results reveal novel roles for profilin as a crucial mediator of cardiomyocyte hypertrophy, as both a signalling molecule and a regulator of myofibrillar and sarcomeric organization.

140

Main Posters 3 3. Myocardial development/cardiomyopathies 3-16

TRANSCRIPTIONAL REGULATION OF FIBROBLAST PLASTICITY IN CARDIAC REMODELING Janet K. Lighthouse, Lissette S. Velasquez, Eric M. Small University of Rochester The heart undergoes hypertrophic growth in response to both physiological and pathological stimuli. Pathological hypertrophy results from various humoral, mechanical, or ischemic insults, and often leads to fetal gene activation, cardiac fibrosis, and heart failure. In contrast, physiological hypertrophy is an adaptive response to the excessive demands of exercise or pregnancy and does not lead to fibrosis. We hypothesized that cardiac fibroblasts, the main cellular source of extracellular matrix in the heart, exhibit distinct gene expression programs in physiological and pathological remodeling that influence the divergent fibrotic responses. To investigate these differences we used RNA-sequencing to obtain the transcriptional profile of cardiac fibroblasts isolated from control mice, or mice subjected to swim training (physiological hypertrophy) or pressure-overload induced cardiac remodeling (pathological hypertrophy). Here, we present evidence that the Serum response factor (SRF)/myocardin-dependent fetal gene program is suppressed in fibroblasts from swim trained hearts. Indeed, a small subset of genes, including a nuclear long non-coding RNA, display antithetical regulation in pathological versus physiological remodeling, consistent with dysregulation in human heart failure samples. This study uncovers a robust cardioprotective network stimulated by exercise that might lead to novel approaches to ameliorate maladaptive cardiac remodeling.

141

Main Posters 3 3. Myocardial development/cardiomyopathies 3-17

TMEM2 REGULATES EXTRACELLULAR MATRIX ORGANIZATION DURING CARDIAC AND SKELETAL MUSCLE MORPHOGENESIS Lucile Ryckebusch, Lydia Hernandez, Deborah Yelon University of California, San Diego The organization of the extracellular matrix (ECM) is essential to maintain the morphology of both cardiac and skeletal muscle, but the pathways that manage the ECM are not fully understood. Here, we implicate the transmembrane protein Tmem2 in the regulation of ECM organization in the zebrafish embryo. Maternal and zygotic depletion of tmem2 (MZtmem2) function causes dystrophy of both fast and slow muscle fibers, in association with impaired laminin organization and ineffective fibronectin degradation at the vertical myoseptum. Reminiscent of the defects observed in skeletal muscle, we find disorganized deposition of laminin and fibronectin surrounding MZtmem2 mutant cardiomyocytes, which could account for the hindered migration of these cells during heart tube formation. Irregular ECM deposition also occurs in conjunction with excessive Wnt signaling during atrioventricular canal differentiation in zygotic tmem2 mutants. Structure-function analysis indicates specific domains within the extracellular portion of Tmem2 that are crucial for its functions. Together, our data suggest a model in which these domains interact with ECM molecules in order to modulate their deposition and organization, thereby facilitating key aspects of heart and muscle morphogenesis.

142

Main Posters 3 3. Myocardial development/cardiomyopathies 3-18

MODELING CERAMIDE-INDUCED LIPOTOXIC CARDIOMYOPATHY IN THE DROSOPHILA HEART Stanley M. Walls, Karen Ocorr, Greg L. Harris, Rolf Bodmer Sanford Burnham Medical Research Institute Lipotoxic cardiomyopathy (LCM) is a form of cardiac dysfunction associated with obesity and Type II Diabetes that results from abnormal myocardial lipid accumulation in the heart. While elevated cardiac levels of the bioactive lipid ceramide have been observed in a variety of LCM models, its direct role in the etiology of LCM has yet to be elucidated. Here, we utilize the Drosophila heart to model ceramide-induced LCM. Our results show that both genetic and pharmacological ceramide accumulation promotes classic hallmarks of LCM, including heart chamber dilation, contractile dysfunction and reduced fractional shortening. Additionally, these data show that blocking de novo ceramide synthesis, through genetic or pharmacological inhibition of serine palmitoyltransferase, in ceramide-accumulating flies prevents LCM. Interestingly, we show that genetic accumulation of the downstream intermediate, sphingosine-1-phosphate (S1P) or pharmacological administration of the S1P analog, FTY720P, rescues ceramide-induced LCM. Finally, we explore new approaches for discovering novel ceramide-protein interactions in vitro, and how we can utilize the genetically tractable Drosophila model to screen the ceramide-protein interactome for novel participants in the etiology of LCM in vivo.

