Embryonic Cloaca Research Articles

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.

Challenges in the management of caudal duplication syndrome

Caudal duplication syndrome (CDS) is a rare developmental anomaly in which embryonic cloaca and notochord structures are duplicated [1]. Due to the diverse clinical manifestation and rarity of CDS, it is crucial to report every case and to share experience and outcomes of individually adjusted management plans. We report here the case of a 2-year-old girl born with duplication of the urogenital (bladder, urethra, uterus, vagina, vulva) and gastrointestinal (gallbladder, appendix vermiformis) systems. Additionally, coccygeal agenesis, lipomyelomeningocele and vertical talus were present. A thorough examination and urological reconstructive surgery were performed. While there may be a desire from patients, parents and healthcare specialists to modify all malformations to an anatomically correct state, the current opinion is that only anatomical variants that influence function should be modified. Consent from patients should be sought for decisions regarding more sensitive matters such as vulva surgery for cosmetic reasons or correction of anatomical variants without functional consequences.

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.

Mutations of MYH14 are associated to anorectal malformations with recto-perineal fistulas in a small subset of Chinese population.

Anorectal malformations (ARMs) are among the most commonly congenital abnormalities of distal hindgut development, ranging from anal stenosis to anal atresia with or without fistulas and persistent cloaca. The etiology remains elusive for most ARM cases and the majority of genetic studies on ARMs were based on a candidate gene approach. In all eight family members of a non-consanguineous Chinese family, we performed whole-exome sequencing. Subsequently, exome sequencing of MYH14 in 72 unrelated probands with ARMs was performed. The accurate distribution of non-muscle myosin II heavy chain (NMHC II) was investigated by immunohistochemistry in serial sagittal sections of E11.5-13.5 mouse cloacal regions. A homozygous mutation in MYH14 was identified in the two siblings of family 1. Compound heterozygous MYH14 changes were identified in an unrelated individual. Immunohistochemical analysis suggest stronger NMHC IIC localization in the epithelium of the murine embryonic cloaca, urorectal septum and hindgut compared with another two NMHC II isoforms. This is the first identification of mutations in MYH14 as a cause of ARMs. The stronger localization of NMHC IIC in E11.5-13.5 mouse cloacal regions further supports the role of MYH14 in anorectal development.

Developmental and Evolutionary Origins of the Amniote Phallus.

An intromittent phallus is used for sperm transfer in most amniote taxa; however, there is extensive variation in external genital morphology within and among the major amniote clades. Amniote phalluses vary in number (paired, single, or rudimentary), spermatic canal morphology (closed tube or open sulcus), and mode of transition between resting and tumescent states (inflation, rotation, eversion, or muscle relaxation). In a phylogenetic context, these varying adult anatomies preclude a clear interpretation for the evolutionary history of amniote external genitalia; as such, multiple hypotheses have been presented for the origin(s) of the amniote phallus. In combination with historic embryological studies, recent comparative developmental analyses have uncovered evidence that, despite extensive morphological variation in adult anatomy, embryonic patterning of the external genitalia is similar among amniotes and begins with emergence of paired swellings adjacent to the cloaca. External genital development in mammals, squamates (lizards, snakes, and amphisbaenians), Rhyncocephalians (tuataras), turtles, crocodilians (alligators, crocodiles, and gharials), and birds proceeds by iterative sequences of budding and fusion events, initiated by emergence of paired swellings adjacent to the embryonic cloaca. Conservation of the embryonic origins, morphogenetic processes, and molecular genetic mechanisms involved in external genital development across Amniota supports derivation from the common ancestor of amniotes, and suggests that lineage-specific divergence of later patterning events underlies the variation observed in extant adult amniote phallus morphology.

Pathology of cloaca anomalies with case correlation

During the fourth week of human embryo development, a transient common channel known as a cloaca is formed from which three cavities with three external orifices arises. Cloaca anomalies occur when there is failure of separation of the rectum, vagina, and urethra channel resulting in a single drain into the perineum. In our previous institutional studies, Runck et al.1 compared human and mouse cloaca development and found early mis-patterning of the embryonic cloaca deranged hedgehog and bone morphogenetic proteins (BMP) signaling. Also, our group reported the embryological correlation of the epithelial and stromal histology found in step sections of the common channel in 14 cloaca malformations in humans.2 In this review, we present the pathology of a 4-year-old female with a cloaca and VACTERL complex, and summarize our current knowledge of cloaca pathology. Furthermore, we suggest that careful pathological examination of cloaca specimens in conjunction with surgical orientation may result in a better understanding of the etiology of this condition.

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.

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.

