Genital Tubercle Research Articles

Bmp4 is an essential growth factor for the initiation of genital tubercle (GT) outgrowth.

The external genitalia are appendage organs outgrowing from the posterior body trunk. Murine genital tubercle (GT), anlage of external genitalia, initiates its outgrowth from embryonic day (E) 10.5 as a bud structure. Several growth factors such as fibroblast growth factor (FGF), Wnt and Sonic hedgehog (Shh) are essential for the GT outgrowth. However, the mechanisms of initiation of GT outgrowth are poorly understood. We previously identified bone morphogenetic protein (Bmp) signaling as a negative regulator for GT outgrowth. We show here novel aspects of Bmp4 functions for GT outgrowth. We identified the Bmp4 was already expressed in cloaca region at E9.5, before GT outgrowth. To analyze the function of Bmp4 at early stage for the initiation of GT outgrowth, we utilized the Hoxa3-Cre driver and Bmp4 flox/flox mouse lines. Hoxa3 Cre/+ ; Bmp4 flox/flox mutant mice showed the hypoplasia of GT with reduced expression of outgrowth promoting genes such as Wnt5a, Hoxd13 and p63, whereas Shh expression was not affected. Formation of distal urethral epithelium (DUE) marked by the Fgf8 expression is essential for controlling mesenchymal genes expression in GT and subsequent its outgrowth. Furthermore, Fgf8 expression was dramatically reduced in such mutant mice indicating the defective DUE formation. Hence, current results indicate that Bmp4 is an essential growth factor for the initiation of GT outgrowth independent of Shh signaling. Thus, Bmp4 positively regulates for the formation of DUE. The current study provides new insights into the function of Bmp signaling at early stage for the initiation of GT outgrowth.

Alternative (backdoor) androgen production and masculinization in the human fetus.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (<1 ng/mL), while in female fetuses, there are significantly lower levels of androsterone and testosterone. In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes. This is consistent with transcript levels of enzymes involved in the alternate pathway (steroid 5α-reductase type 1 [SRD5A1], aldo-keto reductase type 1C2 [AKR1C2], aldo-keto reductase type 1C4 [AKR1C4], cytochrome P450 17A1 [CYP17A1]), as measured by quantitative PCR (qPCR). These data identify androsterone as the predominant backdoor androgen in the human fetus and show that circulating levels are sex dependent, but also that there is little de novo synthesis in the testis. Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues. Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle. Our findings also provide a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans.

Dihydrotestosterone induces minor transcriptional alterations in genital skin fibroblasts of children with and without androgen insensitivity.

Endogenous and exogenous androgens induce masculinization of external genitalia through binding to the androgen receptor (AR). The target genes of androgens in external genitalia remain to be determined, although previous studies have shown that the apolipoprotein D gene (APOD) was significantly upregulated by dihydrotestosterone (DHT), the most potent androgen in humans. In the present study, we performed microarray analysis for genital skin fibroblasts obtained from four boys with buried penis (the control individuals) and a patient with partial androgen insensitivity syndrome (PAIS) due to a hypomorphic mutation in AR (the PAIS patient). We identified 24 transcripts that were upregulated or downregulated by DHT in all samples of control individuals and, to a lesser extent, in the sample of the PAIS patient. Differences between DHT-treated and -untreated samples were small; the results of 24 transcripts did not reach statistical significance. The 24 transcripts included CYP1B1, a gene possibly involved in the development of genital tubercle in mice, and APOD, as well as several genes that have been reported as androgen targets in prostate or other tissues. The results of this study indicate that androgen-mediated masculinization of external genitalia is unlikely to depend on massive transcriptional changes in specific AR target genes. Rather, minor transcriptional changes of several genes, and/or a complex molecular network may play a major role in penile development. Importantly, our data suggest the possible involvement of CYP1B1 in human genital development and confirm the clinical importance of APOD as a biomarker for AR function.