143

Main Posters 3 3. Myocardial development/cardiomyopathies 3-19

MUSCLE LIM PROTEIN MEDIATES CARDIOMYOPATHY IN PROLINE HYDROXYLASE DOMAIN DEFICIENT MICE WH Davin Townley-Tilson, Liang Xie Baylor College of Medicine Ischemic heart disease is due to insufficient blood and oxygen flow to the heart. Prolyl hydroxylase domain (PHD) proteins represent three of the over 200 oxygen-sensor proteins that mediate the physiological response oxygen concentration. The muscle LIM protein (MLP) provides structural support within cardiomyocytes, bridging the sarcomere and sarcolemma, ensuring efficient contraction. Recent evidence reveals that MLP also mediates cardiomyocyte function via metabolic pathways and mitochondrial function. Our data shows that MLP directly associates with, and is hydroxylated by, PHD2 and PHD3 in cardiomyocytes. Mice with long-term (8 week) PHD2 and PHD3 deletion display atrial and ventricular dilated cardiac hypertrophy, and consequently show impaired hemodynamic function. The mitochondria and sarcomeres of PHD2/3-/mice are disorganized, similar to what is observed in MLP-/- mice. These data indicate the MLP has a direct effect in hearts devoid of PHD2 and PHD3, and that MLP may play a role beyond mediated structural integrity within cardiomyocytes. Future work on the interaction between MLP and the fusion/fission/biogenesis machinery of the mitochondria will determine the precise modality of MLP-mediated dysfunction. The link between oxygen concentration, myocyte contraction, and cell metabolism may yield insights into how the heart mitigates pathophysiology through oxygen-sensing proteins, and new therapeutic treatments of ischemic heart disease.

144

Main Posters 3 3. Myocardial development/cardiomyopathies 3-20

CYCLOPHILIN D REGULATES MITOCHONDRIAL ACTIVATION IN THE EMBRYONIC HEART Gisela Beutner, Morgan L. Albert, Kambiz N. Alavian, Elizabeth A. Jonas, George A. Porter, Jr. University of Rochester Medical Center BACKGROUND: Mitochondria regulate cardiac development. Between embryonic day (E) 9.5 and 11.5, mitochondrial maturation causes a fall in cellular oxidative stress, which enhances further myocyte differentiation. This occurs due to closure of the mitochondrial permeability transition pore (mPTP), which is regulated by the protein cyclophilin D (CyPD) and which we recently showed to be derived from ATP synthase. OBJECTIVE: Determine how CyPD controls mPTP activity in the embryonic heart. METHODS: We used hearts and primary myocytes cultures from E9.5, E11.5, E13.5, and adult C57BL/6N wild type (WT) and CyPD null mice for transfection, immunoprecipitation (IP), immunoblotting (IB), immunofluorescence labeling (IF), and electrophysiology experiments. RESULTS: CyPD expression increased with heart development and was highest in the ventricles at E9.5 and in the walls at E13.5 by IF. CyPD’s activity is known to be regulated by lysine acetylation, and IP experiments revealed that CyPD was acetylated at E9.5, but not at older ages. Transfection of CyPD null myocytes with WT or mutant CyPD demonstrated that acetylation of lysine 166 regulates mitochondrial maturation and myocyte differentiation. Finally, CyPD disassembled ATP synthase and opened the mPTP, as measured by patch clamping and IP/IB experiments. CONCLUSION: These data suggest deacetylation of CyPD causes assembly of ATP synthase to close the mPTP and promote myocyte differentiation in the early embryonic heart.

145

Main Posters 3 3. Myocardial development/cardiomyopathies 3-21

REGULATION OF MITOCHONDRIAL REMODELING TO PREVENT HEART DAMAGE R. Nieto-Arellano, R. Acin-Perez, A. Gonzalez-Guerra, I. Carrascoso, A.V. Lechuga, A.V. Alonso-Lopez, L.J. Jimenez-Borreguero, C. Torroja, J.A. Enríquez CNIC Metabolic remodeling in response to a variety of heart insults determines the successful recovery or the progressive deterioration of cardiac performance. Mitochondria participate in both the generation of damage and the proper metabolic adaptation. Therefore, its physiology and biogenesis, the production of reactive oxygen species (ROS), its role in energy supply and in Ca2+ handling are critical at the initial steps and in the progression of the disease. We have investigated the consequences and the different response of heart work overload in mouse models where mitochondrial dynamics, ROS handling and OXPHOS performance has been genetically manipulated. We show that, when mitochondrial remodeling is prevented, a dramatic protection to work overload is induced.