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

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

Caudal duplication syndrome

Caudal duplication syndrome is a rare entity in which structures derived from the embryonic cloaca and notochord are duplicated to various extents. Its prevalence at birth is less than 1 per 100,000. The term caudal duplication encompasses a spectrum of anomalies and is often used to describe incomplete separation of monovular twins or referred to as part of the spectrum of anomalies associated with conjoined twinning. It usually includes multiple rare malformations and duplications of distal organs derived from the hindgut, neural tube, and caudal mesoderm. It was postulated that the disorder is related to misexpression of one or more of the distal HOX genes, potentially HOX10 or HOX11, leading to abnormal proliferation of caudal mesenchyme. The malformations are usually diagnosed by anomaly scan in the second trimester. Here we report the case of a baby presenting on the first day of life with complete duplication of caudal structures below the dorsolumbar level.

BMP7 directs epithelial cell fate choice and dorsal–ventral partitioning of the embryonic cloaca by collaborating with the PCP pathway

BMP7 directs epithelial cell fate choice and dorsal–ventral partitioning of the embryonic cloaca by collaborating with the PCP pathway

Embryology of the hindgut

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.

Reply to: The Development of the Human Anorectum

Reply to: The Development of the Human Anorectum

Applicability of lectin histochemistry in a test system with in ovo treatment for detecting androgenic and antiandrogenic effects of chemicals in Japanese quail (Coturnix japonica)

The aim of the current study was to examine whether analysis of the appearance of specific lectin-positive substances in the quail embryonic cloacal gland would be useful for evaluating the androgenic and antiandrogenic effects of chemicals. Fertilized Japanese quail eggs were injected with 0 to 75 µg of cyproterone acetate (CA), an antiandrogenic compound, on d 12 of incubation (d 12), followed by injection of 0 to 300 µg of testosterone propionate (TP) on d 13. Experimental groups consisted of a control group (corn oil injections on d 12 and 13), a TP-L group [corn oil and a low dose (L; 30 µg) of TP], a TP-H group [corn oil and a high dose (H; 300 µg) of TP], a CA-L + TP-H group [a low dose (L;7.5 µg) of CA + TP-H], and a CA-H + TP-H group [a high dose (H; 75 µg) of CA + TP-H]. The cloacal tissues were collected on d 16, processed into paraffin sections, and stained using 14 different biotinylated lectins. The Vicia villosa (VVA) lectin most strongly stained the developing cloacal glandular cells in TP-H. Western blotting analysis showed 1 VVA-positive band of approximately 75 kDa. The ratio of VVA-positive areas per unit square examined microscopically by image analysis was significantly greater in the TP-H group than in the control group in both males and females. The ratio was significantly decreased in the CA-L + TP-H and CA-H + TP-H groups compared with the TP-H group in both males and females. Furthermore, the ratio was smaller in females than in males within a TP-L or TP-H treatment group. These results suggest that lectin histochemistry on quail embryonic cloaca using VVA is useful for evaluating the androgenic and antiandrogenic effects of chemical compounds.

Embryology of anorectal malformations

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.

Multiphasic and tissue-specific roles of sonic hedgehog in cloacal septation and external genitalia development

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.

Endopelvic Fascia in Women

The endopelvic fascia is a confluent suspensory apparatus of the female pelvic organs. The aim of the study was to construct a three-dimensional model of the endopelvic fascia, defining its shape and its connections to the surrounding parietal structures. We created a three-dimensional multiple-source computer model to simultaneously visualize and analyze all the structures within the female pelvic floor. This model integrates data from magnetic resonance imaging of 15 nulliparas under age 30 with no symptoms of pelvic floor dysfunction. The model also includes data from direct observation in the dissection laboratory and in surgical rooms, together with the relevant scientific literature. The endopelvic fascia has the shape of a semifrontally oriented septum, which surrounds the vagina and part of the uterine cervix and divides the pelvic floor into the anterior and posterior compartments. This confluent septum has specific connections to the pubic bone, anterior perineal membrane, perineal body, and superior fascia of the levator ani muscle. Additionally, the uterosacral part of the septum has three subdivisions- the "vascular part," the "neural part," and the true uterosacral ligament. Each of these subdivisions has a different physical link to the parietal structures. Three-dimensional illustrations and schemes were created to facilitate the understanding of the anatomy of these complex structures. Connecting descriptions of the geometry of the organs visible by magnetic resonance imaging with descriptions of their individual connections to the endopelvic fascia gave us unique information about the three-dimensional representation of the anatomy of the female lesser pelvis. The endopelvic fascia divides the lesser pelvis in a manner that is similar to the way the urorectal septum divides the embryonic cloaca.

