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Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development., (© 2024. The Author(s).)

Congenital anomalies of external genitalia affect approximately 1 in 125 live male births. Development of the genital tubercle, the precursor of the penis and clitoris, is regulated by the urethral plate epithelium, an endodermal signaling center. Signaling activity of the urethral plate is mediated by Sonic hedgehog (SHH), which coordinates outgrowth and patterning of the genital tubercle by controlling cell cycle kinetics and expression of downstream genes. The mechanisms that govern Shh transcription in urethral plate cells are largely unknown. Here we show that deletion of Foxa1 and Foxa2 results in persistent cloaca, an incomplete separation of urinary, genital, and anorectal tracts, and severe hypospadias, a failure of urethral tubulogenesis. Loss of Foxa2 and only one copy of Foxa1 results in urethral fistula, an additional opening of the penile urethra. Foxa1/a2 participate in an autoregulatory feedback loop with Shh, in which FOXA1 and FOXA2 positively regulate transcription of Shh in the urethra, and SHH feeds back to negatively regulate Foxa1 and Foxa2 expression. These findings reveal novel roles for Foxa genes in development of the urethral tube and in division of the embryonic cloaca., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cloacal malformations are characterized by the confluence of the lower urinary tract, the female reproductive tract, and the rectum to create a common channel with a single opening on the perineum. The presence of a cloaca is a normal phase of early human embryological development. Between the 4 th and 7 th weeks of gestation, the cloaca undergoes subdivision to form the hindgut and urogenital sinus. Failure of this process results in the congenital anomaly termed persistent cloaca (PC). The term urorectal septum malformation sequence (URSMS) is also used to describe this anomaly. The classic description of this process which is still cited in many standard textbooks dates from the 19 th century. However, this has been increasingly called into question by the findings of studies using modern scientific methodology. Urogenital sinus anomalies are defined by the confluence of the urethra and vagina to form a common channel of varying length with a single perineal opening. In this condition, the anorectal canal opens separately on the perineum. The presence of a urogenital sinus represents a transient phase of the normal development of the lower genital tract in the female fetus. However, the form of urogenital sinus most commonly encountered in the developed world is a feature of disordered sexual differentiation and does not arise simply from the persistence of the anatomical structure which is a feature of normal fetal development., Competing Interests: None

Subdivision of cloaca into urogenital and anorectal passages has remained controversial because of disagreements about the identity and role of the septum developing between both passages. This study aimed to clarify the development of the cloaca using a quantitative 3D morphological approach in human embryos of 4-10 post-fertilisation weeks. Embryos were visualised with Amira 3D-reconstruction and Cinema 4D-remodelling software. Distances between landmarks were computed with Amira3D software. Our main finding was a pronounced difference in growth between rapidly expanding central and ventral parts, and slowly or non-growing cranial and dorsal parts. The entrance of the Wolffian duct into the cloaca proved a stable landmark that remained linked to the position of vertebra S3. Suppressed growth in the cranial cloaca resulted in an apparent craniodorsal migration of the entrance of the Wolffian duct, while suppressed growth in the dorsal cloaca changed the entrance of the hindgut from cranial to dorsal on the cloaca. Transformation of this 'end-to-end' into an 'end-to-side' junction produced temporary 'lateral (Rathke's) folds'. The persistent difference in dorsoventral growth straightened the embryonic caudal body axis and concomitantly extended the frontally oriented 'urorectal (Tourneux's) septum' caudally between the ventral urogenital and dorsal anorectal parts of the cloaca. The dorsoventral growth difference also divided the cloacal membrane into a well-developed ventral urethral plate and a thin dorsal cloacal membrane proper, which ruptured at 6.5 weeks. The expansion of the pericloacal mesenchyme followed the dorsoventral growth difference and produced the genital tubercle. Dysregulation of dorsal cloacal development is probably an important cause of anorectal malformations: too little regressive development may result in anorectal agenesis, and too much regression in stenosis or atresia of the remaining part of the dorsal cloaca., (© 2018 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

The epithelial Wolffian duct (WD) inserts into the cloaca (primitive bladder) before metanephric kidney development, thereby establishing the initial plumbing for eventual joining of the ureters and bladder. Defects in this process cause common anomalies in the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). However, developmental, cellular, and molecular mechanisms of WD-cloaca fusion are poorly understood. Through systematic analysis of early WD tip development in mice, we discovered that a novel process of spatiotemporally regulated apoptosis in WD and cloaca was necessary for WD-cloaca fusion. Aberrant RET tyrosine kinase signaling through tyrosine (Y) 1062, to which PI3K- or ERK-activating proteins dock, or Y1015, to which PLC γ docks, has been shown to cause CAKUT-like defects. Cloacal apoptosis did not occur in RetY1062F mutants, in which WDs did not reach the cloaca, or in RetY1015F mutants, in which WD tips reached the cloaca but did not fuse. Moreover, inhibition of ERK or apoptosis prevented WD-cloaca fusion in cultures, and WD-specific genetic deletion of YAP attenuated cloacal apoptosis and WD-cloacal fusion in vivo Thus, cloacal apoptosis requires direct contact and signals from the WD tip and is necessary for WD-cloacal fusion. These findings may explain the mechanisms of many CAKUT., (Copyright © 2018 by the American Society of Nephrology.)

