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Defining the molecular pathologies in cloaca malformation: similarities between mouse and human.

Runck LA, Method A, Bischoff A, Levitt M, Peña A, Collins MH, Gupta A, Shanmukhappa S, Wells JM, Guasch G - Dis Model Mech (2014)

Bottom Line: The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca.Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling.These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.

ABSTRACT
Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.

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Molecular defects in the cloaca epithelium of Shh knockout embryos. (A–D) Whole-mount immunofluorescence showing early patterning of the wild-type (WT) cloaca epithelium in E10.5 (A), E11.5 (B), E12.5 (C) and E13.5 (D) embryos stained with Sox2 (red), keratin 8 (green) and FoxA2 (blue). A summary of the Sox2 staining is depicted below each staining. See also supplementary material Movie 1A,B. (E,F) Whole-mount immunostainings of E11.5 WT (E) and KO (F) embryos, showing a normal FoxA2 (blue) staining in the WT and KO, and a decrease or absence of keratin 8 (green) and Sox2 (red) expression in the KO cloaca compared with the WT. See also supplementary material Movie 2A,B. (G–J) Immunofluorescence analysis for the indicated markers of E11.5 WT and KO embryos. (G,H) WT cloaca epithelium (G) expresses keratin 19 (green) and Sox2 (red) at the distal region of the cloaca, in contrast to the KO, which shows no expression of keratin 19 or Sox2 (H). (I,J) CDX2 (red) is expressed at the proximal region of the cloaca epithelium in WT (I) and KO (J). All of the stainings have been performed on at least three WT and three KO littermates. A representative example for each antibody combination is shown. (K,L) H&E-stained sections of E11.5 WT (K) and Shh knockout (KO) (L) littermate embryos. (K) WT epithelium mostly consists of columnar epithelium, which, at two places, gradually transitions into pseudostratified columnar epithelium at two opposite ends. (L) The cloaca epithelium of Shh KO at E11.5 is predominantly pseudostratified columnar epithelium. Higher magnifications of the epithelium are shown in the inset. Scale bars: 100 μm (A), 50 μm (B–D), 5.000e4 μm (E,F), 20 μm (G–J), 10 μm (K,L), 5 μm (inset in K,L). The asterisk denotes autofluorescence. The dotted lines mark the epithelia. Arrows in E point to the nuclear expression of Sox2 and in I,J point to the nuclear expression of CDX2. Abbreviations: int, intestine; ec, ectoderm; cl, cloaca; hg, hindgut; ur, urethra; ugs, urogenital sinus.
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f1-0070483: Molecular defects in the cloaca epithelium of Shh knockout embryos. (A–D) Whole-mount immunofluorescence showing early patterning of the wild-type (WT) cloaca epithelium in E10.5 (A), E11.5 (B), E12.5 (C) and E13.5 (D) embryos stained with Sox2 (red), keratin 8 (green) and FoxA2 (blue). A summary of the Sox2 staining is depicted below each staining. See also supplementary material Movie 1A,B. (E,F) Whole-mount immunostainings of E11.5 WT (E) and KO (F) embryos, showing a normal FoxA2 (blue) staining in the WT and KO, and a decrease or absence of keratin 8 (green) and Sox2 (red) expression in the KO cloaca compared with the WT. See also supplementary material Movie 2A,B. (G–J) Immunofluorescence analysis for the indicated markers of E11.5 WT and KO embryos. (G,H) WT cloaca epithelium (G) expresses keratin 19 (green) and Sox2 (red) at the distal region of the cloaca, in contrast to the KO, which shows no expression of keratin 19 or Sox2 (H). (I,J) CDX2 (red) is expressed at the proximal region of the cloaca epithelium in WT (I) and KO (J). All of the stainings have been performed on at least three WT and three KO littermates. A representative example for each antibody combination is shown. (K,L) H&E-stained sections of E11.5 WT (K) and Shh knockout (KO) (L) littermate embryos. (K) WT epithelium mostly consists of columnar epithelium, which, at two places, gradually transitions into pseudostratified columnar epithelium at two opposite ends. (L) The cloaca epithelium of Shh KO at E11.5 is predominantly pseudostratified columnar epithelium. Higher magnifications of the epithelium are shown in the inset. Scale bars: 100 μm (A), 50 μm (B–D), 5.000e4 μm (E,F), 20 μm (G–J), 10 μm (K,L), 5 μm (inset in K,L). The asterisk denotes autofluorescence. The dotted lines mark the epithelia. Arrows in E point to the nuclear expression of Sox2 and in I,J point to the nuclear expression of CDX2. Abbreviations: int, intestine; ec, ectoderm; cl, cloaca; hg, hindgut; ur, urethra; ugs, urogenital sinus.

