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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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The common channel of Shh mutant mice shows differentiation defects resulting in an indeterminate epithelium. (A–H) Immunofluorescence analysis of WT and Shh knockout (KO) E18.5 embryo for the indicated markers. (A,B) Keratin 5 (green) marks the anal epithelium in WT (A) and the distal common channel in KO (B). CDX2 (red) is absent in the distal common channel (B). (C,D) Sox2 (red) marks the anal epithelium in WT as denoted by the white arrow (C) and is absent in the distal common channel (D). Keratin 19 (green) marks the WT rectum (C) and the suprabasal layer of the distal common channel (D). (E,F) Keratin 8 (green) labels the rectum epithelium in WT embryo (E) and the distal common channel in KO (F), whereas keratin 7 (red), normally found in urothelium, is absent in the distal common channel. (G,H) FoxA2 (red) marks the anal transition zone and the rectal epithelium in the E18.5 WT embryo (G) and the proximal common channel in the KO, but is absent in the distal region (H). 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. The dotted lines mark the epithelia. Abbreviations: RE, rectal epithelium; AE, anal epithelium; CC, common channel; TZ, transition zone. All scale bars: 20 μm. The asterisk denotes autofluorescence.
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f4-0070483: The common channel of Shh mutant mice shows differentiation defects resulting in an indeterminate epithelium. (A–H) Immunofluorescence analysis of WT and Shh knockout (KO) E18.5 embryo for the indicated markers. (A,B) Keratin 5 (green) marks the anal epithelium in WT (A) and the distal common channel in KO (B). CDX2 (red) is absent in the distal common channel (B). (C,D) Sox2 (red) marks the anal epithelium in WT as denoted by the white arrow (C) and is absent in the distal common channel (D). Keratin 19 (green) marks the WT rectum (C) and the suprabasal layer of the distal common channel (D). (E,F) Keratin 8 (green) labels the rectum epithelium in WT embryo (E) and the distal common channel in KO (F), whereas keratin 7 (red), normally found in urothelium, is absent in the distal common channel. (G,H) FoxA2 (red) marks the anal transition zone and the rectal epithelium in the E18.5 WT embryo (G) and the proximal common channel in the KO, but is absent in the distal region (H). 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. The dotted lines mark the epithelia. Abbreviations: RE, rectal epithelium; AE, anal epithelium; CC, common channel; TZ, transition zone. All scale bars: 20 μm. The asterisk denotes autofluorescence.

Mentions: To help further determine the origin of the indeterminate epithelium in mice and humans, we used markers of each type of epithelium: stratified squamous (anal and vaginal epithelium), simple (rectum) and transitional. In mouse, keratin 5 is found in normal stratified squamous epithelium with keratinization (anal canal) (Fig. 4A) and the simple-epithelium-type cytokeratins keratin 8 and 19 are found in normal rectum (Fig. 4C,E). Interestingly, both the stratified-epithelium-type keratin 5 and the simple-epithelium-type keratin 8 and 19 are expressed in the common channel of Shh knockout mice (Fig. 4B,D,F). Keratin 7, normally present in urethra, is not expressed in the common channel in these knockouts (Fig. 4F). Moreover, Sox2 is normally present in the anal canal of WT mice (Fig. 4C) but is absent from the common channel in the knockouts (Fig. 4D). Finally, the distal common channel did not express the endodermal marker FoxA2, normally present in the anal transition zone (Fig. 4G), whereas it was expressed in the proximal part in the knockouts (Fig. 4H), which suggests that this epithelium has undergone maturation and specification. These data indicate that the common channel in Shh deficient mice does not share the molecular characteristics of a normal anal canal, rectum, vagina or urethra.


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)

The common channel of Shh mutant mice shows differentiation defects resulting in an indeterminate epithelium. (A–H) Immunofluorescence analysis of WT and Shh knockout (KO) E18.5 embryo for the indicated markers. (A,B) Keratin 5 (green) marks the anal epithelium in WT (A) and the distal common channel in KO (B). CDX2 (red) is absent in the distal common channel (B). (C,D) Sox2 (red) marks the anal epithelium in WT as denoted by the white arrow (C) and is absent in the distal common channel (D). Keratin 19 (green) marks the WT rectum (C) and the suprabasal layer of the distal common channel (D). (E,F) Keratin 8 (green) labels the rectum epithelium in WT embryo (E) and the distal common channel in KO (F), whereas keratin 7 (red), normally found in urothelium, is absent in the distal common channel. (G,H) FoxA2 (red) marks the anal transition zone and the rectal epithelium in the E18.5 WT embryo (G) and the proximal common channel in the KO, but is absent in the distal region (H). 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. The dotted lines mark the epithelia. Abbreviations: RE, rectal epithelium; AE, anal epithelium; CC, common channel; TZ, transition zone. All scale bars: 20 μm. The asterisk denotes autofluorescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974458&req=5

f4-0070483: The common channel of Shh mutant mice shows differentiation defects resulting in an indeterminate epithelium. (A–H) Immunofluorescence analysis of WT and Shh knockout (KO) E18.5 embryo for the indicated markers. (A,B) Keratin 5 (green) marks the anal epithelium in WT (A) and the distal common channel in KO (B). CDX2 (red) is absent in the distal common channel (B). (C,D) Sox2 (red) marks the anal epithelium in WT as denoted by the white arrow (C) and is absent in the distal common channel (D). Keratin 19 (green) marks the WT rectum (C) and the suprabasal layer of the distal common channel (D). (E,F) Keratin 8 (green) labels the rectum epithelium in WT embryo (E) and the distal common channel in KO (F), whereas keratin 7 (red), normally found in urothelium, is absent in the distal common channel. (G,H) FoxA2 (red) marks the anal transition zone and the rectal epithelium in the E18.5 WT embryo (G) and the proximal common channel in the KO, but is absent in the distal region (H). 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. The dotted lines mark the epithelia. Abbreviations: RE, rectal epithelium; AE, anal epithelium; CC, common channel; TZ, transition zone. All scale bars: 20 μm. The asterisk denotes autofluorescence.
Mentions: To help further determine the origin of the indeterminate epithelium in mice and humans, we used markers of each type of epithelium: stratified squamous (anal and vaginal epithelium), simple (rectum) and transitional. In mouse, keratin 5 is found in normal stratified squamous epithelium with keratinization (anal canal) (Fig. 4A) and the simple-epithelium-type cytokeratins keratin 8 and 19 are found in normal rectum (Fig. 4C,E). Interestingly, both the stratified-epithelium-type keratin 5 and the simple-epithelium-type keratin 8 and 19 are expressed in the common channel of Shh knockout mice (Fig. 4B,D,F). Keratin 7, normally present in urethra, is not expressed in the common channel in these knockouts (Fig. 4F). Moreover, Sox2 is normally present in the anal canal of WT mice (Fig. 4C) but is absent from the common channel in the knockouts (Fig. 4D). Finally, the distal common channel did not express the endodermal marker FoxA2, normally present in the anal transition zone (Fig. 4G), whereas it was expressed in the proximal part in the knockouts (Fig. 4H), which suggests that this epithelium has undergone maturation and specification. These data indicate that the common channel in Shh deficient mice does not share the molecular characteristics of a normal anal canal, rectum, vagina or urethra.

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