Vagal neural crest contribution to the chick embryo cloaca

Intrinsic innervation of the developing chick cloaca is provided by the enteric nervous system, a network of neurons and glia that lies within its walls. The enteric nervous system originates from neural crest cells that migrate from the vagal and sacral regions of the neural tube during the early stages of development. Abnormal cloacal development can cause a number of anorectal anomalies including persistent cloaca. Our study aimed to investigate the contribution of vagal neural crest cells to the total population of enteric neurons and glia within the chick embryo cloaca, using quail-chick chimeras. Chicken embryos were incubated until the 10-12 somite stage (ss). The vagal neural tube, corresponding to somites 1-7, was then microsurgically ablated in ovo and isochronic and isotopic quail grafts were performed. The eggs were then reincubated until embryos were harvested at E12. Whole embryos were fixed in Bouin's fluid, embedded in paraffin wax and sectioned. Immunohistochemistry was carried out using the HNK-1 antibody to label all neural crest cells, and the quail-specific antibody, QCPN, to label quail cells. QCPN-immunoreactive cells were seen to make up a large proportion of enteric neurons and glia within the walls of the embryonic cloaca. HNK-1 labelled all neural crest cells in the myenteric and submucosal plexuses as well as the sacral crest-derived nerve of Remak, while QCPN-positive cells were evident in both plexuses but mostly in the submucosal plexus, where they appeared to make up the majority of neurons. Results show that the chick embryo cloaca is primarily innervated by vagal neural crest cells. Further studies to investigate the contribution of sacral neural crest cells to the same region will give further insight into the development of the enteric nervous system within the embryonic cloaca.

Differences in nitrergic innervation of the developing chick cloaca and colorectum

The intrinsic innervation of the developing gut has long been a subject of investigation, but little is known regarding that of the embryonic cloaca. The cloaca, like the rest of the gastrointestinal tract, is intrinsically innervated by the enteric nervous system. Nitrergic neurons and fibres make up a large part of this system, thus, their distribution provides us with a useful insight into its development. Cloacal and colorectal tissue specimens were removed from chick embryos at embryonic days 11 (E11), E15 and E19. NADPH-diaphorase (NADPH-d) histochemistry was carried out using whole mount tissue preparations. Ganglia density, the number of NADPH-d-positive cells per ganglia in the myenteric plexus and cell size were calculated and statistical analysis was performed to compare both regions of the gut (P<0.001). There were significant differences in the ganglia density in the cloaca compared to the colorectum at E11 (P<0.05) and E15 (P<0.01), with the colorectum having a much denser network. In both the cloaca and the colorectum, ganglia density significantly decreased with age (P<0.001), while significant differences were observed in the number of NADPH-d-positive cells per ganglia in both regions through development. Total cell size was similar in both the cloaca and colorectum at each stage and increased in both regions through development, predominantly due to an increase in the cytoplasm. Results reveal striking differences in innervation between the chick embryo cloaca and colorectum. The sparse network of innervation evident within the cloaca in contrast to the dense network within the colorectum emphasizes the individuality of both regions. These results highlight the need for a further in-depth analysis of the enteric nervous system's development within the embryonic cloaca.

The morphogenesis of the exstrophy-epispadias complex: a new concept based on observations made in early embryonic cases of cloacal exstrophy

The term exstrophy-epispadias complex (EEC) has been coined for a group of congenital malformations that includes epispadias, bladder exstrophy and cloacal exstrophy. It is usually thought that these malformations develop against a similar embryological background. This background, however, is still obscure. This is mainly due to the lack of availability of abnormal human or non-human embryos showing the crucial developmental steps in the morphogenesis of EEC malformations. In this paper, we present chick embryos that show cloacal exstrophy at early developmental stages. To the best of our knowledge, this is the first documentation of this rare malformation in young embryos. Embryos with cloacal exstrophy (n=4) were found among embryos from two experimental series (n=50) that were primarily performed to document the early morphogenesis of facial and cardiovascular malformations. The malformations were induced by the administration of suramin according to established protocols. Suramin can induce a spectrum of malformations including facial clefts, heart defects, and cloacal exstrophy. Besides the presence of an abnormal opening into the cloaca, all embryos were characterised by an abnormal broadening of the caudal trunk at the level of the leg buds, which, in the youngest embryos, was associated with the abnormal presence of large aneurysmatic swellings of the dorsal aortae at this side. We postulate that these aneurysmatic swellings might be the primary defects leading to the development of EEC malformations. These space-occupying anomalies seem to cause abnormal distensions of the developing pelvis and of the infra-umbilical portion of the developing body wall. In consequence, the mid-portion of the developing ventral body wall between the origin of the umbilical cord and the cloacal plate becomes stretched and thinned out. Tension and thinning of the ventral body wall might ultimately lead to its rupture with exposure of the lumen of the embryonic cloaca and allantois. This new concept on the morphogenesis of the EEC is the first not to be inferred from the conditions seen in fetal or postnatal human cases but is based entirely on data from malformed embryos.