J. Rivera-Feliciano, C.J. Tabin / Developmental Biology 295 (2006) 580–588
To further examine the AVC myocardium of Bmp2Nkx2.5del hearts, we analyzed the expression of myocardial restricted genes by section in situ hybridization. We verified that Bmp2 was inactivated by Nkx2.5Cre in the heart, as Bmp2 transcripts
Nkx2.5del Nkx2.5del were not detected in AVC myocardium of Bmp2 but were present in controls (Fig. 5A, Bmp2 hearts , control).
Section in situ hybridization for the chamber myocardium markers Nppa and the small muscle-specific protein Smpx
Fig. 6. Bmp2 is necessary to induce the endothelial cell competence to transform. (A–C) Section in situ hybridization for control and Bmp2Nkx2.5del hearts showing that Id1 (A), Notch1 (B), and Gata4 (C), endothelial specific markers, are downregulated from the putative endocardial endothelium. Black arrowheads indicate atrioventricular canal endocardium. Insets in panels B and C are sections through the outflow portion of the same heart. Magnification 200×.
(Palmer et al., 2001) confirmed the expansion of transcripts into the heart-valve field (compare black arrowhead pairs in Figs. 5B and C) and atria (Figs. 5B and C insets). Similar analysis for Tbx2 verified its downregulation in the AVC (Fig. 5D). Similar to Bmp2 and Tbx2, Msx2 (muscle segment homeobox 2) expression is restricted to the AVC myocardium in controls (Abdelwahid et al., 2001) (Fig. 5E, control), and downregulated in the myocardium abutting the chambers in mutants (Fig. 5E,
). Taken together, expansion of Nppa and Smpx
and absence of Tbx2 and Msx2 expression suggest that chamber myocardium is specified at the expense of AVC myocardium in Bmp2-deficient embryos.
Bmp2 maintains the molecular competence of the cardiac endothelium, which is required to form heart-valve mesenchyme
Fig. 5. Bmp2 is required for the AV canal myocardium to retain its identity as primary myocardium. (A–E) Section in situ hybridization for control and Bmp2Nkx2.5del hearts showing that the atrioventricular canal fails to be specified in the absence of Bmp2. Note that AVC restricted transcripts are absent in
hearts: Bmp2 (A), Tbx2 (D), Msx2 (E) and the upregulation of the
chamber specific transcripts Nppa (B) and Smpx (C) to the putative atrioventricular canal myocardium. Black arrowheads indicate atrioventricular canal myocardium. Insets in (B–D) are sections through the outflow portion of the same heart. Magnification 200×. a, atria; v, ventricle; ot, outflow tract; sec, superior endocardial cushion; iec, inferior endocardial cushion.
Because AVC myocardium induces the adjacent endotheli- um to undergo EMT and form the valves, we examined the expression of Id1, Notch1, and Gata4, genes that are either present in AVC endothelium during EMT (Jen et al., 1996; Timmerman et al., 2004; Zeisberg et al., 2005) or have been implicated in the process (Timmerman et al., 2004) (Figs. 6A– C, control). Expression of Id1, a negative regulator of basic helix–loop–helix transcription factors (Jen et al., 1996), was