Mouse Genital Tubercle Research Articles

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.

Requirement for basement membrane laminin α5 during urethral and external genital development

Hypospadias, a congenital malformation of the penis characteristic of an abnormal urethral orifice, affects 1 in every 125 boys, and its incidence is rising. Herein we test the hypothesis that the basement membrane protein laminin α5 (LAMA5) plays a key role in the development of the mouse genital tubercle, the embryonic anlage of the external genitalia. Using standard histological analyses and electron microscopy, we characterized the morphology of the external genitalia in Lama5 knockout (LAMA5-KO) mouse embryos during both androgen-independent genital tubercle development and androgen-mediated sexual differentiation. We compared regulatory gene expression between control and LAMA5-KO by in situ hybridization. We also examined the epithelial structure of the mutant genital tubercle using immunofluorescence staining and histological analyses of semi-thin sections. We found that Lama5 was expressed in both ectodermal and endodermal epithelia of the cloaca. The LAMA5-KO displayed a profound external genital malformation in which the genital tubercle was underdeveloped with a large ectopic orifice at the proximal end. In older embryos, the urethra failed to form a tubular structure and was left completely exposed. These defects were not associated with a significant alteration in regulatory gene expression, but rather with a defective ectodermal epithelium and an abnormal disintegration of the cloacal membrane. We conclude that LAMA5 is required in the basement membrane to maintain normal architecture of the ventral ectoderm during genital tubercle development, which is essential for the formation of a tubular urethra. Perturbation of LAMA5, and possibly other basement membrane components, may cause hypospadias in humans.

Molecular Characterization of the Genital Organizer: Gene Expression Profile of the Mouse Urethral Plate Epithelium

Lower urinary tract malformations are among the most common congenital anomalies in humans. Molecular genetic studies of mouse external genital development have begun to identify mechanisms that pattern the genital tubercle and orchestrate urethral tubulogenesis. The urethral plate epithelium is an endodermal signaling region that has an essential role in external genital development. However, little is known about the molecular identity of this cell population or the genes that regulate its activity. We used microarray analysis to characterize differences in gene expression between urethral plate epithelium and surrounding tissue in mouse genital tubercles. In situ hybridizations were performed to map gene expression patterns and ToppCluster (https://toppcluster.cchmc.org/) was used to analyze gene associations. A total of 84 genes were enriched at least 20-fold in urethral plate epithelium relative to surrounding tissue. The majority of these genes were expressed throughout the urethral plate in males and females at embryonic day 12.5 when the urethral plate is known to signal. Functional analysis using ToppCluster revealed genetic pathways with known functions in other organ systems but unknown roles in external genital development. Additionally, a 3-dimensional molecular atlas of genes enriched in urethral plate epithelium was generated and deposited at the GUDMAP (GenitoUrinary Development Molecular Anatomy Project) website (http://gudmap.org/). We identified dozens of genes previously unknown to be expressed in urethral plate epithelium at a crucial developmental period. It provides a novel panel of genes for analysis in animal models and in humans with external genital anomalies.

Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies

Congenital penile anomalies (CPAs) are among the most common human birth defects. Reports of CPAs, which include hypospadias, chordee, micropenis, and ambiguous genitalia, have risen sharply in recent decades, but the causes of these malformations are rarely identified. Both genetic anomalies and environmental factors, such as antiandrogenic and estrogenic endocrine disrupting chemicals (EDCs), are suspected to cause CPAs; however, little is known about the temporal window(s) of sensitivity to EDCs, or the tissue-specific roles and downstream targets of the androgen receptor (AR) in external genitalia. Here, we show that the full spectrum of CPAs can be produced by disrupting AR at different developmental stages and in specific cell types in the mouse genital tubercle. Inactivation of AR during a narrow window of prenatal development results in hypospadias and chordee, whereas earlier disruptions cause ambiguous genitalia and later disruptions cause micropenis. The neonatal phase of penile development is controlled by the balance of AR to estrogen receptor α (ERα) activity; either inhibition of androgen or augmentation of estrogen signaling can induce micropenis. AR and ERα have opposite effects on cell division, apoptosis, and regulation of Hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling in the genital tubercle. We identify Indian hedgehog (Ihh) as a novel downstream target of AR in external genitalia and show that conditional deletion of Ihh inhibits penile masculinization. These studies reveal previously unidentified cellular and molecular mechanisms by which antiandrogenic and estrogenic signals induce penile malformations and demonstrate that the timing of endocrine disruption can determine the type of CPA.