Mentions: Shh deficiency in mice results in cloaca malformation (Mo et al., 2001; Perriton et al., 2002; Seifert et al., 2009), although it is not known when defects in cloacal development are first apparent. Because little is known about cloacal development prior to septation, we generated a map of markers expressed during cloacal development from E10.5 through E13.5 (Fig. 1A–D). Because of the complex morphogenesis that accompanies the formation of cloacal derivatives, we analyzed the expression of markers using whole-mount immunofluorescence to generate three-dimensional images of the developing embryonic cloaca. We found that the cloaca at E10.5 expresses the simple-epithelium-type keratin 8 throughout the epithelium, whereas a population of cells positive for the transcription factor Sox2 is restricted to the dorsal side (Fig. 1A; supplementary material Fig. S1A;whole-mount supplementary material Movie 1A). Sox2 becomes more broadly expressed in the cloaca at E11.5 (Fig. 1B; supplementary material Fig. S1B) and will mark the future urethra and anal canal at later stages (Fig. 1C,D; supplementary material Fig. S1C,D; whole-mount supplementary material Movie 1B). Next, we analyzed molecularly and histologically this region in mouse at E11.5, before septation occurs and when the embryonic cloaca is still a cavity, to determine whether loss of Shh can affect differentiation of the cloaca epithelium. At that stage, Shh is strongly expressed in the wild-type (WT) cloaca epithelium (supplementary material Fig. S1E), in contrast to the knockout (supplementary material Fig. S1F). Molecularly, the WT embryonic cloaca epithelium was already compartmentalized, with the expression of Sox2 at the distal region (Fig. 1E,G) and the expression of CDX2 at the proximal region (Fig. 1I). We show that, in the Shh knockout epithelium, Sox2 is absent in the embryonic cloaca (Fig. 1F,H; whole-mount supplementary material Movie 2A,B), whereas CDX2 expression is unaffected (Fig. 1J). Moreover, the knockout embryonic cloaca epithelium shows an additional defect in keratin expression: keratin 8 is not uniformly expressed in these mutants, whereas it is in the WT (Fig. 1E compared with 1F; supplementary material Fig. S1G compared with S1H; whole-mount supplementary material Movie 2B), and keratin 19 is absent from the mutant epithelium (Fig. 1G compared with 1H). Histologically, whereas the WT cloaca epithelium is mostly comprised of columnar epithelium that, at two places, gradually transitions into pseudostratified columnar epithelium at two opposite ends (Fig. 1K), the Shh knockout cloaca shows predominantly pseudostratified columnar epithelium (Fig. 1L). These results indicate that Shh deficiency affects the molecular identity of the embryonic cloaca epithelium prior to septation.


Defining the molecular pathologies in cloaca malformation: similarities between mouse and human.

Runck LA, Method A, Bischoff A, Levitt M, Peña A, Collins MH, Gupta A, Shanmukhappa S, Wells JM, Guasch G - Dis Model Mech (2014)