The cloaca is an embryonic cavity that is divided into the urogenital sinus and rectum upon differentiation of the cloacal epithelium triggered by tissue-specific transcription factors including CDX2. Defective differentiation leads to persistent cloaca in humans (PC), a phenotype recapitulated in Cdx2 mutant mice. PC is linked to hypo/hyper-vitaminosis A. Although no gene has ever been identified, there is a strong evidence for a genetic contribution to PC. We applied whole-exome sequencing and copy-number-variants analyses to 21 PC patients and their unaffected parents. The damaging p.Cys132* and p.Arg237His de novo CDX2 variants were identified in two patients. These variants altered the expression of CYP26A1, a direct CDX2 target encoding the major retinoic acid (RA)-degrading enzyme. Other RA genes, including the RA-receptor alpha, were also mutated. Genes governing the development of cloaca-derived structures were recurrently mutated and over-represented in the basement-membrane components set (q-value < 1.65 × 10-6). Joint analysis of the patients' profile highlighted the extracellular matrix-receptor interaction pathway (MsigDBID: M7098, FDR: q-value < 7.16 × 10-9). This is the first evidence that PC is genetic, with genes involved in the RA metabolism at the lead. Given the CDX2 de novo variants and the role of RA, our observations could potentiate preventive measures. For the first time, a gene recapitulating PC in mouse models is found mutated in humans., (© The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This study examined the expression patterns of proprotein convertase subtilisin/kexin type 5 (Pcsk5) during anorectal development in normal and anorectal malformations (ARM) rat embryos, determine the possible role of Pcsk5 in the pathogenesis of ARM. An ARM rat model was developed by the administration of ethylenethiourea gestational day 10 (GD10). Embryos were harvested by surgical excision from GD13 to GD16, and the spatiotemporal expression of Pcsk5 was evaluated, using immunohistochemistry staining, Western blotting and real time RT-PCR. Immunohistochemistry staining in normal embryos revealed that Pcsk5 was abundantly expressed on the epithelium of the cloaca (CL) on GD13. On GD14 and GD15, positive cells were noted on the urorectal septum and the thin anal membrane. However, the epithelium of the CL of ARM embryos only faintly expressed Pcsk5 from GD13 to GD15. Western blotting and real time RT-PCR showed time-dependent increase of Pcsk5 expression in the developing hindgut. Pcsk5 expression levels were lower in the ARM group from GD14 to GD16 (p ≤ 0.05). These results indicate that downregulation of Pcsk5 during cloaca development into the rectum and urethra might be related to the formation of ARMs.

Septation of the cloaca is a unique mammalian adaptation that required a novel reorganization of the perineum-the caudal portion of the trunk body wall not associated with the hindlimb. Fish, the basal vertebrates, separate ventrolateral body wall musculature of the trunk into two discrete layers, while most tetrapods expand this pattern in the thorax and abdomen into four. Mammals, the only vertebrate group to divide the cloaca into urogenital and anorectal portions, exhibit complex muscle morphology in the perineum. Here we describe how perineal morphology in a broad sample of mammals fits into patterning of trunk musculature as an extension of the four-layer ventrolateral muscular patterning of the thorax and abdomen. We show that each perineal muscle layer has a specific function related to structures formed by cloacal septation. From superficial to deep, there is the subcutaneous layer, which regulates orifice closure, the external layer, which supplements both erectile and micturition function, the internal layer, which provides primary micturition and defecation regulation, and the transversus layer, which provides structural support for pelvic organs. We elucidate how the four-layer body wall pattern, restricted to the non-mammal tetrapod thorax and abdomen, is observed in the mammalian perineum to regulate function of unique perineal structures derived from cloacal septation.

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals., (© 2017 Society for Reproduction and Fertility.)

The developmental process through which the cloaca transforms from one hollow structure to two separated urinary and digestive outlets remains controversial and speculative. Here, we use high-resolution episcopic microscopy to examine a comprehensive series of normal and mutant mouse cloaca in which the detailed 3-dimensional (3-D) morphological features are illuminated throughout the development. We provide evidence that the dorsal peri-cloacal mesenchyme (dPCM) remains stationary while other surrounding tissues grow towards it. This causes dramatic changes of spatial relationship among caudal structures and morphological transformation of the cloaca. The 3-D characterizations of Dkk1 mutants reveal a hyperplastic defect of dPCM, which leads to a significant anterior shift of the caudal boundary of the cloaca, premature occlusion of the cloaca and, imperforate anus phenotype. Conversely, Shh knockout causes a severe hypoplastic defect of cloaca mesenchyme including dPCM and persistent cloaca. Collectively, these findings suggest that formation of the dPCM is critical for cloacal morphogenesis and furthermore, growth and movement of the mesenchymal tissues towards the dPCM lead to the cloaca occlusion and separation of the urinary and digestive outlets., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT-pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT-pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The division of the embryonic cloaca is the most essential event for the formation of digestive and urinary tracts. The defective development of the cloaca results in anorectal malformations (ARMs; 2-5 per 10,000 live births). However, the developmental and pathogenic mechanisms of ARMs are unclear. In the current study, we visualized the epithelia in the developing cloaca and nephric ducts (NDs). Systemic stereoscopic analyses revealed that the ND-cloaca connection sites shifted from the lateral-middle to dorsal-anterior part of the cloaca during cloacal division from E10.5 to E11.5 in mouse embryos. Genetic cell labeling analyses revealed that the cells in the ventral cloacal epithelium in the early stages rarely contributed to the dorsal part. Moreover, we revealed the possible morphogenetic movement of endodermal cells within the anterior part of the urogenital sinus and hindgut. These results provide the basis for understanding both cloacal development and the ARM pathogenesis.