Expression Analysis of DGKK during External Genitalia Formation

Genetic variants in diacylglycerol kinase κ (DGKK) have been strongly associated with risk of hypospadias. We investigated the expression pattern of Dgkk during development of mouse external genitalia to better understand its function and mechanism in the etiology of hypospadias. We performed Dgkk expression analysis via indirect immunofluorescence in histological sections of CD-1 mouse embryonic and postnatal male, female and diethylsilbestrol treated external genitalia. Histological findings were supplemented with DGKK expression analysis using quantitative real-time polymerase chain reaction assays. In mouse external genitalia Dgkk was expressed in the membrane and cytoplasm of differentiating squamous epithelial cells of urethral plate/groove and epidermis but not in the undifferentiated epithelial cells of preputial lamina or basal layer of urethral groove epithelium. CD-1 gestation day 18male mouse genital tubercle treated with oil or diethylstilbestrol showed similar patterns of Dgkk expression despite many morphological differences, including formation of preputial cleft observed in diethylsilbestrol treated mice. Dgkk appears to be a marker or mediator of squamous cell differentiation during development of mouse external genitalia. However, no association exists between Dgkk expression and formation of preputial cleft in the genital tubercle of diethylsilbestrol treated mice, suggesting that these 2 events may follow independent pathways in mice. Further studies are needed to elucidate the role of DGKK in hypospadias.

Hypospadias and Genes Related to Genital Tubercle and Early Urethral Development

We determined whether variants in genes associated with genital tubercle (the anlage for the penis) and early urethral development were associated with hypospadias in humans. We examined 293 relatively common tag single nucleotide polymorphisms in BMP4, BMP7, FGF8, FGF10, FGFR2, HOXA13, HOXD13, HOXA4, HOXB6, SRY, WT1, WTAP, SHH, GLI1, GLI2 and GLI3. The analysis included 624 cases (81 mild, 319 moderate, 209 severe, 15 undetermined severity) and 844 population based nonmalformed male controls born in California from 1990 to 2003. There were 28 single nucleotide polymorphisms for which any of the comparisons (ie overall or for a specific severity) had a p value of less than 0.01. The homozygous variant genotypes for 4 single nucleotide polymorphisms in BMP7 were associated with at least a twofold increased risk of hypospadias regardless of severity. Five single nucleotide polymorphisms for FGF10 were associated with threefold to fourfold increased risks, regardless of severity. For 4 of them the results were restricted to whites. For GLI1, GLI2 and GLI3 there were 12 associated single nucleotide polymorphisms but results were inconsistent by severity and race/ethnicity. For SHH 1 single nucleotide polymorphism was associated with a 2.4-fold increased risk of moderate hypospadias. For WT1 6 single nucleotide polymorphisms were associated with approximately a twofold increased risk, primarily for severe hypospadias. This study provides evidence that single nucleotide polymorphisms in several genes that contribute to genital tubercle and early urethral development are associated with hypospadias risk.

Functional analysis of ectodermal β-catenin during external genitalia formation.

β-catenin is a molecule belonging to the armadillo family of proteins that is a crucial core-component of cellular adherens junctions, and a component of the canonical Wnt-signaling pathway. We attempted to analyze the functional significance of ectodermal-derived β-catenin during the development of the mouse genital tubercle, a mammalian anlage of the external genitalia. For this purpose, the conditional loss of function mouse mutant Wnt7a-Cre;β-cat(f/f) was utilized. Loss of ectodermal β-catenin leads to the formation of urethral cleft during preputial uprising. Although expression of E-cadherin was retained in the genital tubercle ectoderm of mutants, probably through plakoglobin compensatory expression, expression of other crucial adherens junction components such as α-catenin and F-actin in the cell-cell border were distinctly reduced. We also showed that β-catenin is necessary for the expression of its transcriptional downstream target Lef-1 which was localized in the basal layer of the preputial ectoderm, excluding the midventral region at E15.5. Such specialized region was observed to possess cytoplasmic β-catenin expression at this stage. Coincidentally, mitotically active cells were also found in the basal layer of the preputial ectoderm excluding the midventral region. In mutant genital tubercle, cell proliferation in the preputial ectoderm was decreased. Taken together, we suggest that ectodermal β-catenin is necessary not only to maintain adherens junction integrity, but also to regulate cell proliferation possibly through Lef-1 functions. Thus, β-catenin is shown to perform dual functions, initially as an adhesion molecule and later on as a possible transcription factor.