Chronology of Conceptus Development and Gestational Age Determination in Dromedary Camels Between 15-83 Days of Pregnancy Using Ultrasonography

The aims of the current study were to determine the chronology of development of some embryo/fetal parts and determine gestational age through measurement of different fetal parts from the day of pregnancy detection to 83rd day of gestation using B- mode ultrasonography. First day of detection of embryonic vesicle was as early as 15 days post-mating (mean day 17.7±2.3), embryo proper and fetal membranes on day 23.0± 1.0, fetal heart beat detected on day 26, C-shape embryo appeared on day 31 ±1.7, limb buds on day 47.3 ±1.5, ossification of limbs, head, ribs and vertebrae were first detected on day 51±.2.6, 53±1.0, 53.7±2.1 and 53.7±2.1 respectively. The kidneys, stomach, spinal tube, jugular/carotid, intestines and urinary bladder were first detected on days 42.7±1.1, 43.7±0.6, 43.7±1.5, 54.3±0.6, 62± 1.0 and 63 ±1.7 respectively. The genital tubercle located with a high degree of confidence on day 81-82 of gestation. All measurements were found highly correlated to the gestational age and R2 of CRL, EV, AD, EBD, BPD, KIL and STL were 0.943, 0.970, 0.989, 0.931, 0.943, 0.924 and 0.972 respectively. Simple regression equations were established to predict early fetal age in the dromedary camel. In conclusion, ultrasonography proved to be very useful tool to assess conceptus development and estimate fetal age based on appearance and fetometry in the dromedary camel.

Developmental mutant mouse models for external genitalia formation.

Development of external genitalia and perineum is the subject of developmental biology as well as toxicology and teratology researches. Cloaca forms in the lower (caudal) end of endoderm. Such endodermal epithelia and surrounding mesenchyme interact with various signals to form the external genitalia. External genitalia (the anlage termed as genital tubercle: GT) formation shows prominent sexually dimorphic morphogenesis in late embryonic stages, which is an unexplored developmental research field because of many reasons. External genitalia develop adjacent to the cloaca which develops urethra and corporal bodies. Developmental regulators including growth factor signals are necessary for epithelia-mesenchyme interaction (EMI) in posterior embryos including the cloaca and urethra in the genitalia. In the case of male type urethra, formation of tubular urethra proceeds from the lower (ventral) side of external genitalia as a masculinization process in contrast to the case of female urethra. Mechanisms for its development are not elucidated yet due to the lack of suitable mutant mouse models. Because of the recent progresses of Cre (recombinase)-mediated conditional target gene modification analyses, many developmental regulatory genes become increasingly analyzed. Conditional gene knockout mouse approaches and tissue lineage approaches are expected to offer vital information for such sexually dimorphic developmental processes. This review aims to offer recent updates on the progresses of these emerging developmental processes for the research field of congenital anomalies.

Differential cell proliferation and cell death during the urethral groove formation in guinea pig model.

Urethral groove (UG) formation is an important step in penile formation. Because commonly used animal models do not have UG, the mechanisms of UG formation have never been discovered. We aim to discover the cellular mechanism of the UG formation using guinea pig model. Histology was used to study the ontogeny of UG. BrdU immunofluorescence was used to label proliferating cells, cell death was determined using LysoTracker Red and TUNEL staining, and stereology was used for quantification. To reveal Shh mRNA expression patterns, in situ hybridization was performed in guinea pig genital tubercles (GTs) and ShhGFPcre-LacZ-reporter mice were used for comparison. Cell proliferation in the outer layers and programmed cell death in the inner layers of urethral epithelium played key roles during urethral canal movement from dorsal to ventral aspect and final opening to form UG. Shh mRNA expression domain shifted out to the ventral surface of GT from proximal throughout to distal in guinea pigs, but was excluded from the ventral surface epithelium in midshaft and distal of mouse GT. Differential cell proliferation and cell death in developing urethral epithelium lead to UG formation and Shh expression in ventral surface epithelium of GT may play an important role.