Within amniotes, external copulatory organs have undergone extensive morphological diversification. One of the most extreme examples is squamate (lizards and snakes) hemipenes, which are paired copulatory organs that extend from the lateral margins of the cloaca. Here, we describe the development of hemipenes in a basal snake, the ball python (Python regius). Snake hemipenes arise as a pair of lateral swellings on either side of the caudal part of the cloaca, and these paired outgrowths persist to form the left and right hemipenes. In non-squamate amniotes, external genitalia form from paired swellings that arise on the anterior side of the cloaca, which then fuse medially to form a single genital tubercle, the anlagen of the penis or clitoris. Whereas in non-squamate amniotes, Sonic hedgehog (Shh)-expressing cells of the cloacal endoderm form the urethral or sulcus epithelium and are required for phallus outgrowth, the hemipenes of squamates lack an endodermal contribution, and the sulcus does not express Shh. Thus, snake hemipenes differ from the genital tubercles of non-squamate amniotes both in their embryonic origins and in at least part of patterning mechanisms, which raises the possibility that hemipenes may not be direct homologs of the unpaired amniote penis. Nonetheless, we find that some developmental genes show similar expression patterns in snake hemipenes buds and non-squamate genital tubercles, suggesting that homologous developmental mechanisms are involved in aspects of external genital development across amniotes, even when these structures may have different developmental origins and may have arisen independently during evolution.

In most animals, reproduction by internal fertilization is facilitated by an intromittent organ, such as the penis in amniote vertebrates. Recent progress has begun to uncover the mechanisms of mammalian external genital development; however, comparatively little is known about the development of the reptilian penis and clitoris. Here, we describe the development of the phallus and cloaca in the American alligator, Alligator mississippiensis. The embryonic precursor of the penis and clitoris is the genital tubercle, which forms by the budding of genital mesenchyme beneath the ventral body wall ectoderm, adjacent to the cloacal membrane. The cloacal lips develop from another pair of outgrowths, the lateral swellings. Early development of the alligator phallus, cloaca, and urogenital ducts generally resembles that of other reptiles, suggesting that differences in adult reptilian phallus and cloacal anatomy arise at later stages. The phallic sulcus is derived from the cloacal endoderm, indicating that the crocodilian sulcus is functionally and developmentally homologous to the mammalian urethra. Initial external genital outgrowth and patterning occur prior to temperature-dependent sex determination. Our analysis of alligator phallus and cloaca development suggests that modifications of an ancestral program of urogenital development could have generated the morphological diversity found in the external genitalia of modern amniotes.

In most amniotes, the intromittent organ is a single phallus; however, squamates (lizards, snakes, and amphisbaenians) have paired hemiphalluses. All amniotes studied to date initiate external genital development with the formation of paired genital swellings. In mammals, archosaurs, and turtles, these swellings merge to form a single genital tubercle, the precursor of the penis and clitoris; however, in squamates, the paired genital buds remain separate, giving rise to the hemiphalluses (hemipenes in males and hemiclitores in females). Although the molecular genetics and sexual differentiation of the genital tubercle have been investigated in mammals and birds, little is known about hemiphallus development. Here we describe development of the cloaca and hemiphallus in the green anole, Anolis carolinensis. Each hemiphallus originates as a protuberance that emerges at the ventral base of the hindlimb bud. Development of the hemipenes resembles penis development; however, differences exist in their tissue composition, morphogenesis, and gene expression patterns. These findings reveal aspects of phallus development that appear to be evolutionarily labile, both within squamates and more broadly among reptiles, and identify features that are conserved across amniotes. Our results, together with parallel studies in other reptilian taxa, suggest potential mechanisms for the diversification of external genital form.

Development of a phallus occurs in almost all amniotes; however, considerable variation in phallus morphology among different amniote lineages has contributed to the debate about their structural homology. Mammals are the only amniotes that form a closed urethral tube within the penis. In contrast, the phallus of reptiles and birds has an open urethral groove, or sulcus spermaticus, that facilitates directional flow of sperm along the penis. One condition of structural homology is that the organs should share a common developmental origin; de novo development from different embryonic progenitors would indicate that the structure re-evolved in a new position. Although a common developmental origin does not itself demonstrate homology, different origins could indicate a lack of homology. To further understand how development of external genitalia evolved in amniotes, we examined this in the turtle Trachemys scripta. We found that phallus development in the turtle closely resembles that of mice at the tissue, cellular, and molecular levels, consistent with the hypothesis that their phalluses are homologous structures. We find that acquisition of specialized characters, such as a closed urethral tube, involved lineage-specific specialization of the common plan for amniote phallus development.

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.

The anorectal and urogenital systems arise from a common embryonic structure termed cloaca. Subsequent development leads to the division/septation of the cloaca into the urethra, urinary bladder, vagina, anal canal, and rectum. Defective cloacal development and the resulting anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. In the most severe form in females, the rectum, vagina, and urethra fail to develop separately and drain via a single common channel known as a cloaca into the perineum. In this review, we summarize our current knowledge of embryonic cloaca development and malformation, and compare them to what has already been described in the literature. We describe the use of mouse models of cloaca malformation to understand which signaling pathways and cellular mechanisms are involved in the process of normal cloaca development. We also discuss the embryological correlation of the epithelial and stromal histology found in step sections of the common channel in 14 human cloaca malformations. Finally, we highlight the significance of these findings, compare them to prior studies, and discuss their implications for the pediatric surgeons. Understanding and identifying the molecular basis for cloaca malformation could provide foundation for tissue engineering efforts that in the future would reflect better surgical reconstruction and improved quality of life for patients.