RWDD1 interacts with the ligand binding domain of the androgen receptor and acts as a coactivator of androgen-dependent transactivation

During embryogenesis, the development of the male genital is dependent on androgens. Their actions are mediated by the androgen receptor (AR), which functions as a transcription factor. To identify AR coregulators that support AR action during the critical time window of androgen-dependent development in the genital tubercle of male mice, we performed yeast two-hybrid screenings with cDNA libraries of genital tubercles from male mouse embryos using human AR as bait. RWD domain containing 1 (RWDD1) was identified as an AR-interacting protein from three independent libraries of the embryonic days E15, E16 and E17. The interaction between the AR and RWDD1 was confirmed in vitro and in vivo and the ligand binding domain of the AR was shown to be sufficient to mediate the interaction. RWDD1 enhanced AR-dependent transactivation in reporter assays with promoters of different complexity and in different cell lines. These results suggest that RWDD1 functions as a coactivator of androgen-dependent transcription.

Morphology of Mouse External Genitalia: Implications for a Role of Estrogen in Sexual Dimorphism of the Mouse Genital Tubercle

We examined the role of androgens and estrogens in mammalian sexual differentiation by morphological characterization of adult wt and mutant mouse external genitalia. We tested the hypothesis that external genitalia development depends on androgen and estrogen action. We studied serial sections of the external genitalia of the CD-1 and C57BL6 wt strains of adult mice (Charles River Laboratories, Wilmington, Massachusetts). We recorded linear measurements of key structures in each specimen, including the urethra, erectile tissue, bone and cartilage. We used similar methodology to analyze mice mutant for estrogen receptor α (αERKO) and androgen receptor (X(Tfm)/Y) (Jackson Laboratory, Bar Harbor, Maine). Morphology in X(Tfm)/Y adult murine external genitalia was remarkably similar to that in wt females. Bone and clitoral length was similar in wt females and X(Tfm)/Y mice. Conversely the αERKO clitoris was 59% longer and bone length in αERKO females was many-fold longer than that in female wt mice or X(Tfm)/Y mutants. The αERKO clitoris contained cartilage, which is typical of the wt penis but never observed in the wt clitoris. Serum testosterone was not increased in female αERKO mice 10 days postnatally when sex differentiation occurs, suggesting that masculinization of the αERKO clitoris is not a function of androgen. Masculinization of the αERKO clitoris suggests a role for estrogen in the development of female external genitalia. We propose that normal external genital development requires androgen and estrogen action.

MP-01.05: Organotypic Mouse Genital Tubercle Culture as a Model for the Investigation of the Effect of Estrogen on Fetal Penis and Urethral Development

The genital tubercle (GT), an anlage of the external genitalia, differentiates into the penis in males and the clitoris in females. Estrogen plays an important role in the development and differentiation of various organs that are hormonally sensitive. Maternal exposure to synthetic estrogen has been shown to disrupt urethral seam closure in male offspring, causing hypospadias. The purpose of this paper was to develop an organotypic genital tubercle culture system in vitro and to use it to investigate the direct effects of hyperestrogenic state on fetal mouse penile and urethral development.

Estrogen Effects on Fetal Penile and Urethral Development in Organotypic Mouse Genital Tubercle Culture

We developed an organotypic genital tubercle culture system in vitro and used it to investigate the direct effects of the hyperestrogenic state on fetal mouse penile and urethral development. Genital tubercles were dissected from embryonic day 14.5 C57B/L6 male mouse fetuses and cultured using an air-liquid interface on a microporous membrane support soaked in synthetic medium. Cultures were separated into 4 groups. Groups 1 to 3 were supplied with 10 nM dihydrotestosterone, estradiol and 10 nM dihydrotestosterone plus estradiol, respectively. Group 4 was cultured in hormone-free medium. After 36 to 72-hour culture morphological, histological, proliferation, apoptosis, androgen signaling and activating transcription factor 3 analyses were done. The physiological concentration of 10 nM dihydrotestosterone was essential for genital tubercle growth in vitro. Androgen induced growth and urethral development were significantly suppressed by high dose estrogen. Concurrently we observed increased apoptosis and decreased proliferation in the mesenchyma. Androgen signaling was disrupted and activating transcription factor 3, a factor related to hypospadias genesis, was up-regulated. High dose estrogen suppressed male genital tubercle development in vitro. The organotypic genital tubercle culture system in vitro consisting of urethral epithelial and mesenchymal cells can recapitulate the hormonal sensitivity of fetal penile and urethral development. This method is potentially useful for studying the effects of various factors, particularly endocrine disruptors.

