Transgenically ectopic expression of Bmp4 to the Msx1 mutant dental mesenchyme restores downstream gene expression but represses Shh and Bmp2 in the enamel knot of wild type tooth germ

Bmp4 is a downstream gene of Msx1 in early mouse tooth development. In this study, we introduced the Msx1-Bmp4 transgenic allele to the Msx1 mutants in which tooth development is arrested at the bud stage in an effort of rescuing Msx1 mutant tooth phenotype in vivo. Ectopic expression of a Bmp4 transgene driven by the mouse Msx1promoter in the dental mesenchyme restored the expression of Lef-1 and Dlx2 but neither Fgf3 nor syndecan-1 in the Msx1 mutant molar tooth germ. The mutant phenotype of molar but not incisor could be partially rescued to progress to the cap stage. The Msx1-Bmp4 transgene was also able to rescue the alveolar processes and the neonatal lethality of the Msx1 mutants. In contrast, overexpression of Bmp4 in the wild type molar mesenchyme down-regulated Shh and Bmp2 expression in the enamel knot, the putative signaling center for tooth patterning, but did not produce a tooth phenotype. These results indicate that Bmp4 can bypass Msx1 function to partially rescue molar tooth development in vivo, and to support alveolar process formation. Expression of Shh and Bmp2 in the enamel knot may not represent critical signals for tooth patterning.     

Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis

Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the anterior foregut where the tracheal primordium originates and targeted ablation of Bmp4 (Bmp4^cko) resulted in a loss-of-trachea phenotype that closely resembles the Floyd type II pathology, the most common form of TA in humans. In Bmp4^cko embryos, tracheal specification was not affected; however, its outgrowth was severely impaired due to reduced epithelial and mesenchymal proliferation. In agreement, we also observed significant reduction in the expression of Cyclin D1, a key cell cycle regulator associated with cellular proliferation. However, the proliferative effect of Bmp signaling appears to be independent of Wnt signaling. Interestingly, we found significantly reduced expression of activated extracellular signal-regulated kinase (Erk) in the Bmp4^cko ventral foregut, suggesting that Bmp signaling promotes Erk phosphorylation which has been associated with cellular proliferation. This study provides the first evidence linking Bmp signaling to tracheal formation by regulating the proliferative response of the anterior ventral foregut. Our finding sheds light on human tracheal malformations by providing a novel mouse model implicating Bmp signaling, non-canonical Erk activation and cellular proliferation.     

HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis

In humans and mice, loss of HOXA13 function causes defects in the growth and patterning of the digits and interdigital tissues. Analysis of Hoxa13 expression reveals a pattern of localization overlapping with sites of reduced Bmp2 and Bmp7 expression in Hoxa13 mutant limbs. Biochemical analyses identified a novel series of Bmp2 and Bmp7 enhancer regions that directly interact with the HOXA13 DNA-binding domain and activate gene expression in the presence of HOXA13. Immunoprecipitation of HOXA13-Bmp2 and HOXA13-Bmp7 enhancer complexes from the developing autopod confirm that endogenous HOXA13 associates with these regions. Exogenous application of BMP2 or BMP7 partially rescues the Hoxa13 mutant limb phenotype, suggesting that decreased BMP signaling contributes to the malformations present in these tissues. Together, these results provide conclusive evidence that HOXA13 regulates Bmp2 and Bmp7 expression, providing a mechanistic link between HOXA13, its target genes and the specific developmental processes affected by loss of HOXA13 function.     

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.     

Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.     

Morphogenesis of the trachea and esophagus: current players and new roles for noggin and Bmps

The development of the anterior foregut of the mammalian embryo involves changes in the behavior of both the epithelial endoderm and the adjacent mesoderm. Morphogenetic processes that occur include the extrusion of midline notochord cells from the epithelial definitive endoderm, the folding of the endoderm into a foregut tube, and the subsequent separation of the foregut tube into trachea and esophagus. Defects in foregut morphogenesis underlie the constellation of human birth defects known as esophageal atresia (EA) and tracheoesophageal fistula (TEF). Here, we review what is known about the cellular events in foregut morphogenesis and the gene mutations associated with EA and TEF in mice and humans. We present new evidence that about 70% of mouse embryos homozygous null for Nog, the gene encoding noggin, a bone morphogenetic protein (Bmp) antagonist, have EA/TEF as well as defects in lung branching. This phenotype appears to correlate with abnormal morphogenesis of the notochord and defects in its separation from the definitive endoderm. The abnormalities in foregut and lung morphogenesis of Nog null mutant can be rescued by reducing the gene dose of Bmp4 by 50%. This suggests that normal foregut morphogenesis requires that the level of Bmp4 activity is carefully controlled by means of antagonists such as noggin. Several mechanisms are suggested for how Bmps normally function, including by regulating the intercellular adhesion and behavior of notochord and foregut endoderm cells. Future research must determine how Noggin/Bmp antagonism fits into the network of other factors known to regulate tracheal and esophagus development, both in mouse or humans.     