The vesico-ureteric junction (VUJ) forms through a complex developmental program that connects the primordium of the upper urinary tract [the nephric duct (ND)] with that of the lower urinary tract (the cloaca). The signals that orchestrate the various tissue interactions in this program are poorly understood. Here, we show that two members of the EphA subfamily of receptor tyrosine kinases, EphA4 and EphA7, are specifically expressed in the mesenchyme surrounding the caudal ND and the cloaca, and that Epha4(-/-);Epha7(+/-) and Epha4(-/-);Epha7(-/-) (DKO) mice display distal ureter malformations including ureterocele, blind and ectopically ending ureters with associated hydroureter, megaureter and hydronephrosis. We trace these defects to a late or absent fusion of the ND with the cloaca. In DKO embryos, the ND extends normally and approaches the cloaca but the tip subsequently looses its integrity. Expression of Gata3 and Lhx1 and their downstream target Ret is severely reduced in the caudal ND. Conditional deletion of ephrin B2 from the ND largely phenocopies these changes, suggesting that EphA4/EphA7 from the pericloacal mesenchyme signal via ephrin B2 to mediate ND insertion. Disturbed activity of this signaling module may entail defects of the VUJ, which are frequent in the spectrum of congenital anomalies of the kidney and the urinary tract (CAKUT) in human newborns., (© 2014. Published by The Company of Biologists Ltd.)

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.

Cloacal malformations are a spectrum of congenital pelvic malformations that result from abnormal cloacal division during early embryogenesis. Depending on the timing of the developmental arrest, a spectrum of abnormalities can result, ranging from urogenital sinus malformations to cloacal dysgenesis. This case highlights the unique imaging features of cloacal dysgenesis, which is an extremely rare variant of this malformation spectrum. This variant is also the most severe manifestation of the cloacal malformation spectrum.

Purpose: The aims of this study were to identify the mutation gene of a Chinese family with anorectal malformation (ARM) associated with split hand-foot malformation and to determine the spatiotemporal expression of the mutated gene during hindgut and anorectum development in human embryos., Method: A Chinese family with intrafamilial clinically variable manifestation was analyzed and primers were designed for exons 3-14 of P63, DLX5, DLX6, DAC, and HOXD13 as candidate genes and direct sequence analysis of the exons was performed. Immunohistochemical study of mutated gene in the hindgut and anorectum of human embryos of 4th-10th weeks was performed., Result: Affected individuals were found to have an Arg227Gln P63 gene mutation. From the 4th-10th weeks of gestation of the human embryo, the P63-positive cells were mainly located on the epithelium of the apical urorectal septum, hindgut, and cloacal membrane. After the anorectum ruptured during the 8th week, the P63 remained strongly immunoreactive on the epithelium of the anal canal and urethra, but the mucous membrane of the rectum exhibited no reaction., Conclusions: The mutation identified strongly suggests a causal relationship between the ARM phenotype and P63. The expression of P63 was persistently active during the dynamic and incessant septation of the cloaca and hindgut, suggesting that P63 may play a pivotal role in the morphogenesis of the hindgut and anorectum.

Separating digestive and urinary outlets is a critical step during mammalian embryogenesis. However, the natural history of these structures is poorly studied, and little is known about their embryonic origin. Here, we show that peri-cloacal mesenchymal (PCM) progenitors are the major source of these structures. Surprisingly, PCM progenitors also contribute to perineum, a structural barrier separating the urinary and digestive tracts, suggesting a potential role of PCM progenitors in establishing independent urinary and digestive outlets. We demonstrate that Six1 and Six2 are complementarily but asymmetrically expressed in the PCM progenitors. Deletion of these genes results in decreased cell survival and proliferation, and consequently in agenesis of the perineum and severe hypoplasia of the genital tubercle. Together, these findings suggest that PCM progenitors are the unexpected source of perineum and genital tubercle, and establish a basic framework for investigating normal and abnormal development of anorectal and genitourinary structures.

Cloacal malformation is a rare but important anomaly. Prenatal diagnosis is possible with knowledge of the distinctive imaging features. The purpose of this case series is to illustrate characteristic prenatal sonographic and magnetic resonance imaging features of cloacal malformation using imaging from 6 cases seen at a single academic center to augment published data. The imaging feature common to all cases was a central cystic pelvic mass containing a characteristic fluid-fluid level. Additional anomalies include uterine and vaginal duplication, hydronephrosis, and lumbosacral anomalies. Prenatal magnetic resonance imaging showed the absence of the normal T1-hyperintense meconium-filled rectum in all cases. Prenatal diagnosis may affect immediate neonatal care (eg, immediate drainage of hydrocolpos) with an ultimate improved outcome.

Objective: To identify the pitfalls in accurate antenatal diagnosis of bladder exstrophy (BE) and cloacal exstrophy (CE), and thus understand the challenges for antenatal counselling., Methods: A prospectively maintained bladder exstrophy database of antenatal and live born referrals for BE/CE was used to identify patients. Data were collected about the antenatal scan findings and the outcome of pregnancy., Results: Between 2003 and 2009, 40 new babies with BE/CE were referred and of them 10 had an antenatal diagnosis. Five patients did not have a diagnosis despite suspicious findings noted on antenatal scans and another three had a wrong diagnosis of BE/CE. Of the 16 referrals with antenatal suspicion of BE/CE, 5 opted for termination. At the 20-week scan, it was possible to identify the gender of the fetus in 3/16 cases only., Conclusions: Only a quarter of the babies born with BE/CE had received an antenatal diagnosis. Raising awareness about the condition amongst radiographers, and facilitating further scanning by a specialist fetal management unit if suspicious findings are noted, is crucial for improving the rate of detection. An antenatal diagnosis may not be reliable, and difficulty in identifying gender at the 20-week scan adds to the complexity of antenatal counselling. Magnetic resonance imaging and karyotyping may provide additional helpful information., (Copyright © 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