Di‐(2‐ethylhexyl) Phthalate Upregulates ATF3 Expression and Suppresses Apoptosis in Mouse Genital Tubercle

To investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) on the expression of activating transcription factor 3 (ATF3) and apoptosis of fetal mouse genital tubercle (GT). In this developmental toxicity study, pregnant C57BL/6 mice were exposed to corn oil or DEHP (100 or 500 mg/kg/day) from embryonic day 12 (ED12) to ED16. Apoptosis was characterized by Terminal transferase dUTP nick end labeling (TUNEL) assay. Using RT-PCR and western blot, the expressions of ATF3 and apoptosis-related genes (P53, Bcl-2 and Bax) were investigated. Apoptosis of fetal mouse GT cells notably decreased after DEHP treatment. DEHP activated ATF3 both at the mRNA and protein levels in GT. Furthermore, pro-apoptotic P53 was downregulated and the ratio of anti-apoptotic (Bcl-2)/pro-apoptotic (Bax) was not significantly changed. These results suggest that DEHP may induce external genital defects via a mechanism involving apoptosis, which might correlate with the regulation of ATF3 and P53 expressions.

Progesterone Receptors in the Developing Genital Tubercle: Implications for the Endocrine Disruptor Hypothesis as the Etiology of Hypospadias

In fetal mice genital tubercles the ontogenetic expression of progesterone receptors and the effect of in utero estrogen and testosterone exposure were investigated. To evaluate ontogenetic progesterone receptor expression genital tubercles from untreated fetuses at gestational days 12, 14, 16 and 18, and newborn pups were prepared for real-time reverse transcriptase-polymerase chain reaction or immunohistochemistry. To evaluate estrogen and testosterone effects pregnant dams were gavaged once daily with corn oil (vehicle), ethinyl estradiol or testosterone propionate from gestational days 12 through 17. At gestational day 19 the genital tubercles of delivered fetuses were harvested for morphological examination and then pooled for real-time reverse transcriptase-polymerase chain reaction. Progesterone receptor protein was first detected at gestational day 12 in the urethral plate and mesenchyma. At later stages staining intensity increased with a greater progesterone receptor signal, especially in the urethra. Progesterone receptor mRNA expression showed different increasing patterns in each sex until birth. However, no difference was noted between male and female genital tubercles in terms of the distribution and quantity of progesterone receptor expression. In utero ethinyl estradiol led to 8.2, 9.7 and 5.2-fold increases in progesterone receptor mRNA in females and in males with and without hypospadias, respectively. Testosterone propionate significantly decreased progesterone receptor mRNA levels in females and males. Progesterone receptors are expressed in developing genital tubercles, suggesting a direct role of progesterone in normal genital tubercle patterning. Their increasing expression until birth also implies increasing sensitivity of the genital tubercles to the effects of estrogenic and progestogenic endocrine disruptors during fetal life. Ethinyl estradiol and testosterone propionate lead to opposing effects on progesterone receptor expression, in addition to their opposing morphological effects on the genital tubercles. These findings expand our knowledge of genital tubercle morphogenesis and provide important information for understanding the effects of endocrine disruptors.