glypican-3 controls cellular responses to Bmp4 in limb patterning and skeletal development

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.     

BMP7 acts in murine lens placode development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.     

Essential role of Bmp7 (snailhouse) and its prodomain in dorsoventral patterning of the zebrafish embryo

Bone morphogenetic proteins (Bmps) are signaling molecules that have been implicated in a variety of inductive processes. We report here that zebrafish Bmp7 is disrupted in snailhouse (snh) mutants. The allele snh(st1) is a translocation deleting the bmp7 gene, while snh(ty68) displays a Val->Gly exhange in a conserved motif of the Bmp7 prodomain. The snh(ty68) mutation is temperature-sensitive, leading to severalfold reduced activity of mutant Bmp7 at 28 degrees C and non-detectable activity at 33 degrees C. This prodomain lesion affects secretion and/or stability of secreted mature Bmp7 after processing has occurred. Both snh(st1) and snh(ty68) mutant zebrafish embryos are strongly dorsalized, indicating that bmp7 is required for the specification of ventral cell fates during early dorsoventral patterning. At higher temperature, the phenotype of snh(ty68) mutant embryos is identical to that caused by the amorphic bmp2b mutation swirl swr(ta72) and similar to that caused by the smad5 mutation somitabun sbn(dtc24). mRNA injection studies and double mutant analyses indicate that Bmp2b and Bmp7 closely cooperate and that Bmp2b/Bmp7 signaling is transduced by Smad5 and antagonized by Chordino.     

Digit regeneration is regulated by Msx1 and BMP4 in fetal mice

The regeneration of digit tips in mammals, including humans and rodents, represents a model for organ regeneration in higher vertebrates. We had previously characterized digit tip regeneration during fetal and neonatal stages of digit formation in the mouse and found that regenerative capability correlated with the expression domain of the Msx1 gene. Using the stage 11 (E14.5) digit, we now show that digit tip regeneration occurs in organ culture and that Msx1, but not Msx2, mutant mice display a regeneration defect. Associated with this phenotype, we find that Bmp4 expression is downregulated in the Msx1 mutant digit and that mutant digit regeneration can be rescued in a dose-dependent manner by treatment with exogenous BMP4. Studies with the BMP-binding protein noggin show that wild-type digit regeneration is inhibited without inhibiting the expression of Msx1, Msx2 or Bmp4. These data identify a signaling pathway essential for digit regeneration, in which Msx1 functions to regulate BMP4 production. We also provide evidence that endogenous Bmp4 expression is regulated by the combined activity of Msx1 and Msx2 in the forming digit tip; however, we discovered a compensatory Msx2 response that involves an expansion into the wild-type Msx1 domain. Thus, although both Msx1 and Msx2 function to regulate Bmp4 expression in the digit tip, the data are not consistent with a model in which Msx1 and Msx2 serve completely redundant functions in the regeneration response. These studies provide the first functional analysis of mammalian fetal digit regeneration and identify a new function for Msx1 and BMP4 as regulators of the regenerative response.     

Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia

In humans and mice, mutations in Hoxa13 cause malformation of limb and genitourinary (GU) regions. In males, one of the most common GU malformations associated with loss of Hoxa13 function is hypospadia, a condition defined by the poor growth and closure of the urethra and glans penis. By examining early signaling in the developing mouse genital tubercle, we show that Hoxa13 is essential for normal expression of Fgf8 and Bmp7 in the urethral plate epithelium. In Hoxa13(GFP)-mutant mice, hypospadias occur as a result of the combined loss of Fgf8 and Bmp7 expression in the urethral plate epithelium, as well as the ectopic expression of noggin (Nog) in the flanking mesenchyme. In vitro supplementation with Fgf8 restored proliferation in homozygous mutants to wild-type levels, suggesting that Fgf8 is sufficient to direct early proliferation of the developing genital tubercle. However, the closure defects of the distal urethra and glans can be attributed to a loss of apoptosis in the urethra, which is consistent with reduced Bmp7 expression in this region. Mice mutant for Hoxa13 also exhibit changes in androgen receptor expression, providing a developmental link between Hoxa13-associated hypospadias and those produced by antagonists to androgen signaling. Finally, a novel role for Hoxa13 in the vascularization of the glans penis is also identified.     