Embryology of the Female Genital System

Until the seventh week of human embryonic development of both sexes have very similar primordia of genitalia represented by two undifferentiated gonads two mesonephric ducts, which originate the male genital tract and two paramesonephric ducts develop the female genital tract. Genital tubercle, two labiouretrales folds and two labioscrotal folds: Externally the same basic elements that are distinguished in both sexes. From SRY gene expression that occurs during the eighth week a series of morphophysiological events leading establishing a clear sexual dimorphism starts. If the resulting gonad is a testis produced hormones induce masculinization of internal and external genitalia, as well as outline the breast. However, if an ovary is formed or not formed gonads, internal and external genitalia develop in female sense. Genetic sex is not always related to the differentiation of external genitalia or genital tract that is why we consider separately each. This article explores the morphological differentiation into male and female connection, as well as the molecular regulation of the gonads, genital tract and external genitalia.

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.

Fetal sexing in small ruminants through visualization of the genital tubercle

SUMMARY The objective of this study was to verify the accuracy of sexing and fetal number estimation in small ruminants by ultrasonographic examination. Fifty fetuses from 36 sheep and 23 fetuses from 11 goats were evaluated. In the case of sheep, twenty-four, ten, and two pregnancy were single, double, and triple, respectively. Regarding the goats, three, five, two, and one simple pregnancy were single, double, triple, and quadruple, respectively. The evaluations were performed on days 55 and 65 of gestation, by means of transrectal ultrasonographic examination, using a dual-frequency linear transducer, (6.0 and 8.0 MHz). Fetal sex was diagnosed by the location of the genital tubercle or visualization of external genitalia. The accuracy of fetal sexing was evaluated using the Chi-square test (X2) considering a 5% significance level. The gestational period interfered in the accuracy of fetal sexing in both species (P &lt;0.05). Obtained results showed 30% and 82.61% accuracy on day 55 of gestation, and 90% and 95.83% on day 65 for goats and sheep, respectively. The fetal sex and the type of pregnancy did not interfere in the sexing accuracy in both species during the evaluated periods (P&gt; 0.05). Thus, B-mode ultrasonography is an efficient method to perform an early diagnosis of fetal sex and to determine the number of fetuses in the studied small ruminants on day 65 of gestation. In addition, the accuracy of fetal sexing is not influenced by the type of pregnancy or fetal sex.

Development of the human penis and clitoris

The human penis and clitoris develop from the ambisexual genital tubercle. To compare and contrast the development of human penis and clitoris, we used macroscopic photography, optical projection tomography, light sheet microscopy, scanning electron microscopy, histology and immunohistochemistry. The human genital tubercle differentiates into a penis under the influence of androgens forming a tubular urethra that develops by canalization of the urethral plate to form a wide diamond-shaped urethral groove (opening zipper) whose edges (urethral folds) fuse in the midline (closing zipper). In contrast, in females, without the influence of androgens, the vestibular plate (homologue of the urethral plate) undergoes canalization to form a wide vestibular groove whose edges (vestibular folds) remain unfused, ultimately forming the labia minora defining the vaginal vestibule. The neurovascular anatomy is similar in both the developing human penis and clitoris and is the key to successful surgical reconstructions.

Single nucleotide polymorphisms of HAAO and IRX 6 genes as risk factors for hypospadias