We report a covered urethral duplication in a girl presenting prenatally with an enlarged fluid-filled vulvar cyst, genital duplication, and urogenital sinus revealed by fetal magnetic resonance imaging (MRI) and serial ultrasounds. Physical examination revealed an enlarged vulvar mass covering the clitoris, a single orifice, and normally sited anus. Congenital adrenal hyperplasia was ruled out at birth. MRI in addition showed an accessory duct between the sinus and the urine-filled vulvar pouch with a bifid clitoris. A total urogenital sinus mobilization with resection of the accessory urethra and vulvoplasty was performed with uneventful follow-up., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

Cloacal dysgenesis sequence (CDS) is a rare and lethal malformation. We report such a case of long-term survival, currently to 12 years of age. In the fetal period, she received a timely placement of vesico-amniotic shunt for a megabladder due to a severe urethral obstruction. Postnatally, cystostomy and colostomy were created because of no perineal opening of urethra, vagina, and anus. Anorectoplasty, construction of efferent conduit, and colostomy closure were performed at 4 years of age. Ileovaginoplasty and ileovesicostomy which was a Mitrofanoff-type of conduit, and labioplasty were performed at the age of 11 years. To the best of our knowledge, only five survivors with CDS over 1 year of age have been reported.

The evolutionarily conserved Six1-Eya1 transcription complex is central to mammalian organogenesis, and deletion of these genes in mice results in developmental anomalies of multiple organs that recapitulate human branchio-oto-renal (BOR) and DiGeorge syndromes. Here, we report that both Six1 and Eya1 are strongly expressed in the peri-cloacal mesenchyme (PCM) surrounding the cloaca, the terminal end of hindgut dilation. Six1 and Eya1 are absent from the intra-cloacal mesenchyme (ICM), a cell mass that divides the cloaca into dorsal hindgut and ventral urogenital sinus. Deletion of either or both Six1 and Eya1 genes results in a spectrum of genitourinary tract defects including persistent cloaca - hypoplastic perineum tissue between external urogenital and anorectal tracts; hypospadias - ectopic ventral positioning of the urethral orifice; and hypoplastic genitalia. Analyses of critical signaling molecules indicate normal expression of Shh in the cloaca and cloaca-derived endodermal epithelia. Using a Cre/loxP genetic fate mapping strategy, we demonstrate that Six1-positive PCM progenitors give rise to the most caudal structures of the body plan including the urogenital and anorectal complex, and the perineum region. Thus, Six1 and Eya1 are key regulators of both upper and lower urinary tract morphogenesis. Results from this study uncover essential roles of the PCM progenitors during genitourinary tract formation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Persistent cloaco is the most severe type of anorectal malformation encountered in children. Patients with cloacal anomalies have a high incidence of associated anomalies most commonly: urinary tract and spinal. Persistent cloaca remains a difficult reconstructive challenge but it is now possible to anatomically correct the defect with surgery in the majority of patients. This review discusses embryology, prenatal diagnosis, neonatal physical and radiological findings. A summary of early management and investigation is provided and the commonest surgical reconstruction techniques are discussed. The main goals of surgical reconstruction are the achievement of bowel and bladder control for the child and normal sexual function in adult life. Although the majority of cloaca patients can achieve faecal and urinary continence with the surgical reconstructive procedures performed today, many require additional/multiple urological procedures to achieve continence, treat bladder dysfunction and to protect renal function. One half of patients will develop renal failure, so regular and life long surveillance is mandatory. Due to the high number of associated gynaecological problems all patients should be assessed by a paediatric gynaecologist at puberty.

NPHP4 mutations cause nephronophthisis, an autosomal recessive cystic kidney disease associated with renal fibrosis and kidney failure. The NPHP4 gene product nephrocystin-4 interacts with other nephrocystins, cytoskeletal and ciliary proteins; however, the molecular and cellular functions of nephrocystin-4 have remained elusive. Here we demonstrate that nephrocystin-4 is required for normal cloaca formation during zebrafish embryogenesis. Time-lapse imaging of the developing zebrafish pronephros revealed that tubular epithelial cells at the distal pronephros actively migrate between the yolk sac extension and the blood island towards the ventral fin fold to join the proctodeum and to form the cloaca. Nphp4-deficient pronephric duct cells failed to connect with their ectodermal counterparts, and instead formed a vesicle at the obstructed end of the pronephric duct. Nephrocystin-4 interacts with nephrocystin-1 and Par6. Depletion of zebrafish NPHP1 (nphp1) increased the incidence of cyst formation and randomization of the normal body axis, but did not augment cloaca malformation in nphp4-deficient zebrafish embryos. However, simultaneous depletion of zebrafish Par6 (pard6) aggravated cloaca formation defects in nphp4-depleted embryos, suggesting that nphp4 orchestrates directed cell migration and cloaca formation through interaction with the Par protein complex.