Estrogen Receptor-α and β are Differentially Distributed, Expressed and Activated in the Fetal Genital Tubercle

We examined the ontogenic and sex specific expression of estrogen receptor-alpha and beta in mouse genital tubercles and assessed the effects of in utero estrogen exposure on these parameters. Expression of the 2 genes was detected in mouse genital tubercles from fetuses collected on gestational days 12, 14, 16 and 18, and from newborns using immunohistochemistry and quantitative polymerase chain reaction. Pregnant dams were exposed to ethinyl estradiol or corn oil as the control. Estrogen receptor-alpha and beta proteins first appeared on gestational days 12 and 14, respectively. The 2 proteins were expressed in the urethral plate and mesenchyma. Staining intensity was more prominent in the mesenchyma for estrogen receptor-alpha and in the urethral plate for estrogen receptor-beta. Female genital tubercles expressed more estrogen receptor-alpha than male genital tubercles (p <0.01), while estrogen receptor-alpha expression increased gradually in the 2 sexes until birth. Estrogen receptor-beta expression did not differ between males and females, and it showed no notable variation during fetal life. Ethinyl estradiol led to a 2.1 and 3.8-fold increase in estrogen receptor-alpha expression in females and in males with hypospadias (p = 0.002 and 0.04, respectively). Estrogen receptor-beta expression did not change in response to ethinyl estradiol. This study provides in vivo evidence that estrogen receptor-alpha expression in the genital tubercles of each sex increases until parturition but estrogen receptor-beta expression does not, implying genital tubercle sensitivity to estrogen increases during fetal life. Exogenous administration of estrogens results in a response of increased expression of estrogen receptor-alpha but not of estrogen receptor-beta. These differential findings for estrogen receptor-alpha and beta imply that the 2 receptors may have different roles in normal or anomalous genital tubercle development.

Gene expression profiles in mouse urethral development

To analyse the gene expression profiles of the mouse genital tubercle (GT) during urethral tube development at embryonic (E) days E14, E15, E16 and E17, as the aetiology of hypospadias, one of the most common congenital anomalies, remains unknown. During GT development the urethral folds fuse to form an epithelial seam; subsequently, the epithelial seam disappears, resulting in the normal tubular urethra. Abnormalities in urethral seam formation and remodelling might explain hypospadias, and elucidating the molecular developmental mechanisms underlying normal penile development might provide the basis for understanding hypospadias. Total RNA was isolated from the genital tubercle at embryonic days E14, E15, E16, and E17. Together with reference RNA, sample RNA was labelled with Cy-3 and Cy-5 respectively and hybridized to a 16 000-mouse gene array that included the Incyte GEM2.1 and NIA 7k sets. Candidate genes were analysed by immunohistochemistry and real-time polymerase chain reaction. Using cDNA microarrays, we identified the up-regulation of genes involved in the transforming growth factor (TGF)-beta and Wnt-Frizzled pathways, and of thrombospondin (TSP) 4, a member of a cell-migration molecule family, all candidates for involvement in urethral tube formation. Immunohistochemistry showed TGFbeta1, TGFbeta receptor III, and Frizzled1 were expressed exclusively in E14-E17 urethral epithelium. TSP4 was expressed in the mesenchymal basal layer underlying E17 GT skin epidermis. Many signalling pathways are involved in late GT development, and cell migration molecules might have an important role in urethral tube formation.

Ontogeny of Androgen Receptor and Disruption of Its mRNA Expression by Exogenous Estrogens During Morphogenesis of the Genital Tubercle

The ontogeny of androgen receptor expression in male and female mouse genital tubercles, and the effects of in utero ethinyl estradiol exposure on androgen receptor mRNA expression in the hypospadias model were studied. Androgen receptor mRNA expression was measured in mouse genital tubercles from fetuses and pups collected on gestational days 12, 14, 16 and 18, and from newborns using immunohistochemistry and real-time quantitative polymerase chain reaction. Pregnant dams were exposed to ethinyl estradiol or corn oil as controls from gestational days 12 to 17. Genital tubercles of gestational day 19 fetuses were then examined by further quantitative polymerase chain reaction analysis after identification of the seam area using a dissecting microscope to diagnose hypospadias in males. Androgen receptor protein was detected in genital tubercles by gestational day 14. Androgen receptor mRNA expression increased gradually in each sex during normal development. However, female genital tubercles expressed a higher level of androgen receptor mRNA throughout development compared to male genital tubercles (p <0.0001). In utero ethinyl estradiol exposure led to a 5.4 and 4.5-fold increase in androgen receptor mRNA in the genital tubercles of female and male embryos (p = 0.004 and 0.001, respectively). Hypospadiac male genital tubercles showed increased androgen receptor mRNA expression compared to control males (p = 0.006). Levels in hypospadiac males did not differ from those in control females but they were less than those in ethinyl estradiol treated females (p >0.05 and 0.01, respectively). Androgen receptor protein is expressed abundantly in male and female genital tubercles. Androgen receptor mRNA levels are higher in female than in male genital tubercles through development and they increase in response to in utero ethinyl estradiol exposure with ethinyl estradiol treated females having the highest levels of expression, followed by ethinyl estradiol treated hypospadiac males. We infer that higher estrogen in genital tubercles results in a physiological response of increased androgen receptor mRNA expression. We found no direct association between changes in androgen receptor mRNA expression and the presence or absence of hypospadias in males, suggesting that alterations in the expression of proteins other than or in addition to androgen receptor result in anomalous urethral development. This finding supports the idea that the etiology of hypospadias is multifactorial in origin.