PurposeWe evaluated the association of hypospadias with 17 susceptibility loci previously identified by a European genome-wide association study (GWAS) in Japanese patients. We also examined the expression of candidate genes in male mouse embryos to discuss the possible underlying mechanisms of this disease. Materials and MethodsWe enrolled 169 Japanese patients (mean age at surgery: 3.7 years) who underwent repair of hypospadias. Genotyping of 17 SNPs was performed by using a multiplex-polymerase chain reaction (PCR) invader assay. We also performed in situ hybridization to determine whether candidate genes were expressed in the male genital tubercle during embryonic development of the external genitalia in mice. ResultsSNP rs3816183 of HAAO was significantly associated with susceptibility to hypospadias in general (p=0.0019) and both anterior/middle hypospadias (p=0.0283) and posterior hypospadias (p=0.0226), while SNP rs6499755 of IRX6 showed an association with susceptibility to anterior/middle hypospadias (p=0.0472). In mouse embryos, there was no significant upregulation of Haao expression in the developing male external genitalia. Irx3 and Irx5, which are linked to Irx6 within the IrxB cluster, were expressed in the mesenchyme remote from the urethral plate epithelium during the critical embryonic period for masculinization. Irx6 was expressed in the ectodermal epithelium, showing prominent dorsal ectodermal expression without expression in ventral ectoderm adjacent to the urethral plate during the same period. ConclusionsGenetic variations of HAAO and IRX6 influence susceptibility to hypospadias in Japanese population, although further study will be needed to clarify the mechanism by which contributes to the pathogenesis of hypospadias.

Maternal exposure to di-n-butyl phthalate (DBP) promotes epithelial-mesenchymal transition via regulation of autophagy in uroepithelial cell

Maternal exposure to di-n-butyl phthalate (DBP) induces hypospadias, but the underlying mechanisms remain elusive. Here we hypothesize that aberrant activation of autophagy and epithelial-mesenchymal transition (EMT) are the leading cause of DBP-related hypospadias. Pregnant rats received DBP orally at a dose of 750 mg/kg/day during gestational days 14–18. In DBP-induced hypospadiac male offspring, immunohistochemistry (IHC) staining and Western blot showed increased expression of autophagy and EMT markers in genital tubercle (GT) tissue compared to the control. In addition, lower testosterone levels and androgen receptor (AR) expression in GT tissue were detected. In vitro studies revealed that impaired AR signaling was involved in DBP-induced autophagy and autophagy activation furthermore promoted EMT in urethral epithelial cells. DBP combined with chloroquine, an autophagy inhibitor, reduced the expression of EMT markers compared with DBP treatment alone, while DBP combined with the autophagy inducer rapamycin elevated the expression of EMT markers. The autophagy-lysosomal pathway inhibitor CQ but not proteasome inhibitor MG-132 rescued the decrease of E-cadherin after DBP treatment, which indicated autophagy-induced E-cadherin degradation contributes to DBP-related EMT. Taken together, our findings show that prenatal exposure to DBP induces abnormal autophagy and EMT that may play important roles in hypospadias development.

In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

The human genital tubercle is steroidogenic organ at early pregnancy.

It is generally accepted that androgens produced by fetal Leydig cells (FLC) control proper masculinization of the male external genitalia. Here, we hypothesized that the human genital tubercle (GT) has potential to synthesize androgens independently of FLC at early pregnancy. We observed that human GT of both genders have capacity to synthesize steroids of the Δ4, Δ5 and alternative pathway of DHT synthesis including the androgen itself. The presence of steroids in the GT was associated with the expression of corresponding steroidogenic enzymes. Levels of steroids and the expression of steroidogenic enzymes were similar in the GT from male and female fetuses. In contrast to the GT, the human fetal testis synthesized DHT from testosterone but not via the alternative pathway. Our findings strongly suggest that the human GT at early pregnancy can synthesize DHT via the alternative pathway, which may play an important role in organogenesis of the urethra.

Equine foetal gender determination in mid- to late gestational mares: A practical inquiry.

In recent years, the interest in equine foetal gender determination (FGD) during gestation increased remarkably. Ultrasonographic FGD can be performed in two different periods during gestation. The earliest examination can take place at a gestational age of 60-70days, whereby the genital tubercle is used to differentiate between male and female foeti. The time window of the second approach is wider (120-210days), and there are more characteristics to take into consideration. In this article, the feasibility and accuracy of ultrasonographic FGD in mid- to late gestation are evaluated. One hundred twenty-one mares from different breeds with a pregnancy stage between 120 and 270days were examined once, using B-scale ultrasonography (Esaote MyLab™ClassC). None of the mares were sedated nor shaved, and the procedure was completed within 15min. Diagnosis was firstly based on the gonads. The final judgement was made based on all visible foetal reproductive organs. In three cases with a pregnancy stage beyond 257days, FGD was not possible. All of the examined mares in which a FGD could be performed gave birth to a healthy foal. In 98% of the examinations (116/118), the diagnosis was correctly made. In both cases of misdiagnosis, only one characteristic was seen during the procedure and wrongly interpreted. Beyond 210days of pregnancy, the extremities can preclude a good visualization of the inguinal region. In conclusion, equine FGD in mid- to late gestation is an accessible and accurate technique, although a good ultrasound device is a prerequisite and experience and expertise is necessary.

