Genital Tubercle Research Articles

Deletion of fibroblast growth factor receptor 2b (Fgfr2b) gene results in anorectal malformation in mouse

Introduction: Anorectal malformations occur in 1 per 4,000 live births, and represent a surgical challenge. Fgfr2b is known to serve a key role in the development of other organ systems including parts of the gastrointestinal tract (GIT). We sought to evaluate the role of Fgfr2b in the development of normal anorectal structures. Methods:Fgfr2b expression in wild type (Wt) embryos was evaluated using whole mount in-situ hybridization. Wt and Fgfr2b-/- embryos were harvested from timed pregnant mothers at E10.5 through E18.5 and were analyzed for anorectal phenotype. Results: FGFR2 is expressed throughout the structures of the hindgut (data not shown). Fgfr2b-/- mutants demonstrate a genetically reproducible severe anorectal malformation, characterized by a shortened perineal body, an absence of pelvic rectum, and no visible distal recto-urinary fistula as shown in figure. Figure: E18.5 landscape view of perineum in Wt (a) and Fgfr2b-/- (b), transected tail (T), anus (A), genital tubercle (GT); Sagittal section of pelvis in Wt (c) and Fgfr2b-/- (d) bladder (B) urethra (U), genital tubercle (GT), rectum (R) and Anus (A). Conclusions: FGFR2 is expressed early in distal hindgut indicating an active role in development of normal anorectal continuity. Furthermore, Fgfr2b invalidation (Fgfr2b-/- mutant), results in a severe anorectal malformation as demonstrated in both landscape and cross sectional views. Fgfr2b function is required for development of anorectal continuity. Further mechanistic investigation of Fgfr2b in hindgut and anus interactions may lead to a better understanding of the mechanism of development these anomalies in humans.

The Impact of Prenatal Androgens on Vaginal and Urogenital Sinus Development in the Female Mouse

In females abnormal urogenital virilization can occur secondary to prenatal exposure to exogenous or endogenous androgens. We studied the effects of different doses of prenatal androgens on urogenital sinus development and the location of the vaginal confluence in a mouse model. Timed pregnant C57/6 mice were exposed to 2, 5 and 10 mg testosterone propionate on gestational days 14 through 18. On gestational day 19 the genital tubercles and internal genitalia were examined grossly and histologically for the presence of virilization. Three-dimensional computer reconstruction was done and plastic cast injection molds of the urogenital sinus were made in select specimens. Microscopic analysis confirmed the spectrum of virilization, which occurred in 98% of testosterone propionate treated female fetuses. Plastic cast injection showed that affected females had a longer urogenital sinus, more proximal confluence and shorter vagina in a dose dependent manner. Histological sections and 3-dimensional reconstruction revealed that the bladder neck moved proximal under the pubic bone, also in a dose dependent manner. Prenatal exposure to increasing levels of androgen causes urogenital sinus elongation in a female mouse fetus. In the mouse model the confluence area moves proximally together with the bladder neck in a dose dependent manner.

The urethral plate—does it grow into the genital tubercle or within it?: E.C. Penington and J.M. Hutson. Br J Urol Int 89:733–739, (May) 2002

The urethral plate—does it grow into the genital tubercle or within it?: E.C. Penington and J.M. Hutson. Br J Urol Int 89:733–739, (May) 2002

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.

Induction of hypospadias in a murine model by maternal exposure to synthetic estrogens

We tested the hypothesis that maternal exposure to synthetic estrogen can cause hypospadias in male offspring and defined the morphological changes in the disrupted urethral seam. Timed pregnant C57/6 mice were exposed to synthetic estrogens. The genital tubercles were examined for the presence of hypospadias using histology, three-dimensional computer reconstruction, and plastic cast injection molds of the urethra. Microscopic serial analysis confirmed the presence of hypospadias, which occurred in ∼50% of the synthetic-estrogen-treated male fetuses. No effect was seen in the female embryos. Plastic cast injection showed that affected males had a shorter total urethral length and loss of male anatomic features such as the prostatic utricle. Exposure to synthetic estrogens during pregnancy affects the normal development of the urethra in the mouse. We conclude that endocrine disrupters play an important role in genital tubercle anomalies.

