Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

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.

Foxa1 and Foxa2 orchestrate development of the urethral tube and division of the embryonic cloaca through an autoregulatory loop with Shh.

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.

The embryology of persistent cloaca and urogenital sinus malformations.

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 4th and 7th 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 19th 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.

The embryology of persistent cloaca and urogenital sinus malformations / 亚洲男科学杂志(英文版)

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 4th and 7th 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 19th 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.

The development of the cloaca in the human embryo.

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.

Reciprocal Spatiotemporally Controlled Apoptosis Regulates Wolffian Duct Cloaca Fusion.

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 PLCγ 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.

De novo mutations in Caudal Type Homeo Box transcription Factor 2 (CDX2) in patients with persistent cloaca.

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.

Spatiotemporal expression of proprotein convertase subtilisin/kexin type 5 in the development of anorectal malformations in fetal rats.

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.

Reorganization of mammalian body wall patterning with cloacal septation.

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.

Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus.

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.

Clarification of mammalian cloacal morphogenesis using high-resolution episcopic microscopy.

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.

Systematic stereoscopic analyses for cloacal development: The origin of anorectal malformations.

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.

WNT3 involvement in human bladder exstrophy and cloaca development in zebrafish.

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.

Morphogenesis and patterning of the phallus and cloaca in the American alligator, alligator mississippiensis.

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.

Development of the cloaca, hemipenes, and hemiclitores in the green anole, Anolis carolinensis.

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.

Phallus development in the turtle Trachemys scripta.

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.

Development of hemipenes in the ball python snake Python regius.

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.

A relative shift in cloacal location repositions external genitalia in amniote evolution.

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 great divide: septation and malformation of the cloaca, and its implications for surgeons.

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.

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.

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.