Lrp4 Regulates Initiation of Ureteric Budding and Is Crucial for Kidney Formation – A Mouse Model for Cenani-Lenz Syndrome

Background

Development of the kidney is initiated when the ureteric bud (UB) branches from the Wolffian duct and invades the overlying metanephric mesenchyme (MM) triggering the mesenchymal/epithelial interactions that are the basis of organ formation. Multiple signaling pathways must be integrated to ensure proper timing and location of the ureteric bud formation.

Methods and Principal Findings

We have used gene targeting to create an Lrp4 null mouse line. The mutation results in early embryonic lethality with a subpenetrant phenotype of kidney agenesis. Ureteric budding is delayed with a failure to stimulate the metanephric mesenchyme in a timely manner, resulting in failure of cellular differentiation and resulting absence of kidney formation in the mouse as well as comparable malformations in humans with Cenani-Lenz syndrome.

Conclusion

Lrp4 is a multi-functional receptor implicated in the regulation of several molecular pathways, including Wnt and Bmp signaling. Lrp4^−/− mice show a delay in ureteric bud formation that results in unilateral or bilateral kidney agenesis. These data indicate that Lrp4 is a critical regulator of UB branching and lack of Lrp4 results in congenital kidney malformations in humans and mice.     

Distinct developmental programs require different levels of Bmp signaling during mouse retinal development

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, we have used a Cre-loxP strategy to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina.     

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4^flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4^flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.     

Zili antagonizes Bmp signaling to regulate dorsal-ventral patterning during zebrafish early embryogenesis

Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-βsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.     

Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4 activity during forebrain development

Twisted gastrulation (Tsg) is a secreted protein that regulates Bmp signaling in the extracellular space through its direct interaction with Bmp/Dpp and Chordin (Chd)/Short gastrulation (Sog). The ternary complex of Tsg/Chd/Bmp is cleaved by the metalloprotease Tolloid (Tld)/Xolloid (Xld). Studies in Drosophila, Xenopus and zebrafish suggest that Tsg can act both as an anti-Bmp and as a pro-Bmp. We have analyzed Tsg loss-of-function in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. We provide evidence that Tsg interacts genetically with Bmp4. When only one copy of Bmp4 is present, a requirement of Tsg for embryonic development is revealed. Tsg-/-;Bmp4+/- compound mutants die at birth and display holoprosencephaly, first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development.     

Bmp7 functions via a polarity mechanism to promote cloacal septation

Background

During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood.

Methodology/Principal Findings

We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse.

Conclusion/Significance

Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.     

Noggin regulates Bmp4 activity during pituitary induction

Bone morphogenetic protein (Bmp) signaling is critical for the development and patterning of the mouse pituitary from the initial induction of Rathke's pouch to cell specification in the anterior lobe. We examined the regulation of Bmp signaling during pituitary development by analyzing null embryos for noggin, a Bmp 2 and 4 antagonist. Noggin is expressed in the ventral diencephalon during Rathke's pouch induction, in the underlying cartilage plate during cell specification and in the adult anterior pituitary gland. Noggin null embryos have a variable pituitary phenotype, which ranges from a rostrally displaced Rathke's pouch to induction of secondary pituitary tissue. While cell specification in the anterior pituitary appears normal, patterning in the ventral diencephalon is disrupted; Bmp4 activity is expanded resulting in Fibroblast growth factor 10 repression and in a rostral shift in the boundary between the Bmp4 and Sonic hedgehog expression domains. The expanded domain of Bmp4 activity also results in additional invaginations of oral ectoderm and can shift the position of Rathke's pouch or create secondary pituitary tissue. This work demonstrates the importance of attenuating the activity of Bmp signaling during pituitary induction in order to maintain the proper balance of signaling factors necessary for pituitary organogenesis.     

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

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.KEY WORDS: Anorectal malformation, Cloaca, Patterning, Epithelial differentiation, Sonic hedgehog