J. Rivera-Feliciano, C.J. Tabin / Developmental Biology 295 (2006) 580–588
Bmp2Nkx2.5del, and Bmp2Nkx2.5del (Figs. 2A–C, PECAM) + 200 ng/ml BMP2 explants . In control explants, SMA is
In the absence of Bmp2, the AV canal myocardium loses its identity as primary myocardium
expressed in invasive mesenchymal cells after EMT but not in endocardial cells (Sugi et al., 2004) (Fig. 2A, α-SMA). No SMA-expressing mesenchymal cells were detected in
explants (Fig. 2B, α-SMA), and upon addition
of BMP2 to the media, SMA-positive invasive mesenchymal cells reappeared. Co-expression of SMA and PECAM1 only occurs in activated endothelial cells (asterisks in Figs. 2A and C, merged) and transformed mesenchymal cells, and it is indicative of EMT (Camenisch et al., 2002b) (yellow
We have determined that the morphology of the region between atrial and ventricular foramens in Bmp2Nkx2.5del embryos is rather straight. The scarce matrix and close proximity to the myocardium suggests that the endothelium in this malformed area morphologically resembles that of the atria and ventricles. We also have shown that the defect in heart morphogenesis occurs prior to the increase in extracel- lular matrix deposition at the cushion-forming region, as the
arrowheads in Fig. 2A, merged). Bmp2Nkx2.5del
cardiac jelly never swells in Bmp2Nkx2.5del
never exhibit co-expression of these markers, indicating that BMP2 is required for AVC endothelium to undergo EMT (e in Fig. 2B, merge). Addition of BMP2 to Bmp2Nkx2.5del explants rescues this activation and transformation (yellow arrowheads, Fig. 2C, merge). In order to better characterize the extent of rescue, we counted the number of invasive cells with mesenchyme cell morphology in all three conditions.
explants never undergo EMT, we find
that addition of 200 ng/ml of BMP2 to Bmp2Nkx2.5del explants results in a 30% rescue of invasive mesenchyme formation relative to controls, with a P value of 4 × 10−6 (Fig. 3). Although we cannot rule out the possibility that BMP2 may have a role in controlling proliferation of the AVC endothelium, we speculate that a mere increase in endothelial cell numbers will not necessarily result in a proportional increase in the formation of invasive mesen- chyme. Local proliferation in the absence of EMT may result in formation of a multilayered non-invasive endothelium (Dor et al., 2001). Nevertheless, our experiments support our initial hypothesis that BMP2 is sufficient and necessary for inducing AVC EMT within the forming endocardial endothelium.
ally, we have demonstrated that Bmp2 is both essential and sufficient to transform AV canal endocardial cells to mesenchyme. Taken together, the defects we observed in
morphogenesis and AVC patterning in Bmp2Nkx2.5del
suggest that Bmp2 may also be required for specification of segmental heart patterning. To investigate this, embryos were examined at 9.5 dpc when cushion endothelium is undergoing EMT (Camenisch et al., 2002a). Compared to controls, Bmp2Nkx2.5del hearts have a straight morphology on the left (compare red arrowheads Fig. 4A) and have defects with atrial rotation. However, the OT is present and looped. To determine if loss of Bmp2 alters gene expression in the putative AVC region, expression of Nppa, a gene exclusive to chamber myocardium (Christoffels et al., 2000), was examined. In 9.5 dpc control embryos, Nppa is expressed in myocardium of the outer curvature of the atrial and ventricular compartments as they differentiate and balloon out (Christoffels et al., 2000). The inner curvature forms a continuity of myocardium with IT, AVC, and OT, but it does not express Nppa and remains undifferentiated (Christoffels et al., 2000) (Fig. 4F, control, red bars). In Bmp2-deficient hearts, Nppa expression is expanded (Figs. 4C–F;
) into the atria, IT and inner curvature of the
heart (red bars in Fig. 4C, red bars in D–F), suggesting that loss of Bmp2 causes primitive inner curvature myocardium to acquire the identity of differentiated outer curvature myocar- dium. Additionally, the expression of Nppa in the putative AVC region suggests that this heart-valve forming region has also adopted chamber myocardium identity.
A mechanism has been proposed wherein site-specific AVC formation results from localized repression of chamber differentiation in the heart-valve region (Habets et al., 2002). In this model, Tbx2, a transcriptional repressor (Jacobs et al., 2000), forms a complex with Nkx2.5 on T-box and NK-class elements within the Nppa promoter and suppresses Nppa activation in the AVC and OT (Habets et al., 2002). Therefore, we examined if misexpression of Nppa in atria, IT, and inner curvature of Bmp2-deficient hearts could result from the loss of Tbx2 expression. Tbx2 was expressed normally in AVC and OT of control hearts (Christoffels et al., 2004; Habets et al., 2002)
Fig. 3. Addition of BMP2 protein can rescue invasive mesenchyme formation. Quantitative analysis of invasive mesenchyme formation at 48 h. One representative experiment in shown for control (blue, n = 5), Bmp2Nkx2.5del (red, n = 2), and rescued (green, n = 9) heart explants. The 30% rescue in mesenchyme formation in Bmp2Nkx2.5del explants after the addition of 200 ng/ml BMP2 is statistically significant, with a P value of 4 × 10−6.
(Figs. 4G–H, control). In Bmp2Nkx2.5del
hearts, Tbx2 was absent
from the AVC and inner curvature (Fig. 4H). These data suggest that Bmp2 instructs AVC development by regulating Tbx2 expression and is consistent with the model in which Tbx2 represses chamber myocardium fate and permits AVC