146

Main Posters 3 3. Myocardial development/cardiomyopathies 3-22

DIFFERENTIAL LECTIN BINDING PATTERNS IN ZEBRAFISH AND GIANT DANIO HEARTS Trina Manalo, Adam May, Josh Quinn, Pascal J Lafontant DePauw University Lectins are carbohydrate-binding proteins commonly used in the study of mammalian tissues. However they have received little use in fish. Here, we determine the binding patterns of commonly used lectins, wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA lectin), Bandeiraea simplicifolia (BS lectin), Concanavalin A (Con A), Ricinus communis (RCA), and Lycopersicon esculentum (tomato lectin) in zebrafish and giant danio hearts. Our results show that WGA stained cardiac myocytes in patterns similar to that seen in mammalian hearts, with staining markedly stronger in the fish compact hearts. Con A showed broad staining of epicardium, endocardium and coronary vessels in both fish. By contrast, UEA lectin did not react to the vasculature of both fish. Tomato lectin showed particular binding affinity to the zebrafish transitional cardiac myocyte of the heart junctional region, while RCA showed a labile but specific staining of compact hearts. Differential lectin staining was also observed in developing hearts. Interestingly, BS lectin reacted poorly in zebrafish, but strongly stained coronary vessels of the Giant danio. Importantly, enhanced BS lectin biding to vascular endothelium compared to endocardium allowed for 3D coronary angiography in the Giant danio for quantitation of vascular reconstruction during regeneration. In conclusion, our study demonstrates that lectins are simple but important tools for studies of embryonic, adult and regenerating danio hearts.

147

Main Posters 3 3. Myocardial development/cardiomyopathies 3-23

RAC1 REGULATES CARDIAC VENTRICULAR AND OUTFLOW TRACT DEVELOPMENT Rebecca Dodds, Deborah Henderson, Helen Phillips Newcastle University Rac1, a Rho GTPase, is involved in a variety of fundamental cellular processes, including cell proliferation, adhesion and migration. Rac1 is upregulated in cardiovascular disease with a role in NADPH oxidase activation, however its function during cardiovascular development remains unclear. Using Cre-LoxP technology we have identified that Rac1 is essential in the myocardium, but not in the epicardium, for the normal development of the mouse embryonic heart. Myocardial targeted deletion of Rac1, using the TnT-Cre transgenic mouse line, results in cardiac defects including a thinned myocardium, disorganised trabeculae, ventricular septal defects and double outlet right ventricle. Immunohistochemical staining suggests that polarity may be disrupted in Rac1-deficient cardiomyocytes. Ongoing microarray analysis of Rac1;TnTCre hearts will aid in deciphering the essential role of Rac1 in early ventricular and outflow tract development. Approximately one third of all congenital heart defects involve malformations of the outflow tract. Understanding the role of the genes that regulate outflow tract formation could facilitate the development of preventative measures and treatments for congenital heart defects.

148

Main Posters 3 3. Myocardial development/cardiomyopathies 3-24

THE ROLE OF CARDIAC MICROTUBULES IN HEART DEVELOPMENT AND DISEASE Annabelle Williams, Elise R. Pfaltzgraff, David Bader Vanderbilt University Microtubules are highly organized structures with functionally diverse roles within the cell. The orientation and dynamic instability of microtubules are critical in the regulation of the cytoskeleton, maintenance of cell shape and motility, intracellular transport, and chromosome segregation. However, in heart development, the relevance of the microtubule network in cardiogenesis has been largely overlooked. It has only recently been shown that the loss of CENP-F, a microtubule binding protein, results in alteration of cardiac growth, morphogenesis, and function. In CENP-F -/- hearts, intercalated discs, sarcomeres, and costameres of cardiac myocytes are affected and lead to cardiomyopathy, a disease of the myocardium. These data illustrate the need to better understand the role of microtubules during cardiac development and maturation. The current study provides a plan to analyze how microtubules influence cardiac development and their role in mature cardiomyocytes. During key time points in cardiac development we have assayed for the nucleation of tubulin and its eventual localization, as well as analyzed the dynamic distribution of key microtubule associated proteins in order to establish a primer on microtubules in cardiac development. This study will ultimately provide insight on the importance of the microtubule network on heart development, maintenance, and disease.

