Figure 1.
Sirenomelia in Bmp7−/−;Shh−/− and Bmp7−/−;Shh+/− compound mutants.
(A) Ventral view of the caudal body of a wild type newborn. (B) Severe sirenomelia typical of Bmp7−/−;Shh−/− mutants. Note the centrally positioned atrophic hindlimb and also the typical features of Shh mutants including proboscide, cyclopia and truncated forelimbs (insert in B). (C) Milder sirenomelia typical of Bmp7−/−;Shh+/− mutants in which the two hindlimbs can still be distinguished. (D and E) Bmp7−/−;Shh+/− neonates showing different degrees of fusion of the hindlimbs. (F) Bmp7−/−;Shh+/− neonate that does not display a clear sirenomelia but showing midline approximation of the hindlimbs. (G) Normal appearance of the genital tubercle and anus in a wild type neonate. (H) Dorsal view of a double homozygous mutant showing imperforate anus. (I) Imperforate anus and absence of genital tubercle in a Bmp7−/−;Shh+/− newborn. Note the presence of a practically normal tail in Bmp7−/−;Shh+/− newborns. Genotypes indicated at the top of the panels. Blue arrow points to the genital tubercle and the yellow arrow to the anus.
Figure 2.
Skeletal analysis of sirenomelia phenotypes at birth.
(A–C) Ventral views of Victoria Blue cartilage staining of representative wild type (A), Bmp7−/−;Shh−/− homozygous (B) and Bmp7−/−;Shh+/− mutant (C). Note the isquia, proximal femora (arrow) and fibula (f) fusion in double homozygous (B) and the medial position of the fibulae due to the absence of hindlimb rotation (B–C). (D and E) Ventral view, with corresponding scheme below, and lateral view of Alcian Blue and Alizarin Red stained pelvis of wild type (D) and Bmp7−/−;Shh+/− neonates (E). The abnormal morphology of the hip is characterized by medial approximation and fusion of the ischia, truncation of the pubes but normal sacrum (E). Abbreviations: f: femur; fi: fibula; il: illium; is: ischium; pu: pubis; t: tibia. The arrowheads in C point to the patellae.
Figure 3.
Visceral analysis in wild type and mutants.
(A–B) Autopsy preparations of abdominal viscera in newborns. Superficial (left panel), profound (medium panel) and corresponding scheme (right panel) are shown. Mutant embryos exhibit relatively normal stomach and midgut but hypoplastic kidneys and hindgut and absence of bladder. (C–D) H&E-stained mid sagittal sections of wild-type (C) and Bmp7−/−;Shh+/− mutants (D) at the embryonic stage indicated at the top. The yellow arrows indicate the hindgut and the blue arrows the urethra. Note the absence of urogenital duct and septum in mutants. Abbreviations: b: bladder; g: gonad and gonadal duct; hg: hindgut; k: kidney; u: urachus. The urogenital septum is indicated by an asterisk.
Figure 4.
Reduced Bmp7 and Shh signaling in the ventral caudal mesoderm of mutant embryos.
(A–E) Expression of Shh in whole mount ISH and corresponding transverse section (level indicated by bar) at E8.5 (A–B) and E9.5 (C–E). (F–J) Expression of Bmp7 by Bmp7-LacZ reporter in Bmp7+/− embryos at E8.5 (F–G) and E9.5 (H–J). (K) Cartoon showing the pattern of expression of Shh and Bmp7 at E9.5, the area influenced by both signals is encircled. (L–N) Expression of Shh (L), Ptc1 (M) and Msx2 (N) in transverse caudal sections of E9.5 wild type and Bmp7−/−;Shh+/− embryos as indicated at the top of each panel. Abbreviations: c: coelomic cavity; da: dorsal aorta; hg: hindgut; n: notochord; nt: neural tube; o: omphalomesenteric artery.
Figure 5.
Onset of the morphology defect.
Semithin (1 micron thick) transverse serial sections stained with toluidine blue of control and mutant embryos (genotype indicated on the left) at E8.5 (A–C) and E9.5 (D–F′). The level of the section is indicated in the pictures on the right. (A–C) Sections at the tip of the tailbud of E8.5 embryos. No differences between wild type and mutants are observed. (D–F′) Section at two different caudal levels, as indicated on the right. Note the narrower hindgut and the absence of the recurved portions of the dorsal aorta in mutants. (D″–F″) Schematic representation of the sections with the structures marked. Abbreviations: hg:hindgut; da: dorsal aortas; nt: neural tube; c: choelomic cavity; o: omphalomesenteric artery. The presence of red blood cells in the coelomic cavity of mutants is an artifact that sometimes occurs when dissecting the embryo disregarding the genotype.
Figure 6.
Expression of Cadherin5 in wild-type and mutant embryos at E9.5.
(A–E) Ventral views of the caudal region of E9.5 embryos hybridized for Cadh5. (A′–E′) Transverse sections of the hybridized embryos at the level indicated by the horizontal bar. The genotypes are indicted at the top. The red arrowheads point to the poorly local ventral mesoderm vascular plexus in Bmp7−/−;Shh+/− and Bmp7−/−;Shh−/− mutants.
Figure 7.
Spatial distribution of Islet1, Tbx4 and Pitx1 mRNA in wild-type and mutants.
(A–D) Islet1 expression in transverse sections through the caudal region of E9.5 (A–B) and 10.5 (C–D) wild type and mutant embryos. Note the strong downregulation of expression in the midline tissues and in the interlimb mesoderm of mutants. Note the absence of the recurved portions of the dorsal aortas in E10.5 mutant embryos. (E–H) Expression of Tbx4 (E–F) and Pitx1 (G–H) specific hindlimb markers in consecutive sections of E10.5 wild-type and mutant embryos showing the abnormal connection between both hindlimbs. Note the absence of the recurved portions of the aorta in mutants. Genotypes indicated on the left. Da: dorsal aorta; rda: recurved portion of aorta.
Figure 8.
Gene expression analysis in hindlimbs of wild-type and Bmp7−/−;Shh−/− littermates at E10.5.
The expression of limb bud markers is normal in mutant hindlimbs except for the merged phenotype. The domain of expression of Fgf8 (G), Bmp4 (H) and Msx2 (I) in the apical ectodermal ridge of both hindlimb is continuous because of the merging. The expression of Lmx1b reflects the abnormal position of the limbs (J–E).