Faulty ventral openings of the urethra constitute a broad spectrum of malformations that are subsumed under the term "hypospadia." The normal development of the urethra and the genitals critically depends on the following events: (a) formation of the external genitalia, (b) fate of the cloacal membrane, and (c) formation of the distal urethra. The purpose of this study was to demonstrate these events using microsurgical techniques and scanning electron microscopy in staged rat embryos., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Normal and abnormal development of the hindgut is still in debate. Normal development of the hindgut critically depends on the cloacal membrane. In this study, scanning electron microscopy of staged rat embryos between the gestational days 10-15 was performed to show the normal development of the hindgut and the abnormal development in Danforth's short tail (SD) mice. Our studies in normal and abnormal development indicate that the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in females. To explain the abnormal development in anorectal malformations, further studies are mandatory., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aims of Study: Retinoid-mediated signal transduction plays a crucial role in the embryogenesis of various organs. We previously reported the successful induction of anorectal malformations in mice using retinoic acid (RA). Retinoic acid controls the expression of essential target genes for cell differentiation, morphogenesis, and apoptosis through a complicated interaction in which RA receptors form heterodimers with retinoid X receptors. In the present study, we investigated whether the retinoid antagonist, LE135, could prevent the induction of anorectal malformations (ARMs) in mice., Methods: Retinoic acid was intraperitoneally administered as 100 mg/kg of all-trans RA on E9; and then the retinoid antagonist, LE135, was intraperitoneally administered to pregnant ICR strain mice on the eighth gestational day (E8), 1 day before administration of RA (group B) or on E9, simultaneously (group C) with RA administration. All of the embryos were obtained from the uteri on E18. Frozen sections were evaluated for concentric layers around the endodermal epithelium by hematoxylin and eosin staining., Results: In group A, all of the embryos demonstrated ARM with rectoprostatic urethral fistula, or rectocloacal fistula, and all of the embryos showed the absence of a tail. In group B, 36% of the embryos could be rescued from ARM. However, all of the rescued embryos had a short tail that was shorter than their hind limb. The ARM rescue rates in group B were significantly improved compared to those in group A (P < .01). In group C, 45% of the embryos were rescued from ARM, but all of the rescued embryos had short tail. The ARM rescue rate in group C was significantly improved compared to that in group A (P < .01). However, there was no significant difference in the ARM rescue rate between group B and Group C., Conclusion: The present study provides evidence that in the hindgut region, RAR selective retinoid antagonist, LE135, could rescue embryos from ARM. However, the disturbance of all-trans RA acid was limited to the caudal region. Further study to establish an appropriate rescue program for ARM in a mouse model might suggest a step toward protection against human ARM in the future., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.

Cloacal anomaly is a rare malformation with an incidence of 1 in 50,000 births. The definitive prenatal diagnosis of cloacal dysgenesis sequence is difficult. The use of three-dimensional (3-D) ultrasonography helps to describe the perineum and change diagnosis. In our case report, a large median genital structure was visualized, which was initially considered a penis-like structure, but the 3-D technique showed an enlarged bud. The urethral meatus was at the tip of this smooth structure. The lack of anal structure was clearly demonstrated., (Copyright © 2011 S. Karger AG, Basel.)

Variants of cloaca and exstrophy are difficult to explain and manage. We describe a female neonate having partial umbilical exstrophy with a cecal patch in the bladder and an intact distal hindgut that was expressed as a cloaca. The baby was managed by ileostomy and bladder closure without separating the cecal patch from the bladder. The possible embryological origin of this abnormality is discussed, and the literature is reviewed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Today, the normal and abnormal development of the hindgut is still a matter of speculation. However, as the result of recent studies in appropriate animal models, most embryologic events that finally lead to abnormal hindgut development are better known than in the past: (1) the process of maldevelopment starts in early embryonic stages; (2) the cloacal membrane is always too short in its dorsal part, thus, the dorsal cloaca is missing; and (3) as a result, the hindgut remains attached to the sinus urogenitalis, forming the recto-urethral fistula. In the past, an impaired process of septation was believed to be the main cause of abnormal hindgut development. In contrast to this, our results indicate that the development of the septum is more passive than active. Furthermore, the results of our studies in normal and abnormal development indicate that (1) the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in female embryos, and (2) to explain abnormal development, studies in abnormal embryos are mandatory., (Copyright 2010 Elsevier Inc. All rights reserved.)

A newborn boy was brought to us, 2 hours after birth, with a mucosal-lined left hemiperineal lesion associated with classical bladder exstrophy and an anterolaterally displaced anus. Perineal anatomy was restored by excising the mucosa lined lesion. The bladder closure for classical bladder exstrophy was done at the same time. Histologically, gastric, respiratory, and small intestinal epithelia were present in the mucosa. A rectal duplication cyst that had ruptured in utero through the hemiperineum could explain the anomaly. The association of classical bladder exstrophy with ruptured rectal duplication cyst has never previously been described in the literature., (Copyright 2010 Elsevier Inc. All rights reserved.)

Introduction: The enteric nervous system of the chick embryo hindgut is derived from the vagal and sacral neural tube. Nerve cells from these regions produce various neuronal phenotypes, including inhibitory and excitatory nerve cells, providing intrinsic innervation to the chick embryo cloaca. We hypothesised that the vagal and sacral neural tubes provide the cloaca, with phenotypically similar nerve cells. The aim of our study was to investigate the origin of excitatory neurotransmission in the developing cloaca., Materials and Methods: Chicken embryos were incubated until the 10-12 somite stage (ss). To study the vagal neural tube contribution to the cloaca, this region was microsurgically ablated in ovo and replaced with the corresponding region from age-matched quail embryos. To study the sacral neural tube contribution to the cloaca, the vagal neural tube was ablated at the 10-12 ss, but not replaced with quail neural tube, thus, only the sacral neural crest cells remained. All embryos were harvested at E12 and E14, embedded in paraffin wax and serially sectioned. Immunohistochemistry was carried out on all embryos using human natural killer-1, quail non-chick perinuclear, choline acetyltransferase (ChAT) and substance P (SubP) antibodies., Results: Choline acetyltransferase- and SubP-positive neurons were seen to originate in both the vagal and the sacral neural tube. The vagal neural tube provided the majority of the nerve cells to the chick embryo cloaca and expressed both ChAT and SubP in both the myenteric and submucosal plexus. The sacral neural tube contributed a lesser amount of nerve cells to the chick embryo cloaca, but was also seen to produce both ChAT- and SubP-positive nerve cells in both ganglionated plexus., Conclusion: This study shows, for the first time, that the excitatory ChAT- and SubP-expressing neurons in the developing cloaca originate in both the vagal and the sacral neural tube. These results highlight the origin of phenotypically similar nerve cells in both regions of the neural tube, providing new insights into the developmental origin of the intrinsic innervation of the persistent cloaca.

