Malformations of cardiac valvuloseptal structures are prevalent causes of congenital heart diseases (CHDs), which occur in as many as 1% of newborns and remain the leading cause of infant morbidity and mortality. The long term goal of this study is to identify the molecular, cellular, and genetic mechanisms governing normal valvuloseptal morphogenesis and to reveal their contributions to CHDs. Valvuloseptal development in the atrioventricular canal (AVC) region is initiated with cushion formation by regional expansion of extracellular matrix (ECM) at ~E9.0 in mouse embryonic hearts. Shortly thereafter, in response to stimulatory signals released from the myocardium, a subgroup of endocardial cells in the AVC transdifferentiate into mesenchymal cells and migrate into the ECM. The cellularized cushions serve as the primordia of valves and septa, and are further remodeled into final structures through complicated maturation processes. Bone Morphogenic Protein (BMP) pathways play essential roles during AV cushion formation, cellularization, and remodeling;mutations that disturb BMP signaling cause various valvuloseptal defects both in animal models and in human patients. Nevertheless, the downstream genes that mediate the complex activities of BMP signaling remain elusive. To facilitate the application of cellular and molecular approaches to study AV cushion morphogenesis, we developed a temperature sensitive immortal AV cushion mesenchymal cell line, tsA58-AVM. To the best of our knowledge, no other permanent cushion cell line has been reported in the literature. Using this unique cell culture system, we identified Sema6D as a novel regulatory target of BMP signaling in AV cushions. While Semaphorins were initially recognized as phylogenetically conserved neuronal guidance cues, their functions have now been implicated in regulating cell morphology, proliferation, adhesion and migration during various biological/pathological processes. No study in the literature has directly addressed the potential roles of Semaphorin signaling during AV valvuloseptal morphogenesis. Our preliminary studies using in vitro cultured cells and ex vivo cultured AV tissues suggested that Sema6D promotes cushion mesenchyme formation and migration. Moreover, we established a conditional knockout mouse line of Sema6D, and our initial studies using this line supported the essential in vivo role of Sema6D for normal AV cushion cellularization. We hypothesize that Sema6D is a key BMP regulatory target critical for promoting AV cushion mesenchyme formation and migration during valvuloseptal development.
Three specific aims are outlined in this proposal.
In Aim 1, we will determine the cellular activity of Sema6D in endocardial and mesenchymal cells using immortal cell lines.
In Aim 2, we will test the functional interaction between Sema6D and BMP signaling during AV cushion mesenchyme formation and migration using ex vivo cultured AV tissues.
In Aim 3, we will determine the in vivo function of Sema6D during AV cushion morphogenesis through a conditional gene inactivation approach. Successful accomplishment of this study will significantly advance our knowledge of the molecular mechanisms underlying normal valvuloseptal formation and CHDs.

Public Health Relevance

Malformations of cardiac valvuloseptal structures account for a large proportion of congenital heart diseases. BMP signaling plays a critical role in atrioventricular (AV) valvuloseptal development. We have identified Sema6D as a downstream regulatory target of BMP signaling in AV cushions, and will apply multiple complementary approaches to explore the potentially critical function of Sema6D in supporting normal development of AV cushions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL095783-04
Application #
8391711
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Schramm, Charlene A
Project Start
2010-01-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
4
Fiscal Year
2013
Total Cost
$345,183
Indirect Cost
$109,563
Name
University of Alabama Birmingham
Department
Genetics
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Peng, Yin; Song, Lanying; Li, Ding et al. (2016) Sema6D acts downstream of bone morphogenetic protein signalling to promote atrioventricular cushion development in mice. Cardiovasc Res :
Yan, Shun; Jiao, Kai (2016) Functions of miRNAs during Mammalian Heart Development. Int J Mol Sci 17:
Chen, Dongquan; Li, Yufeng; Wang, Lizhong et al. (2015) SEMA6D Expression and Patient Survival in Breast Invasive Carcinoma. Int J Breast Cancer 2015:539721
Peng, Yin; Song, Lanying; Zhao, Mei et al. (2014) Critical roles of miRNA-mediated regulation of TGFβ signalling during mouse cardiogenesis. Cardiovasc Res 103:258-67
Liu, Yuelong; Harmelink, Cristina; Peng, Yin et al. (2014) CHD7 interacts with BMP R-SMADs to epigenetically regulate cardiogenesis in mice. Hum Mol Genet 23:2145-56
Harmelink, Cristina; Peng, Yin; DeBenedittis, Paige et al. (2013) Myocardial Mycn is essential for mouse ventricular wall morphogenesis. Dev Biol 373:53-63
Peng, Yin; Zhao, Shaomin; Song, Langying et al. (2013) Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts. Biochem Biophys Res Commun 441:751-6
Mao, Xia; Debenedittis, Paige; Sun, Yong et al. (2012) Vascular smooth muscle cell Smad4 gene is important for mouse vascular development. Arterioscler Thromb Vasc Biol 32:2171-7
Debenedittis, Paige; Harmelink, Cristina; Chen, Yunjia et al. (2011) Characterization of the novel interaction between muskelin and TBX20, a critical cardiogenic transcription factor. Biochem Biophys Res Commun 409:338-43
Wang, Bing; Sinha, Tanvi; Jiao, Kai et al. (2011) Disruption of PCP signaling causes limb morphogenesis and skeletal defects and may underlie Robinow syndrome and brachydactyly type B. Hum Mol Genet 20:271-85

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