The forkhead box F1 transcription factor is expressed in brain and head mesenchyme during mouse embryonic development

The forkhead box f1 (Foxf1) transcription factor is expressed in septum transversum mesenchyme and splanchnic (visceral) mesoderm, which give rise to mesenchymal cells of gut-derived organs such as liver, gall bladder, lung, stomach, and intestine. Foxf1 −/− embryos die in utero and haploinsufficiency of the Foxf1 gene resulted in a variety of developmental abnormalities of the lung, gall bladder, esophagus, and trachea and caused defects in liver and lung repair. In this study, we used Foxf1 +/− mouse embryos containing the β-Galactosidase (β-Gal) gene knocked into the Foxf1 locus to examine whether Foxf1 is expressed in the developing brain and head region. In addition to previously reported Foxf1 expression patterns, β-Gal staining was also found in the mesenchyme of developing pituitary gland, eye, pancreas, heart, notochord, genital tubercle, Meckel's cartilage, and cartilage primordia of nasal septum and vertebral bodies. In adult Foxf1 +/− mice, β-Gal staining was detected in anterior and posterior pituitary gland, astrocytes of the cerebellum and cerebral cortex, lens and retina of the eye, tracheal cartilage, parathyroid, cortical layer of thymus, capsule of the spleen, coronary arteries, and pancreatic blood vessels.

The role of androgens in development of the scrotum of the grey short-tailed Brazilian opossum ( Monodelphis domestica).

In eutherian mammals, sex differentiation is initiated by expression of the testis-determining gene on the Y chromosome. Subsequent phenotypic development of the reproductive tract and genitalia depends on the production of hormones by the differentiated testis. In marsupials the mechanisms of phenotypic development may vary from this pattern, as differentiation of the scrotal primordia has been shown to occur before that of the gonad. Thus, the development of the scrotum in the marsupial has been regarded as an androgen-independent process. We have sought to clarify the ontogeny of scrotal development and the appearance of androgen receptor immunoreactivity by examining Monodelphis domesticaembryos/pups from 1 day prior to birth until 2 days after birth. We have also used immunocytochemistry to determine the expression of the key steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase as an indicator of when the developing gonad may be capable of synthesizing androgens. Expression of this enzyme was first detected in the gonads and adrenals of both sexes 1 day prior to birth and before the appearance of scrotal bulges. Androgen receptor immunoreactivity was detected in the scrotal anlagen of male opossum pups as early as 1 day following birth. This finding is significantly earlier than previous reports and coincides with the appearance 1 day after birth of distinct scrotal bulges. Androgen receptor immunoreactivity was also observed in the genital tubercles of male pups, but not female pups, 2 days after birth. These results suggest that androgens may play an important role in the development of the male genitalia at a much earlier stage than that indicated by previously published work and that scrotal development in this species may not be androgen-independent.

Reproductive Applications of Ultrasound in the Cow

During the past decade portable ultrasound units that provide excellent image quality have become available to veterinary practitioners. The use of transrectal ultrasonography for evaluation of reproductive structures in cattle extends and enhances the diagnostic capabilities of practitioners beyond the traditional use of palpation per rectum. Ultrasonography of ovaries enables more consistent differentiation of ovarian follicles and corpora lutea. Accurate counting of follicles and precise measurement of follicles or corpora lutea is possible. Sequential monitoring of ovarian structures enables the user to characterize ovarian follicular waves, to identify dominant follicles and to detect ovulation. Images can be used to identify, objectively measure and to characterize the quality of a corpus luteum. Ultrasonography is also a valuable tool for identification of abnormal (cystic) luteal and follicular structures. Pregnancy diagnosis can be performed using ultrasound by day 19 to 24 post-breeding and fetal viability can be verified by visualization of a fetal heartbeat. Aging embryos and fetuses between days 20 and 100 of gestation can be facilitated by measuring crown-rump length (20-50 days) or the diameter of the head or trunk (50- 100 days). Sexing of bovine fetuses is accomplished by visualizing the genital tubercle (penis) and scrotum in males or by the lack of male genetalia and the presence of the genital tubercle (vulva) in females. Sex determination is most practical between days 60 and 85 of gestation. Like other diagnostic tools, ultrasound has limitations. Skill of the ultrasound technician in capturing clear images, differentiating anatomical orientations and interpreting ultrasound images is essential. The effective use of ultrasound technology enhances diagnostic capabilities and increases the services offered by veterinarians.

P057: Fetal gender determination by first trimester sonography

Objective: To determine the accuracy of first trimester sonography in identifying fetal gender. Materials and methods: This study was carried out prospectively in 250 singleton pregnancies who underwent sonography at 11–14 weeks of gestation (confirmed by CRL or BPD). Fetal genitalia was examined in a midsagittal plane and if the angle of genital tubercle to horizontal line through the lumbosacral skin surface was greater than 30′ the gender was assigned as male and when the genital tubercle was parallel or convergent less than 30′, female. The results were checked after birth. Results: In 22 (8.2%) patients fetal sex was not determined. Genital tubercle was identified in 228 (91.2%) patients. Sex determination was correct in 207 (90.8%) and wrong in 21 (9.2%) of cases. The accuracy of sex determination increase with gestational age from 82.7% at 11 weeks, to 90.9% at 12 weeks, 94.7% at 13 weeks and 94.1% at 14 weeks. Most common mistake was to assign male fetuses as female. Conclusion: Sex determination by sonography at first trimester has an important role in case of X-linked anomalies to avoid invasive procedures in female fetuses. The accuracy of correctly identifying fetal gender by sonography at first trimester was 90.8%.