Malformations of the external genitalia are among the most common congenital anomalies in humans. The urogenital and anorectal sinuses develop from the embryonic cloaca, and the penis and clitoris develop from the genital tubercle. Within the genital tubercle, the endodermally derived urethral epithelium functions as an organizer and expresses sonic hedgehog (Shh). Shh knockout mice lack external genitalia and have a persistent cloaca. This identified an early requirement for Shh, but precluded analysis of its later role in the genital tubercle. We conducted temporally controlled deletions of Shh and report that Shh is required continuously through the onset of sexual differentiation. Shh function is divisible into two temporal phases; an anogenital phase, during which Shh regulates outgrowth and patterning of the genital tubercle and septation of the cloaca, and a later external genital phase, during which Shh regulates urethral tube closure. Disruption of Shh function during the anogenital phase causes coordinated anorectal and genitourinary malformations, whereas inactivation during the external genital phase causes hypospadias. Shh directs cloacal septation by promoting cell proliferation in adjacent urorectal septum mesenchyme. Additionally, conditional inactivation of smoothened in the genital ectoderm and cloacal/urethral endoderm shows that the ectoderm is a direct target of Shh and is required for urethral tube closure, highlighting a novel role for genital ectoderm in urethragenesis. Identification of the stages during which disruption of Shh results in either isolated or coordinated malformations of anorectal and external genital organs provides a new tool for investigating the etiology of anogenital malformations in humans.

Cloacal dysgenesis sequence (CDS) is a severe hindgut malformation occurring in 1:50,000 to 250,000 live births (Qureshi et al. Prenatal diagnosis of cloacal dysgenesis sequence: differential diagnosis from other forms of fetal obstructive uropathy. Fetal Diagn Ther 1998;13:69-74; Bargaje et al. Cloacal dysgenesis sequence. Ann Diagn Pathol 2008;12:62-66). It is characterized by a smooth perineum with no urethral, vaginal, or anal openings, and lack of labioscrotal development. Typically, the bladder, vagina, and colon each end blindly, although persistent cloaca without perineal orifice can be seen. With no egress for urine, infants have renal insufficiency and pulmonary hypoplasia, usually making CDS lethal (Sahinoglu Z et al. The prenatal diagnosis of cloacal dysgenesis in six cases: can the termination of pregnancy always be the first choice? Prenat Diagn 2004;24:10-16). Reported survivors have had a persistent urachus or have been spared the effects of oligohydramnios by the presence of a twin (Liang X. Cloacal dysgenesis sequence: observations in four patients, including three fetuses of second trimester gestation. Pediatr Dev Pathol 1998;1:281-288). We report a case of long-term survival, currently to 25 months of age, and renal transplantation in a monochorionic, diamniotic twin girl with CDS.

A specimen of accessory phallic urethra duplication (triplication) in a 32.5-week gestation female was studied histologically. The specimen demonstrated an anterior "phallic urethra" ending inside a penoclitoral organ in a megalourethra-like dilatation with a narrow epispadiac meatus and "anterior urethrocutaneous fistula" and a posterior canal opening with the anal canal. With histology suggestive of a split persistent urogenital sinus, correlation to normal development strongly suggests an error in the formation of the cloacal membrane and developing superficial cloaca at 26 to 29 days' ovulation age (2 to 4 mm), causing an isolated interruption of the membrane by mesenchyme, which by participating in the growth of the early cloaca may extend as far as the vaginal orifice later. Differences in number, position, and size of such mesenchymal interruptions can explain other duplications and triplications and offer a single alternative for the many current theories of pathogenesis, none of which are compatible with new insights in normal development.

Objective: In the normal embryonic development of anorectum, apoptosis plays an important role. To explore the role of apoptosis in anorectal malformations (ARM), this study investigated cell apoptosis during the cloacal embryonic development in ARM embryos., Methods: ARM embryos were induced by intragastric administration of ethylenethiourea (125 mg/kg) for pregnant rats on embryonic day 10 (E10). The distribution of apoptotic cells in the cloaca was ascertained by hematoxylin and eosin and TUNEL staining in the normal control embryos (n=102) and ARM embryos (n=147) on E13, E13.5, E14, E15 and E16., Results: On E13, apoptotic cells were detected in the urorectal septum of rat embryos in the control group. With the development of embryos, the number of apoptotic cells in the mesenchyme of urorectal septum gradually increased and a large number of apoptotic cells were seen in the dorsal rectal mesenchyme. On E14, apoptotic cells appeared at the terminal rectum and the dorsal cloacal membrane. On E15, the urorectal septum fused with the cloacal membrane and apoptotic cells in the urorectal septum mesenchyme continuously extended down to the fusion region. Compared with the control group, apoptotic cells in the urorectal septum, the dorsal rectal mesenchyme and the cloacal membrane of the ARM rat embryos were significantly reduced during the embryonic development. The development of the urorectal septum was delayed and it did not fuse with the cloacal membrane in ARM embryos., Conclusions: During the embryonic development of cloaca, abnormal apoptosis in the urorectal septum, the dorsal rectal mesenchyme and the cloacal membrane may be one of the reasons for anorectal malformations. The proper regulation of cell apoptosis may be one of the key mechanisms for normal development of anorectum in the embryonic stage.

