Abnormalities in valvuloseptal development significantly contribute to congenital heart defects, yet the underlying causes are complex and poorly understood. Early cardiac regulatory genes are differentially expressed during valvuloseptal development, consistent with novel functions during heart chamber formation in chicken and mouse embryos. Distinct valve cell lineages were identified in the leaflets, chordae tendineae, and myotendinous junctions with the papillary muscles based on restricted expression of extracellular matrix molecules. Specific cell types within these structures demonstrate characteristics of chondrogenesis and tendon development, identified by scleraxis, type II collagen, and tenascin expression. In chicken embryos, valve remodeling and maturation accompanies a decrease in mitotic index indicated by reduced bromodeoxyuridine incorporation. Analysis of Tie2-cre ؋ ROSA26R mice demonstrates that mature valve structures, including the atrioventricular and outflow tract semilunar valve leaflets, chordae tendineae, and the fibrous continuity that connects the septal leaflets of mitral and tricuspid valves, arise from endothelial cells of the endocardial cushions. Together, these studies provide novel insights into the origins and cell lineage diversity of mature valve structures in the developing vertebrate heart. Developmental Dynamics 230: 239 -250, 2004.

Abstract-Cardiomyocytes actively proliferate during embryogenesis and withdraw from the cell cycle during neonatal stages. FOXO (Forkhead O) transcription factors are a direct target of phosphatidylinositol-3 kinase/AKT signaling in skeletal and smooth muscle and regulate expression of the Cip/Kip family of cyclin kinase inhibitors in other cell types; however, the interaction of phosphatidylinositol-3 kinase/AKT signaling, FOXO transcription factors, and cyclin kinase inhibitor expression has not been reported for the developing heart. Here, we show that FOXO1 and FOXO3 are expressed in the developing myocardium concomitant with increased cyclin kinase inhibitor expression from embryonic to neonatal stages. Cell culture studies show that embryonic cardiomyocytes are responsive to insulin-like growth factor 1 stimulation, which results in the induction of the phosphatidylinositol-3 kinase/AKT pathway, cytoplasmic localization of FOXO proteins, and increased myocyte proliferation. Likewise, adenoviral-mediated expression of AKT promotes cardiomyocyte proliferation and cytoplasmic localization of FOXO. In contrast, increased expression of FOXO1 negatively affects myocyte proliferation. In vivo myocyte-specific transgenic expression of FOXO1 during heart development causes embryonic lethality at embryonic day 10.5 because of severe myocardial defects that coincide with premature activation of p21 cip1 , p27 kip1 , and p57 kip2 and decreased myocyte proliferation. Transgenic expression of dominant negative FOXO1 in cardiomyocytes does not obviously affect heart development at embryonic day 10.5, but results in abnormal morphology of the myocardium by embryonic day 18.5 along with decreased cyclin kinase inhibitor expression and increased myocyte proliferation. These data support FOXO transcription factors as negative regulators of cardiomyocyte proliferation and promoters of neonatal cell cycle withdrawal during heart development. (Circ Res. 2008;102:686-694.)Key Words: heart development Ⅲ FOXO1 (FKHR) Ⅲ Cip/Kip cyclin kinase inhibitors N ormal heart morphogenesis and development are dependent on highly controlled differential regulation of cell proliferation in specific populations of cardiomyocytes during embryonic, fetal, and neonatal stages. 1 Embryonic cardiomyocytes throughout the primitive heart tube rapidly proliferate to provide sufficient cell numbers to build the working myocardium. 2 At fetal stages, proper formation of the ventricular trabeculae, compact zone, and interventricular septum is dependent on more localized temporal and spatial regulation of cardiomyocyte proliferation. 3,4 Immediately before birth, cardiomyocytes throughout the myocardium undergo a hyperplastic to hypertrophic transition in which cell division slows and cell growth increases. 5 After birth, neonatal cardiomyocytes withdraw from the cell cycle, and growth of the myocardium into adulthood occurs primarily by hypertrophy. 1 The molecular mechanisms that control cardiomyocyte maturation, proliferation, and resultant myocardia...

The atrioventricular heart valve leaflets and chordae tendineae are composed of diverse cell lineages and highly organized extracellular matrices that share characteristics with cartilage and tendon cell types in the limb buds and somites. During embryonic chicken valvulogenesis, aggrecan and sox9, characteristic of cartilage cells, are observed in the AV valve leaflets, in contrast to tendon-associated genes scleraxis and tenascin, present in the chordae tendineae. In the limb buds and somites, cartilage cell lineage differentiation is regulated by BMP2, while FGF4 controls tendon cell fate. The ability of BMP2 and FGF4 to induce similar patterns of gene expression in heart valve precursor cells was examined. In multiple assays of cells from prefused endocardial cushions, BMP2 is sufficient to activate Smad1/5/8 phosphorylation and induce sox9 and aggrecan expression, while FGF4 treatment increases phosphorylated MAPK (dpERK) signaling and promotes expression of scleraxis and tenascin. However, these treatments do not alter differentiated lineage gene expression in valve progenitors from fused cushions of older embryos. Together, these studies define regulatory pathways of AV valve progenitor cell diversification into leaflets and chordae tendineae that share inductive interactions and differentiation phenotypes with cartilage and tendon cell lineages.

Wnt signaling mediated by beta-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VIC) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of beta-catenin and induction of periostin and matrix gla-protein, but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis and pathogenesis.