P63 Coordinates Anogenital Modeling and Epithelial Cell Differentiation in the Developing Female Urogenital Tract

p63 is a p53 homologue required for cutaneous development that is expressed in immature squamous epithelium and reserve cells of the cervix. Humans with p63 mutations exhibit defects in limb, accessory organ (skin appendage, breast, prostate), and genitourinary development. Because p63 expression patterns imply a strong role of the gene in the female genital tract development, newborn female p63-/-, +/-, and +/+ mice were examined in situ, dissected, and compared. Nuclear p63 protein was localized to the skin, vagina, bladder, urethra, and basal columnar cells of the caudal uterus in p63+/+ and +/- animals. p63-/- mice exhibited abnormal genital morphogenesis with hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites that normally undergo squamous and urothelial differentiation. The defects observed support p63-dependent pathways of genital tract development that permit externally, ectodermal basal cell replenishment integral to reciprocal epithelial stromal signaling, urorectal septation, and modeling of the external genitalia; and internally, the emergence of basal epithelial cell populations capable of divergent epithelial cell differentiation in the vagina, cervix, and urinary tract. Defects in the first pathway explain imperforate anus, vaginal septum, genital hypoplasia, and micropenis reported in humans with p63 mutations. The second is necessary for the generation of multipotential reserve cells in the cervix and may be operative in other epithelial stromal interactions integral to the emergence of uterine basal cells later in life.

Sonic hedgehog Signaling from the Urethral Epithelium Controls External Genital Development

External genital development begins with formation of paired genital swellings, which develop into the genital tubercle. Proximodistal outgrowth and axial patterning of the genital tubercle are coordinated to give rise to the penis or clitoris. The genital tubercle consists of lateral plate mesoderm, surface ectoderm, and endodermal urethral epithelium derived from the urogenital sinus. We have investigated the molecular control of external genital development in the mouse embryo. Previous work has shown that the genital tubercle has polarizing activity, but the precise location of this activity within the tubercle is unknown. We reasoned that if the tubercle itself is patterned by a specialized signaling region, then polarizing activity may be restricted to a subset of cells. Transplantation of urethral epithelium, but not genital mesenchyme, to chick limbs results in mirror-image duplication of the digits. Moreover, when grafted to chick limbs, the urethral plate orchestrates morphogenetic movements normally associated with external genital development. Signaling activity is therefore restricted to urethral plate cells. Before and during normal genital tubercle outgrowth, urethral plate epithelium expresses Sonic hedgehog (Shh). In mice with a targeted deletion of Shh, external genitalia are absent. Genital swellings are initiated, but outgrowth is not maintained. In the absence of Shh signaling, Fgf8, Bmp2, Bmp4, Fgf10, and Wnt5a are downregulated, and apoptosis is enhanced in the genitalia. These results identify the urethral epithelium as a signaling center of the genital tubercle, and demonstrate that Shh from the urethral epithelium is required for outgrowth, patterning, and cell survival in the developing external genitalia.

Equine fetal sex determination using a single ultrasonic examination under farm conditions

The aims of this study were to evaluate the reliability, under general farm conditions, of the use of a single transrectal sonogram in pregnant mares to determine fetal sex by locating the genital tubercle, and the feasibility of extending the period of gestation in which this examination can be carried out. This research was done during routine reproductive examinations on three different stud farms. Forty mares between the 54th and the 89th day of gestation were examined once; gestation was calculated by identifying the last day of mating as Day 0. In order to verify the precision of the determination, data regarding the sex of the foals at birth were collected. This information was not available for two of the 40 mares examined. The diagnosis was made without any certainty level or any other methods which might quantify or increase the reliability of the examination. Of 40 mares, a diagnosis was possible in 35 (87%); in five mares (12%), it was not possible due to insufficient data. Twenty-six of 40 sex determinations (65%) were correct, 9 of 40 (22%) were incorrect. The comparison of the percentages obtained using the χ 2-test was P=0.0011. In spite of positive statistical analysis, the results of this study are not sufficient to allow systematic use of this approach due to the high percentage of error.

The urethral plate--does it grow into the genital tubercle or within it?