In mammalian embryos, male and female external genitalia develop from the genital tubercle. Outgrowth of the genital tubercle is maintained by the urethral epithelium, and it has been reported that Fgf8 mediates this activity. To test directly whether Fgf8 is required for external genital development, we conditionally removed Fgf8 from the cloacal/urethral epithelium. Surprisingly, Fgf8 is not necessary for initiation, outgrowth or normal patterning of the external genitalia. In early genital tubercles, we found no redundant Fgf expression in the urethral epithelium, which contrasts with the situation in the apical ectodermal ridge (AER) of the limb. Analysis of Fgf8 pathway activity showed that four putative targets are either absent from early genital tubercles or are not regulated by Fgf8. We therefore examined the distribution of Fgf8 protein and report that, although it is present in the AER, Fgf8 is undetectable in the genital tubercle. Thus, Fgf8 is transcribed, but the signaling pathway is not activated during normal genital development. A phylogenetic survey of amniotes revealed Fgf8 expression in genital tubercles of eutherian and metatherian mammals, but not turtles or alligators, indicating that Fgf8 expression is neither a required nor a conserved feature of amniote external genital development. The results indicate that Fgf8 expression is an early readout of the genital initiation signal rather than the signal itself. We propose that induction of external genitalia involves an epithelial-epithelial interaction at the cloacal membrane, and suggest that the cloacal ectoderm may be the source of the genital initiation signal.

Signaling by Bone morphogenetic proteins (Bmps) has multiple and diverse roles in patterning and morphogenesis of the kidney, eye, limbs and the neural tube. Here, we employed the Bmp7(lacZ) strain to perform a detailed analysis of Bmp7 expression and the null phenotype during development of the mouse urogenital system. The urethral compartment originates in mid-embryogenesis from the ventral part of the cloaca, a transient cavity at the caudal end of the hindgut. At mid-gestation, Bmp7 expression was detected within several specific domains in the cloacal epithelium and mesenchyme. In late embryogenesis, Bmp7 expression was present in the urethra, rectum, the urethral glands, corpus cavernosum, and in the male and female genital ducts. Importantly, loss of Bmp7 resulted in arrest in cloacal septation, and severe defects in morphogenesis of the genital urethra and mesenchyme. Together, our analysis of Bmp7 expression and the null phenotype, indicates that Bmp7 may play an important role in re-organization of the epithelium during cloacal septation and morphogenesis of the genital tubercle.

Purpose: Acetylcholine is the major excitatory neurotransmitter involved in intestinal contraction and therefore plays a pivotal role in normal gastrointestinal motility. Acetylcholinesterase (AchE) is an enzyme involved in hydrolysing acetylcholine during its metabolism. The AchE histochemistry can therefore be used to label acetylcholine-positive nerve cells and fibres, giving an overview of cholinergic innervation in the gut. Persistent cloaca is the most common anorectal malformation seen in female infants. The normal avian embryo contains a cloaca, reminiscent to the human malformation, which acts as a collecting chamber into which the digestive, urinary, and reproductive tracts merge. The aim of our study was to investigate cholinergic innervation in the chick embryo cloaca and colorectum at various stages of development., Methods: Chick embryos were harvested at embryonic days 12 (E12), E14, E16, and E18. The colorectum and cloaca were removed from each embryo and frozen. Frozen transverse sections were then cut and AchE histochemistry was performed. The AchE staining was evaluated and graded using light microscopy., Results: Results of our study show that acetylcholine is increasingly expressed in the chick embryo cloaca and colorectum from E12 to E18, with a stronger expression in the colorectum at each stage. The AchE-positive ganglia and fibres were evident in the submucosal and myenteric plexuses at all stages in both the cloaca and the colorectum. Ganglia size was seen to increase with age, and there was a notable increase in the amount of AchE-positive nerve fibres., Conclusion: Results show an increase in cholinergic innervation in both the cloaca and colorectum during development. As acetylcholine is the major excitatory neurotransmitter in the enteric nervous system, this study provides us with valuable information regarding the regional differences in the development of cholinergic innervation of the embryonic hindgut.

The Hedgehog (Hh) family of signalling molecules is essential for a wide range of developmental processes. Mammalian studies have implicated the Hedgehog pathway in the aetiology of anorectal malformations (ARMs), relatively common congenital anomalies caused by failures in the development of the cloaca. In this study we demonstrate that Hh signalling is absolutely required for the formation of the zebrafish cloaca and that the severity of the posterior gut abnormalities induced by a reduction in Hh activity is dependent on the levels of Hh signal transduction. The complete loss of all Hh activity results in the most severe defects and the critical period for Hh activity is between 34 and 74 hours post fertilisation. Using a range of mutant genotypes that cause notochord and floorplate abnormalities, we show that the source of the Hh signals required for posterior gut formation is the endoderm and not the notochord, as previously postulated in mammalian models of ARMs. We show that Adriamycin, a drug known to cause ARMs in rat, but not chick embryos, has no effect on the development of the zebrafish gastrointestinal tract. These studies establish the zebrafish as a model for ARMs, and for the elucidation of other pathways involved in hindgut developmental processes.