Role of PERK-eIF2α signaling pathway in fetal male rats with hypospadias induced by di-n-butyl phthalate

This study aims to explore the role of PERK-eIF2α signaling pathway in fetal male rats with hypospadias induced by maternal exposure to di-n-butyl phthalate (DBP). DBP was used to treat pregnant SD rats by gastric intubation from gestation day (GD) 14–18 to construct a hypospadias rat model. The amount, weight, anogenital distance (AGD), and hypospadias incidence of rats were recorded and the genital tubercle (GT) of fetal male rats was collected on GD 19. Western blotting was performed to detect the expressions of PERK-eIF2α pathway- and autophagy-related proteins, and cell apoptosis was detected using TUNEL method. Then, GT fibroblasts of fetal rats were obtained and transfected with PERK-siRNA to detect cell apoptosis and autophagy in each transfected group. The incidence of hypospadias was 43.49% in fetal male rats induced by DBP. The fetal rats in DBP group presented the decreased birth weight and anogenital distance (AGD)/body weight ratio than the Control group (all P < 0.05). Further, p-PERK, p-eIF2α and ATF4 protein expressions and the ratio of LC3-II/LC3-I were greatly increased in the GTs of fetal rats, while apoptosis index (AI) and P62 protein expression were evidently decreased (all P < 0.05). In addition, the apoptosis rate was increased in GT fibroblasts after transfection of PERK-siRNA with the increased P62 and reduced LC3-II/LC3-I ratio (all P < 0.05). Activation of PERK-eIF2α signaling pathway can influence the GT development of fetal male rats with hypospadias induced by DBP through activation of autophagy and inhibition of apoptosis.

Contrasting mechanisms of penile urethral formation in mouse and human

This paper addresses the developmental mechanisms of formation of the mouse and human penile urethra and the possibility that two disparate mechanisms are at play. It has been suggested that the entire penile urethra of the mouse forms via direct canalization of the endodermal urethral plate. While this mechanism surely accounts for development of the proximal portion of the mouse penile urethra, we suggest that the distal portion of the mouse penile urethra forms via a series of epithelial fusion events. Through review of the recent literature in combination with new data, it is unlikely that the entire mouse urethra is formed from the endodermal urethral plate due in part to the fact that from E14 onward the urethral plate is not present in the distal aspect of the genital tubercle. Formation of the distal portion of the mouse urethra receives substantial contribution from the preputial swellings that form the preputial-urethral groove and subsequently the preputial-urethral canal, the later of which is subdivided by a fusion event to form the distal portion of the mouse penile urethra. Examination of human penile development also reveals comparable dual morphogenetic mechanisms. However, in the case of human, direct canalization of the urethral plate occurs in the glans, while fusion events are involved in formation of the urethra within the penile shaft, a pattern exactly opposite to that of the mouse. The highest incidence of hypospadias in humans occurs at the junction of these two different developmental mechanisms. The relevance of the mouse as a model of human hypospadias is discussed.

