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. 2009 Mar-Apr;126(3-4):117-27.
doi: 10.1016/j.mod.2008.11.008. Epub 2008 Dec 11.

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Devorah C Goldman  1 , Nathan Donley, Jan L Christian
Affiliations

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Devorah C Goldman et al. Mech Dev. 2009 Mar-Apr.

Abstract

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.

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Figures

Fig. 1
Fig. 1
Skeletal defects in adult Bmp2−/+ mice. (A–D) Dorsal views of skeletal preparations of cervical (A and B) and thoracic (C and D) vertebrae from adults. Arrowheads indicate vertebrae that have not fused, arrows highlight the region missing from the 13th rib of mutant, but not wild type animals.
Fig. 2
Fig. 2
Bmp2 and Bmp4 expression and function in the fetal vasculature of the placenta. (A) Bmp2 expression in E10.5 fetal vessels of the placenta and in the heart (positive control) detected by RT-PCR. The 723 bp Bmp2 band is not detected in the absence of reverse transcriptase (−RT, negative control). (B–G) Immunofluorescence analysis of placentas dissected from Bmp4lacZ/+ embryos at E10.5. PECAM-1 (green) detects endothelial cells (B,D,E and G), nuclear-localized β-galactosidase (red) detects Bmp4 expressing cells (B and C) and pSmad1/5/8 (red) detects BMP-responsive cells (E and F). Sections were counterstained with Hoechst (blue) to visualize nuclei (C,D,F and G). Nuclei in which β-galactosidase or pSmad1/5/8 are expressed appear pink (blue + red = pink). Arrowheads indicate fetal endothelial cells that stain for both PECAM-1 and Bmp4 expression (B–D) or pSmad1/5/8 (E–G). Fetal blood vessels contain nucleated erythrocytes and can thus be distinguished from maternal vessels that do not. (H–J) Wholemount PECAM-1 immunostaining of placentas from Bmp4S2G/S2G (H) Bmp2−/+; Bmp4lacZ/+ (I) and Bmp2−/+; Bmp4S2G/S2G (J) embryos at E10.5.
Fig. 3
Fig. 3
Body wall closure defects in Bmp2 heterozygotes and Bmp2−/+; Bmp4lacZ/+ compound mutants. (A and B) Alcian Blue and Alizarin Red-stained skeletons from adult mice show split xiphoid processes in Bmp2−/+ (asterisk), but not wild type mice. (C and D) Hematoxylin and eosin stained cross sections through the thoracic cavity of E15.5 embryos. Arrowheads indicate lateral edges of the ventral body wall of a Bmp2−/+; Bmp4lacZ/+ mutant which have failed to fuse, leading to externalization of the heart (arrow) and other organs.
Fig. 4
Fig. 4
Defects in heart septation and OFT lengthening in Bmp2−/+; Bmp4lacZ/+ compound mutants. (A–F) Hematoxylin and eosin stained cross sections through the thoracic region of an E15.5 wild type (A–C) or Bmp2−/+; Bmp4lacZ/+ mutant embryo (D–F). The mutant heart shows inappropriate communication between the aorta (Ao) and the right ventricle (RV) (DORV, arrow in E) as well as ventricular (VSD, arrow in F) and atrial septal defects (ASD, arrow head in F). PT, pulmonary trunk; RA, right atrium; LA, left atrium; LV, left ventricle. (G–I) Expression of Nkx2.5 in E9.5 embryos, analyzed by whole mount in situ hybridization to highlight heart structures. The OFT of Bmp2−/+; Bmp4lacZ/+ mutants is shortened relative to that of stage-matched controls. White arrowheads mark the distal and proximal ends of the OFT. Variable expression of Nkx2.5 was observed regardless of genotype (data not shown).
Fig. 5
Fig. 5
AV canal defects in Bmp2−/+; Bmp4lacZ/+ compound mutants. (A and B) Hematoxylin and eosin stained cross sections through the thoracic region of an E12.5 wild type (A) or Bmp2−/+; Bmp4lacZ/+ mutant embryo (B) reveals the complete absence of AV canal septation and the presence of a common set of AV valves (arrowheads) in the mutant heart. Arrows indicate an intact ASP. (C and D) Frontal sections through an E11.5 wildtype (C) or Bmp2−/+; Bmp4lacZ/+ compound mutant embryo (D) reveals reduction of tissue in the AV mesenchymal mass (arrowheads) whereas the ASP is intact (arrows). (E–G) Hoeschst (E) and pSmad1/5/8 immunofluorescent staining (F, low power and G, high power of boxed region in F) of E11.5 frontal section through the heart of a wild type embryo. Extensive pSmad1/5/8 staining is seen in the ASP (arrow) and in the mesenchymal mass (arrowhead) that septates the AV canal.
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