Molecular defects in the cloaca epithelium of Shh knockout embryos. (A–D) Whole-mount immunofluorescence showing early patterning of the wild-type (WT) cloaca epithelium in E10.5 (A), E11.5 (B), E12.5 (C) and E13.5 (D) embryos stained with Sox2 (red), keratin 8 (green) and FoxA2 (blue). A summary of the Sox2 staining is depicted below each staining. See also supplementary material Movie 1A,B. (E,F) Whole-mount immunostainings of E11.5 WT (E) and KO (F) embryos, showing a normal FoxA2 (blue) staining in the WT and KO, and a decrease or absence of keratin 8 (green) and Sox2 (red) expression in the KO cloaca compared with the WT. See also supplementary material Movie 2A,B. (G–J) Immunofluorescence analysis for the indicated markers of E11.5 WT and KO embryos. (G,H) WT cloaca epithelium (G) expresses keratin 19 (green) and Sox2 (red) at the distal region of the cloaca, in contrast to the KO, which shows no expression of keratin 19 or Sox2 (H). (I,J) CDX2 (red) is expressed at the proximal region of the cloaca epithelium in WT (I) and KO (J). All of the stainings have been performed on at least three WT and three KO littermates. A representative example for each antibody combination is shown. (K,L) H&E-stained sections of E11.5 WT (K) and Shh knockout (KO) (L) littermate embryos. (K) WT epithelium mostly consists of columnar epithelium, which, at two places, gradually transitions into pseudostratified columnar epithelium at two opposite ends. (L) The cloaca epithelium of Shh KO at E11.5 is predominantly pseudostratified columnar epithelium. Higher magnifications of the epithelium are shown in the inset. Scale bars: 100 μm (A), 50 μm (B–D), 5.000e4 μm (E,F), 20 μm (G–J), 10 μm (K,L), 5 μm (inset in K,L). The asterisk denotes autofluorescence. The dotted lines mark the epithelia. Arrows in E point to the nuclear expression of Sox2 and in I,J point to the nuclear expression of CDX2. Abbreviations: int, intestine; ec, ectoderm; cl, cloaca; hg, hindgut; ur, urethra; ugs, urogenital sinus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f1-0070483: Molecular defects in the cloaca epithelium of Shh knockout embryos. (A–D) Whole-mount immunofluorescence showing early patterning of the wild-type (WT) cloaca epithelium in E10.5 (A), E11.5 (B), E12.5 (C) and E13.5 (D) embryos stained with Sox2 (red), keratin 8 (green) and FoxA2 (blue). A summary of the Sox2 staining is depicted below each staining. See also supplementary material Movie 1A,B. (E,F) Whole-mount immunostainings of E11.5 WT (E) and KO (F) embryos, showing a normal FoxA2 (blue) staining in the WT and KO, and a decrease or absence of keratin 8 (green) and Sox2 (red) expression in the KO cloaca compared with the WT. See also supplementary material Movie 2A,B. (G–J) Immunofluorescence analysis for the indicated markers of E11.5 WT and KO embryos. (G,H) WT cloaca epithelium (G) expresses keratin 19 (green) and Sox2 (red) at the distal region of the cloaca, in contrast to the KO, which shows no expression of keratin 19 or Sox2 (H). (I,J) CDX2 (red) is expressed at the proximal region of the cloaca epithelium in WT (I) and KO (J). All of the stainings have been performed on at least three WT and three KO littermates. A representative example for each antibody combination is shown. (K,L) H&E-stained sections of E11.5 WT (K) and Shh knockout (KO) (L) littermate embryos. (K) WT epithelium mostly consists of columnar epithelium, which, at two places, gradually transitions into pseudostratified columnar epithelium at two opposite ends. (L) The cloaca epithelium of Shh KO at E11.5 is predominantly pseudostratified columnar epithelium. Higher magnifications of the epithelium are shown in the inset. Scale bars: 100 μm (A), 50 μm (B–D), 5.000e4 μm (E,F), 20 μm (G–J), 10 μm (K,L), 5 μm (inset in K,L). The asterisk denotes autofluorescence. The dotted lines mark the epithelia. Arrows in E point to the nuclear expression of Sox2 and in I,J point to the nuclear expression of CDX2. Abbreviations: int, intestine; ec, ectoderm; cl, cloaca; hg, hindgut; ur, urethra; ugs, urogenital sinus.
Mentions: Shh deficiency in mice results in cloaca malformation (Mo et al., 2001; Perriton et al., 2002; Seifert et al., 2009), although it is not known when defects in cloacal development are first apparent. Because little is known about cloacal development prior to septation, we generated a map of markers expressed during cloacal development from E10.5 through E13.5 (Fig. 1A–D). Because of the complex morphogenesis that accompanies the formation of cloacal derivatives, we analyzed the expression of markers using whole-mount immunofluorescence to generate three-dimensional images of the developing embryonic cloaca. We found that the cloaca at E10.5 expresses the simple-epithelium-type keratin 8 throughout the epithelium, whereas a population of cells positive for the transcription factor Sox2 is restricted to the dorsal side (Fig. 1A; supplementary material Fig. S1A;whole-mount supplementary material Movie 1A). Sox2 becomes more broadly expressed in the cloaca at E11.5 (Fig. 1B; supplementary material Fig. S1B) and will mark the future urethra and anal canal at later stages (Fig. 1C,D; supplementary material Fig. S1C,D; whole-mount supplementary material Movie 1B). Next, we analyzed molecularly and histologically this region in mouse at E11.5, before septation occurs and when the embryonic cloaca is still a cavity, to determine whether loss of Shh can affect differentiation of the cloaca epithelium. At that stage, Shh is strongly expressed in the wild-type (WT) cloaca epithelium (supplementary material Fig. S1E), in contrast to the knockout (supplementary material Fig. S1F). Molecularly, the WT embryonic cloaca epithelium was already compartmentalized, with the expression of Sox2 at the distal region (Fig. 1E,G) and the expression of CDX2 at the proximal region (Fig. 1I). We show that, in the Shh knockout epithelium, Sox2 is absent in the embryonic cloaca (Fig. 1F,H; whole-mount supplementary material Movie 2A,B), whereas CDX2 expression is unaffected (Fig. 1J). Moreover, the knockout embryonic cloaca epithelium shows an additional defect in keratin expression: keratin 8 is not uniformly expressed in these mutants, whereas it is in the WT (Fig. 1E compared with 1F; supplementary material Fig. S1G compared with S1H; whole-mount supplementary material Movie 2B), and keratin 19 is absent from the mutant epithelium (Fig. 1G compared with 1H). Histologically, whereas the WT cloaca epithelium is mostly comprised of columnar epithelium that, at two places, gradually transitions into pseudostratified columnar epithelium at two opposite ends (Fig. 1K), the Shh knockout cloaca shows predominantly pseudostratified columnar epithelium (Fig. 1L). These results indicate that Shh deficiency affects the molecular identity of the embryonic cloaca epithelium prior to septation.

Bottom Line: The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca.Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling.These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.

ABSTRACT
Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.

Show MeSH
Related in: MedlinePlus