Sex-Related Differences in Urethra Development in Human Embryos

Virtually no information on the chronology of prenatal development of the human urinary tract and the sex-related differences in the emergence of urinary tract topography during embryonic development is presently available. The aim of our work was to study sex-related differences in urethra development in human embryos and early fetuses. Forty-nine preparations of human embryos and early fetuses without external signs of anatomical abnormalities were studied in order to achieve the aim and fulfill the objectives of the study. Embryos and early fetuses were divided into six groups according to gestational age and parietococcygeal length. The complex of adequate methods of morphological research used in the study included preparation and microscopy of serial histological and anatomical sections of human embryos, including female and male urinary tracts, preparation of 3D-reconstruction models, and morphometry. The formation of prostatic urethra, a derivative of the urogenital sinus, was shown to occur at the beginning of the ninth week of embryogenesis, and the primordium of the internal sphincter of the urinary tract was formed at the end of the tenth week. Formation of the terminal part of spongy urethra took place during weeks 10–11 and involved funnel-like protrusion of the ectoderm from the top of the balanus towards the urethra lumen. The secondary ventral displacement of the urethral opening does not occur in female fetuses, and, therefore, only the prostatic urethra is a homolog of the female urinary tract. The pelvic part of the urogenital sinus was transformed into the prostatic urethra and the membranous urethra of the male at the end of the first stage of fetal development. Elongation of the genital tubercle (a penis primordium) and formation of the urethral ridge walls that involved the urogenital folds occurred at the same time.

External Genital Development, Urethra Formation, and Hypospadias Induction in Guinea Pig: A Double Zipper Model for Human Urethral Development

To determine whether the guinea pig phallus would be an appropriate model of human penile development, we characterized the embryology and sexual differentiation of guinea pig external genitalia and attended to induce hypospadias in males and tubular urethra formation in females pharmacologically. The external genitalia of guinea pig were collected from genital swelling initiation to newborn stages, and scanning electronic microscopy and histology were performed to visualize the morphology and structure. Immunohistochemistry was used to determine the androgen receptor localization. Bicalutamide and methyltestosterone were given to pregnant dams to reveal the role and timing of androgen in guinea pig penile masculinization. Canalization and dorsal-to-ventral movement of the urethral canal develops the urethral groove in both sexes, and then the males perform distal-opening-proximal-closing to form tubular urethra. More nuclear-localized androgen receptor is found in proximal genital tubercles of males than in females at (E) 29. Antiandrogen treatment at E26-E30 can cause hypospadias, and methyltestosterone administration at E27-E31 can induce tubular urethra formation in females. Fetal development of the guinea pig phallus is homologous to that of humans. Although guinea pig has structures similar to mouse, the urethral groove and the tubular urethra formation are more similar to humans. Antiandrogen treatment causes hypospadias in males and additional androgen induces tubular urethra formation in females. Thus, guinea pig is an appropriate model for further study of cellular and molecular mechanisms involved in distal-opening-proximal-closing in tubular urethra formation and the evaluation of the pathophysiological processes of hypospadias.

Isl1 mediates mesenchymal expansion in the developing external genitalia via regulation of Bmp4, Fgf10 and Wnt5a.

Genital malformations are among the most common human birth defects, and both genetic and environmental factors can contribute to these malformations. Development of the external genitalia in mammals relies on complex signaling networks, and disruption of these signaling pathways can lead to genital defects. Islet-1 (ISL1), a member of the LIM/Homeobox family of transcription factors, has been identified as a major susceptibility gene for classic bladder exstrophy in humans, a common form of the bladder exstrophy-epispadias complex (BEEC), and is implicated in a role in urinary tract development. We report that deletion of Isl1 from the genital mesenchyme in mice led to hypoplasia of the genital tubercle and prepuce, with an ectopic urethral opening and epispadias-like phenotype. These mice also developed hydroureter and hydronephrosis. Identification of ISL1 transcriptional targets via ChIP-Seq and expression analyses revealed that Isl1 regulates several important signaling pathways during embryonic genital development, including the BMP, WNT, and FGF cascades. An essential function of Isl1 during development of the external genitalia is to induce Bmp4-mediated apoptosis in the genital mesenchyme. Together, these studies demonstrate that Isl1 plays a critical role during development of the external genitalia and forms the basis for a greater understanding of the molecular mechanisms underlying the pathogenesis of BEEC and urinary tract defects in humans.