149

Main Posters 3 3. Myocardial development/cardiomyopathies 3-25

CK2 EXPRESSION IN XENOPUS LAEVIS CARDIAC DEVELOPMENT Nicole Yoon, Dr. Isabel Dominguez, Mikaela Oliverio Boston University Medical School CK2 is a highly conserved, ubiquitous serine-threonine kinase involved in biological processes that include embryonic development, cancer, inflammation and circadian rhythms. The enzyme is made of two catalytic α-subunits and two regulatory β-subunits that form relatively stable heterotetramers; the β-subunits allow for optimal expression and regulation of substrate binding (Vilk et. al, 1999). Here we used whole-mount immunohistochemistry followed by sectioning to analyze CK2 protein expression in stages of cardiac development during which organogenesis occurs in Xenopus laevis. Our data showed that CK2 protein expression is concentrated in the myocardial and endocardial tissue during frog heart formation especially during the latter stages, suggesting an important role for CK2 proteins in proliferation and movement. Such results support recent studies that reveal a possible role of CK2 in cell proliferation. Using these results, our interest is turned towards the relationship between Wnt/β-catenin signaling - known to be a critical component of cardiac development - and CK2, a positive regulator of the pathway.

150

Main Posters 3 3. Myocardial development/cardiomyopathies 3-26

MICRORNA REGULATION OF ALTERED CALCIUM HANDLING IN FAMILIAL DILATED CARDIOMYOPATHY Christine Wahlquist, Agustin Rojas-Muñoz, Fabio Cerignoli, Cecilia Hurtado, Mark Mercola Sanford Burnham Medical Research Institute Dilated cardiomyopathy (DCM) is the most common form of heart disease, the third leading cause of heart failure in the U.S., and a leading cause of heart transplantation. As many as 20-35% of DCM cases are estimated to have a genetic etiology, with mutations in contractile proteins and nuclear laminins accounting for most familial forms. The connection between genotype and phenotype is poorly understood, although elegant biophysical studies have shown that some familial DCM mutations, such as in β-MHC and cTn-T, decrease Ca2+ sensitivity or increase the energetic cost of contraction and therefore might make the cardiomyocyte susceptibility to wall tension and dilation. We explored the role of microRNAs in controlling contractility in the context of familial DCM. Since DCM involves impaired Ca2+ uptake resulting from decreased SERCA2a activity, we used functional screening to identify 144 screened for miRNAs that suppress SERCA2a. Of these, 23 are upregulated in familial DCM and might contribute to diminished contractile function in DCM. We also found that mechanical strain and chronic adrenergic stimulation induced a number of these miRNAs, suggesting candidate networks that might link physiological stimuli to decreased calcium handling and sarcomeric protein expression.

151

Main Posters 3 3. Myocardial development/cardiomyopathies 3-27

RECIPROCAL SIGNALING BETWEEN THE MYOCARDIUM AND ENDOCARDIUM REGULATES EMBRYONIC CARDIAC DEVELOPMENT S. Ram Kumar, MD, PhD, Jiang Liu, PhD, Binyun Ma, PhD, Timothy Martens, MD, PhD, Sydney J. Zagger, BS, Parkash S. Gill, MD, Henry M. Sucov, PhD University of Southern California The embryonic ventricular myocardium receives signals from the endocardium and epicardium that regulate development. EphB4, a member of the largest family of receptor tyrosine kinases is expressed in the developing endocardium, along with its cognate ligand EphrinB2. EphrinB2 is also expressed in the embryonic myocardium and expression wanes as the embryo matures. Using cardiac-specific knockout of EphB4 and EphrinB2, we show that disruption of EphB4 expression results in a severely thinned myocardial layer, ventricular non-compaction, and mid-gestation lethality. Mutant hearts show reduced activation of PI3K pathway and impaired proliferation of developing cardiomyocytes. Knockdown of EphB4 alters downstream signaling pathways of significance in cardiac development. In particular, EphB4 expression regulates neuregulin-1 levels, which signals via myocardial ErbB receptor. Conversely, whole heart or, to a lesser extent, endocardial or myocardial-specific, knockdown of EphrinB2 shows similar impairment in cardiomyocyte proliferation and ventricular underdevelopment. Our study, therefore, establishes a novel paradigm in cardiac development. In mid-gestation, following initial cardiac chamber organization, the developing myocardium signals to the endocardium to produce mitogens that then stimulate the myocardium to induce cardiomyocyte proliferation and organization. Disruption of any aspect of this signal loop results in arrested cardiac development and mid-gestation lethality.

