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J. Rivera-Feliciano, C.J. Tabin / Developmental Biology 295 (2006) 580588

587

Nkx2.5del Nkx2.5del absent from Bmp2 heart endothelium compared to controls (Fig. 6A, Bmp2 ), suggesting BMP2 regulates

Id1 expression in AVC endothelium. This finding is consistent with other studies demonstrating that Id1 is induced by BMP2 in osteoblast-like and myoblastic cells (Katagiri et al., 1994; Ogata et al., 1993) and is a direct target of BMP in embryonic

stem cells (Hollnagel et al., 1999).

Since the Notch1 signal transduction pathway has been found to directly regulate genes important for endocardial EMT (Timmerman et al., 2004), and deletion of Notch1 results in hypocellular ECs (Timmerman et al., 2004), we examined if Notch1 expression is altered in the absence of Bmp2. While control hearts exhibit robust Notch1 expression in AVC endothelium and mesenchyme (Fig. 6B, control), Bmp2Nkx2.5del endothelium completely lacks Notch1 transcripts (Fig. 6B,

Bmp2Nkx2.5del

). Although Notch1 makes endocardial endothe-

lium competent to form heart-valve mesenchyme, we find that Bmp2 is necessary for Notch1 expression and inducing this competence.

Gata4, a transcription factor essential for heart formation (Kuo et al., 1997; Molkentin et al., 1997), is robustly expressed at 9.5 dpc in control AVC endocardium and EC mesenchyme, which is mostly derived from AVC endocardium (Kisanuki et al., 2001; Zeisberg et al., 2005) (Fig. 6C, control). Interestingly, myocardial- specific ablation of Gata4 results in the loss of endocardial Gata4 expression, indicating a paracrine signal derived from myocardi- um regulates its endocardial expression (Zeisberg et al., 2005). In

Bmp2Nkx2.5del

hearts, myocardial expression of Gata4 remains

intact while the expression in AV endothelium was lost (Fig. 6C,

Bmp2Nkx2.5del

). Based on this finding, we speculate that this

paracrine signal is Bmp2. Taken together, absence of Id1, Notch1, and Gata4 expression from Bmp2-deficient endothelium suggests that these mutants failed to acquire endothelial cell competence for transformation. It also places Id1, Notch1, and Gata4 epistatic to Bmp2 during heart morphogenesis. Moreover, putative AVC endothelium in Bmp2Nkx2.5del hearts has the molecular signature

of chamber endothelium. Thus, this region in Bmp2Nkx2.5del

hearts

has acquired chamber fates at the expense of primary myocardium progenitors.

Conclusion

The regional specificity of induction and the instructive potential of Bmp2 in the myocardium of the AVC initiate a cascade of events that establish EMT competence in the endothelium. Despite the promiscuity of BMP ligand/receptor binding (Ebisawa et al., 1999; ten Dijke et al., 1994), we find that one signaling molecule, Bmp2, is essential and non- redundant for specification of the heart-valve-forming region. Hence, our results place Bmp2 at the top of a genetic hierarchy essential for formation of the AVC and, consequently, for formation of the heart valves.

Acknowledgments

We are grateful to Vicki Rosen for the use of the Bmp2 floxed allele prior to publication and for the recombinant BMP2

protein. We thank Richard Harvey for the Nkx2.5Cre line, Jenna L. Galloway for critical reading of the manuscript, members of the laboratories of CJT, C. Cepko, S. Dymecki, and J. and C. Seidman for the reagents, discussions, and advice. Many thanks to Michael Peterson and Dane Loeliger for help with the last rounds of genotyping. Heart development research in CJT's laboratory is supported by the NIH (HD0045499). JR- F was supported by a predoctoral National Research Service Award from the NIH.

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