**3.1.4 BMP inhibitors**

100 Congenital Heart Disease – Selected Aspects

mesoderm to undergo cardiac differentiation (Alsan and Schultheiss 2002; Barron, Gao, and Lough 2000; Tirosh-Finkel et al. 2010). *Bmp2* deletion in mice causes embryonic lethality between E7.5-E9.0 (Zhang and Bradley 1996). Some mutant embryos lack hearts altogether and others develop ectopic heart tubes in the exocoelomic cavity, suggesting a critical role of

BMP signaling pathways induce cardiac differentiation through upregulation of cardiogenic genes. Expression of the cardiac transcription factors *Nkx2.5* and *Gata4* is initiated by BMP signaling (Frasch 1995; Schultheiss, Burch, and Lassar 1997; Andrée et al. 1998; Schlange et al. 2000; Jamali et al. 2001; Liberatore et al. 2002; Shi et al. 2000; Lien et al. 2002; Reiter, Verkade, and Stainier 2001; Schultheiss, Xydas, and Lassar 1995). The *Nkx2.5* promoter region contains evolutionary conserved BMP-response elements that are necessary for its cardiac expression (Lien et al. 2002; Liberatore et al. 2002; Brown et al. 2004). BMP signaling also activates the expression of myocardin, a cardiac and smooth muscle-specific transcriptional cofactor for serum response factor, a regulator of cardiac differentiation (Arsenian et al. 1998; Wang et al. 2001; Callis, Cao, and Wang 2005). SMAD1 is also a transcriptional cofactor for myocardin to activate downstream gene expression (Callis, Cao,

The BMP type I receptor ALK3 is widely expressed in mouse embryos and *Alk3* deletion causes embryonic lethality at E8.0 with no mesoderm formation (Mishina et al. 1995; Dewulf et al. 1995). ALK2, another type I receptor, is expressed in Hensen's node and in the primitive streak (Gu et al. 1999; Mishina et al. 1999). Deleting *Alk2* in mouse embryos results in gastrulation defects and embryonic lethality before E9.5 (Gu et al. 1999; Mishina et al. 1999). The third type I receptor, ALK6, is not expressed during early heart development and disrupting its function does not affect mouse cardiogenesis or viability (Dewulf et al. 1995; Yi et al. 2000). Knockout of the type II receptor, BMPR2, which is expressed widely throughout chicken embryos and during mouse cardiomyogenesis, causes embryonic lethality at gastrulation (Ehrman and Yutzey 1999; Stern et al. 1995; Feijen, Goumans, and van den Eijnden-van Raaij 1994; Beppu et al. 2000). In mice, ACVR2A is expressed after cardiomyocyte formation at E9.5 and ACVR2B is ubiquitously expressed during cardiomyogenesis (Feijen, Goumans, and van den Eijnden-van Raaij 1994; Beppu et al. 2000). Disruption of *Acvr2a* alone does not cause heart defects and disruption of *Acvr2b* causes heart defects later in development (Matzuk, Kumar, et al. 1995; Oh and Li 1997). However, deletion of both *Acvr2a* and *Acvr2b* results in embryonic death at gastrulation, suggesting

In chicken embryos, the receptor-regulated SMAD proteins, SMAD1, SMAD5, and SMAD8, are enriched in the heart forming region (Faure et al. 2002). In mice, *Smad1* and *Smad5*  mRNA are expressed in the mesoderm during cardiomyocyte formation (Tremblay, Dunn, and Robertson 2001). *Smad1* disruption in mice results in embryonic lethality at E10.5 from failure of umbilical-placental connections to form (Tremblay, Dunn, and Robertson 2001). Germline deletion of *Smad5* results in defective left-right symmetry with a heart looping abnormality and defective angiogenesis (Chang et al. 2000; Yang et al. 1999). Deletion of *Smad4,* the gene encoding the common SMAD, causes death before E7.5, with reduced size

BMP signaling for heart formation (Zhang and Bradley 1996).

functional redundancy of these type II receptors (Song et al. 1999).

and Wang 2005).

**3.1.2 BMP receptors** 

**3.1.3 SMAD proteins** 

Inhibition of BMP during gastrulation restricts the heart forming fields to discrete territories in the anterior of the embryo. Noggin, chordin, and follistatin are secreted from the notochord and bind BMP ligands, preventing receptor activation (McMahon et al. 1998; Streit et al. 1998; Sasai et al. 1995; Hemmati-Brivanlou, Kelly, and Melton 1994; Fainsod et al. 1997). The responsiveness of pre-cardiac mesoderm to inhibitory signals from the notochord is developmentally regulated. Ectopic application of noggin to stage 4 chick mesendoderm prevents the initiation of the cardiac gene expression and development of the contracting cardiomyocytes (Schultheiss, Burch, and Lassar 1997; Schlange et al. 2000). If noggin is applied to explants a stage later, the cardiac gene expression is initiated without spontaneous contraction of myocytes. If noggin is applied at stage 6, differentiation occurs normally (Nakajima et al. 2002). In mice, deletion of noggin or follistatin individually does not cause heart defects, but deletion of both reverses heart looping (Bachiller et al. 2000; McMahon et al. 1998; Matzuk, Lu, et al. 1995). Deleting chordin causes defects phenocopying those in DiGeorge syndrome (Bachiller et al. 2003).