152

Main Posters 3 3. Myocardial development/cardiomyopathies 3-28

DROSOPHILA AS A VALUABLE MODEL TO STUDY CARDIAC DEVELOPMENT IN HEALTH AND DISEASES STATES Barbara Rotstein, Maik Drechsler, Ariane Wilmes, Heiko Meyer, Achim Paululat University of osnabrueck The fruit fly Drosophila melanogaster is a very valuable system to study cardiac development in health and disease states because it combines the advantages of invertebrate genetics with novel physiological measurement techniques that allow significant comparisons with data from vertebrate model systems. The Drosophila heart possesses a compartmentalized extracellular matrix (ECM) with unique components such as Pericardin (Prc), which is essential to maintaining heart integrity, mainly due to the fact that the Drosophila heart lacks mechanisms for tissue repair or replacement. Consequently, mutations in genes coding for cardiac ECM components lead to severe cardiac defects, e.g. the disassembly of the cardiac matrix and finally to heart collapse. Our lab has recently identified a cardiac specific ECM adapter protein (Lonely heart, Loh), which belongs to the ADAMTS-like family of matricellular proteins. Lonely heart is crucial for recruiting the collagen-like structural Prc protein into the meshwork of the cardiac ECM. We hypothesize that Prc modulates the physical properties of the cardiac ECM by being incorporated and crosslinked to form a defined microarchitecture in a yet undefined manner. Thus it is crucial to provide the correct balance of stiffness versus elasticity, which are critical features for heart tube functionality.

153

Main Posters 3 3. Myocardial development/cardiomyopathies 3-29

OVER-EXPRESSION OF KIF1A CAUSES HEART DEFECTS BY DISRUPTION OF THE ATIN CYTOSKELETON IN DROSOPHILA Takeshi Akasaka, Karen Ocorr, Grant Hogg, Lizhu Lin, M. Benjamin Perryman, Rolf Bodmer, Paul Grossfeld UCSD Left-sided heart defects are one of the most common forms of congenital heart disease, ranging in severity from the relatively minor bicuspid aortic valve to hypoplastic left heart syndrome (HLHS), a uniformly fatal lesion. In this study, we identify a candidate gene for left-sided heart defects, KIF1A, and define a mechanism by which over-expression of KIF1A in Drosophila causes structural heart defects. We identified a patient with left-sided heart defects in association with a chromosomal balanced translocation [t(2,8;q37,p11)] whose breakpoint is in the 5’ non-translated region of the Kif1A gene at 2q37, a locus previously associated with left-sided congenital heart defects, such as HLHS. We hypothesized that dysregulation of KIF1A expression can cause structural heart defects. We generated transgenic fly lines that over-express Kif1A in mesodermal tissue and heart muscle. Over-expression of KIF1A resulted in cardiac myofibril disorganization, dysplastic valve structures, and a hypoplastic ventricular chamber with impaired contraction, all hallmarks HLHS in humans. We also examined the effect of Kif1A in C2C12 myoblasts and determined that Kif1A over-expression disrupts actin but not microtubule organization of the cytoskeleton. In summary, dysregulation of Kif1A expression impairs normal heart development in fruit flies, providing potential new insights into the pathogenesis of some forms of congenital heart disease in humans.

154

Main Posters 3 3. Myocardial development/cardiomyopathies 3-30

INNER ARCHITECTURE OF THE RIGHT VENTRICLE: THE ROLE OF THE TRICUSPID VALVE Lucile Houyel, Marianne Peyre, Bettina Bessières, Meriem Mostefa-Kara, Marie Gonzales Marie-Lannelongue Hospital A major anatomic characteristic of the right ventricle (RV), in addition to the coarseness of the apical trabeculations, is the presence of muscular bands arranged in a semicircular fashion: parietal band and subpulmonary conus, septal band (SB), moderator band (MB). Recent publications show that SB, MB and anterior papillary muscle of the tricuspid valve (APM) could derive from the primitive muscular tricuspid gully. In order to confirm these findings, we reviewed 32 postnatal and 24 fetal human heart specimens with tricuspid atresia (TA). Forty-two hearts had ventriculo-arterial concordance, 14 had D-transposition. There were 51 muscular TA (musTA, including 7 without any RV cavity), and 5 membranous TA (mbTA). All 49 hearts with a RV cavity had a well-developed ventriculo-infundibular fold (VIF). A rudimentary SB (with demonstrable limbs in 3) was present in 7/44 musTA vs 4/5 mbTA (p