J. Rivera-Feliciano, C.J. Tabin / Developmental Biology 295 (2006) 580–588
embryos and discovered that Bmp2 ablation in cardiac progenitors causes death at 11.5 days post coitum (dpc) due to cardiac failure. Although we never recovered embryos past 11.5 dpc, all embryos at 10.5 dpc were alive as assessed by cardiac contractile activity at the time of harvesting. At 10.5 dpc, the AVC is barely visible on the leftside of the control embryos because of the expanding size of the developing chambers (Fig. 1A, left panel). At the same stage,
the vascular phenotype. In contrast, our conditional ablation strategy takes advantage of a heart-specific promoter, which is not expressed in the extracardiac vasculature. And indeed,
embryos have an abnormally patterned heart, and
the region amid the atria and ventricles is readily visible with an uncharacteristic, straight morphology (Fig. 1A, right panel black arrowhead). Some of the Bmp2Nkx2.5del embryos have cardiac edema and pericardial effusion (data not shown). In
addition, the Bmp2Nkx2.5del
embryos are smaller, presumably as
a secondary consequence of their cardiovascular defects. To analyze the morphological difference between control and Bmp2Nkx2.5del hearts, we examined sagittal sections by differ- ential interference contrast microscopy and hematoxylin and eosin staining (Figs. 1B and C, respectively). In control embryos, the forming ECs separate the atria and ventricles, and as the chambers enlarge, the AV constriction appears at the inner curvature of the heart (Fig. 1B and black arrowhead in Fig. 1C). The Bmp2Nkx2.5del hearts lack ECs, EC mesenchyme (red arrowheads in Fig. 1C) and the AV constriction (compare black arrowheads in both panels of Fig. 1C). In contrast, while the receptor tyrosine kinases Erbb2 and Erbb3 knockout mice lack cushion mesenchyme (Camenisch et al., 2002b), they retain endocardial swells and normal AV constriction. The first morphological indication of commitment of AV canal myocar- dium to instruct the formation of the heart valves is the deposition and swelling of the extracellular matrix. Compared to the Erbb2 and Erbb3 defect, our Bmp2Nkx2.5del mutants arrest at a stage prior to matrix deposition, hence before specification of the AVC myocardium occurs, indicating that Bmp2 is required for endocardial swelling in addition to heart-valve mesenchyme formation. In contrast, the outflow tract of the
heart is grossly normal in Bmp2Nkx2.5del
embryos. The high
contribution of neural crest mesenchyme to the development of this structure may contribute to the absence of a phenotype in this region.
Bmp2 is essential for extracellular matrix deposition
We investigated the possibility that Bmp2 could regulate the increase of extracellular matrix deposition at the cushion forming regions, which is necessary for cushion mesenchyme formation (Camenisch et al., 2000). We examined the EC
matrix of Bmp2Nkx2.5del
hearts and found no increase in
matrix deposition (yellow arrowheads, Fig. 1D) as assessed by binding of Alcian Blue to acidic glycosaminoglycans. The lack of matrix deposition in Bmp2Nkx2.5del ECs phenocopies hyaluronan synthase-2 (Has2)-deficient embryos (Camenisch et al., 2000). However, unlike our conditional Bmp2 knockouts, Has2 mutants also display vascular abnormalities and cardiomegaly. Therefore, it is unclear whether the cardiac defects in the Has2 mutant are due to loss of hyaluronan synthase-2 in the ECs or are secondary to
Fig. 1. Bmp2 is required for the formation of the endocardial cushions. (A)
Control and Bmp2Nkx2.5del
embryos during midgestation. Note that the
atrioventricular constriction is absent in the mutant hearts, black arrowhead. h, Heart. (B) Differential interference contrast (DIC) microscopy of control and Bmp2Nkx2.5del heart sagittal sections. Photographs are from the left side of the embryos: atrial foramen (a) to the right, ventricle foramen (v) to the left, outflow tract foramen (ot) on top. otc, outflow tract cushion; sec, superior endocardial cushion; iec, inferior endocardial cushion. Magnification 200×. (C) Hematox- ylin and eosin staining of sections through the heart of control and Bmp2Nkx2.5del hearts. The red arrowheads indicate endocardial cushion mesenchyme in the control and at the predicted location of endocardial cushions in a Bmp2Nkx2.5del heart. Note the presence of endocardial cushion cells in the control embryos and
Nkx2.5del a t r i o v e n t the absence of mesenchyme in Bmp2 r i c u l a r c o n s t r i c t i o n i s a b s e n t i n B m p 2 N k x 2 . 5 d e l hearts. In addition, the hearts (black arrowhead).
(D) Alcian Blue staining of acidic glycosaminoglycans. Bmp2Nkx2.5del hearts fail to deposit extracellular matrix at the heart-valve-forming region and fail to undergo endocardial swelling (compare blue staining, yellow arrowheads).