Anatomical studies of the mouse genital tubercle.

To study the etiology of hypospadias, we propose the use of a mouse model, the embryonic mouse genital tubercle. In this study, we define the development of the mouse genital tubercle with special emphasis on urethral formation demonstrating anatomical similarities to human development. Serial sections of genital tubercles from embryonic male and female mice ages 14 to 21 days gestation from timed pregnant animals, newborn and adult mice were immunohistochemical stained with antibodies to E-cadherin, cytokeratins 7, 10, and 14. Patency of the urethral was assessed by india ink injection via the bladder. Urethral lumen morphology was determined by the creation of plastic resin cast. Surface morphology of the genital tubercle was defined by scanning electron microscopy. India Ink injection into the bladder showed that the urethral lumen was patent from 14 days gestation. Plastic resin casts revealed that the male urethra was characterized by a S shaped curve, the presence of the bulbar urethral gland and a longer length than age matched females. The ontogeny of the genital tubercle development revealed two epithelial edges that subsequently touched and fused into the completed urethra. During development cytokeratin immunohistochemical staining demonstrated that the epithelial cells of the urethral lumen are of bladder origin and the surface cells of skin origin. The functional and developmental anatomy of the mouse genital tubercle provides a useful model to study normal and abnormal human urethral development.

The effect of oestrogen and testosterone on the urethral seam of the developing male mouse genital tubercle.

To describe the effects of exogenous oestrogens and androgens on urethral formation in the mouse, as the development of the mouse and human urethra have significant similarities, and understanding normal male urethral development may help to identify the causes of abnormal development, e.g. hypospadias. Timed-pregnant C57/6 mice were exposed to synthetic oestrogens and androgens. The morphology of the genital tubercles was examined histologically and with three-dimensional computer reconstruction. Specific attention was focused on the developing urethral seam. Microscopic serial analysis confirmed the presence of an arrest in seam formation in about half of oestrogen-treated male fetuses. In contrast, there was acceleration of urethral fold fusion and a longer urethral tube in those treated with androgens. Oestrogen-treated fetuses had a thin periurethral spongiosa, in contrast to androgen-treated fetuses which developed a thicker periurethral spongiosa. The effect of oestrogens on seam area formation did not depend on the dose, but in contrast, in the androgen-treated fetuses it was. Oestrogens and androgens have a direct effect on the fusion of the urethral fold that leads to seam formation. Normal urethral development depends on the delicate balance of these complementary hormones.

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

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

Embryonic development of mouse external genitalia: insights into a unique mode of organogenesis.

The mammalian external genitalia are specialized appendages for efficient copulation, internal fertilization and display marked morphological variation among species. In this paper, we described the embryonic development of mouse genital tubercle (GT), an anlage of the external genitalia utilizing the scanning electron microscope (SEM) analysis. It has been shown that the Distal Urethral Epithelium (DUE) may fulfill an essential role in the outgrowth control of the GT. Our present SEM analysis revealed a small distal protrusion at the tip of the GT of normal embryos as well as some morphological differences between male and female embryonic external genitalia. Previous analysis shows that the teratogenic dose of Retinoic Acid (RA) induces a drastic marformation of the urethral plate, but not gross abnormalities for GT outgrowth. Interestingly, a small distal protrusion at the tip of GT was clearly observed also after RA treatement. Furthermore, we showed that treatment with anti-androgen flutamide resulted in the demasculinization of the GT in males. The unique character of GT development and the sexual dimorphism are discussed.