To determine whether the urethral plate grows into the genital tubercle or develops within it. Sprague-Dawley rat embryos were examined at 11-21 days of gestation and compared with available human embryo sections. Rat embryos were examined by a combination of light microscopy, dissection, serial histological section and immunohistochemistry. In rat embryos the urethral plate extended to the tip of the genital tubercle from the earliest stages of genital tubercle development. The plate formed from adherence in the sagittal plane of the inner walls of the primitive cloacal cavity. The urethral plate continued to extend to the tip of the genital tubercle throughout penile development in the male and developed a lumen ahead of the advancing perineal mesenchyme. In the female the perineal side of the urethral plate disintegrated by massive apoptosis. This loss of perineal substance allowed the genital tubercle to collapse on itself and left the urogenital opening at the base of the genital tubercle. Human embryos also showed the presence of the urethral plate at the tip of the genital tubercle throughout genital tubercle development. The urethral plate forms ab initio within the genital tubercle and extends to the tip; it does not grow into the genital tubercle as is often described. The contribution of ectodermal ingrowth to urethral development at the tip of the penis is minimal.

The cloacal plate: the missing link in anorectal and urogenital development

To determine whether differences in the embryology of the anorectal and urogenital area, previously examined in sheep, pigs, rats, rabbits and guinea pigs, producing varying conclusions, may be secondary to differences in species development. Rat and human embryos were studied at the time of genital tubercle development and cloacal partition by standard serial-section histology, and by immunohistochemistry, dissection and scanning electron microscopy. The images obtained were compared with those previously reported for pig and sheep embryos. The cloacal plate, a vertically orientated midline plate of epithelial cells in the caudal half of the genital tubercle, was the key structure that varied between the different species. In rats the plate maintained a vertical height along its length, while in humans and pigs it reverted to a two-layer membrane dorsally, shortly before it degenerated to expose both the anorectal and urogenital tracts. In sheep the plate was taller ventrally than in the other species but also reverted to a short membrane dorsally that exposed the hindgut when it degenerated. The anterior part of the cloacal plate persisted in all embryos as the urethral plate, which then participated in the formation of the urethra in the male and the vestibule in the female. The animal that most closely resembled humans in anogenital development was the pig. The cloacal plate is the key to understanding early anorectal and urogenital development and yet it is barely recognized in published reports. An appreciation of its relevance helps to explain the variations in the described embryology of the region, and is mandatory when interpreting embryological findings in animal models of human anomalies. Given the similarities between porcine and human development, the pig may be the most legitimate animal model for the study of anorectal and urogenital anomalies in humans.

Expression of the SMADIP1 gene during early human development

The smad binding protein 1 gene ( SMADIP1, MIM 605802) has been recently identified as a disease causing gene in a polytopic embryonic defect (MIM 235730) including midline anomalies, facial dysmorphic features and enteric nervous system malformation (Hirschsprung disease). To confirm the pleiotropic role of SMADIP1 during embryogenesis and investigate its role in neural crest cell derivatives differentiation, we performed RNA in situ hybridization at early stages of human development. According to the spectrum of malformations observed in patients, expression of SMADIP1 is observed in neural crest derived cells (peripheric nervous system, enteric nervous system, facial neurectoderm and cranial nerve ganglia), central nervous system, genital tubercle, muscles and kidneys. Surprisingly, SMADIP1 expression is also found in limbs and developing eye. Although congenital heart defects are frequently observed in patients with either a SMADIP1 large scale deletion or truncating mutation, no SMADIP1 expression could be detected in the developing heart at the stages studied.

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.

Depletion of Mab21l1 and Mab21l2 messages in mouse embryo arrests axial turning, and impairs notochord and neural tube differentiation.

The nematode mab-21 gene specifies sensory ray cell identity and was first isolated because of its mutant sensory ray defects. Vertebrate Mab21 orthologs have since been identified in mammals and amphibians. In this report, we characterized in detail two Mab21 orthologs in mouse, Mab21l1 and Mab21l2. We examined the genomic organizations of Mab21 genes and used northern blot and in situ hybridizations to assay their temporal-spatial expression pattern. Their embryonic functions were revealed by specific attenuation of Mab21 messages with antisense oligos in cultured embryos. Mab21l1 and Mab21l2 have very similar protein make-up and gene structures. Both genes were expressed in overlapping domains of actively differentiating embryonic tissues. In addition, Mab21l1 had unique expression in the lens vesicles and genital tubercle whereas Mab21l2 was expressed in the retinal epithelium and umbilical cord. Mab21l1 and Mab21l2 depleted embryos had severe defects in notochord, neural tube, organogenesis, vasculogenesis, and axial turning. The findings demonstrate that both Mab21 genes are required in developing embryos for embryonic turning, formation of the notochord, neural tube, and other organ tissues.

External Genitalia Formation

The process of fetal external genitalia development might be divided into two processes. The first process accomplishes the initial outgrowth of the anlage, genital tubercle (GT). Previous analysis suggests that the distal urethral epithelium (DUE) of the GT, the Fgf8-expressing region, regulates the outgrowth of the GT. The second process eventually generates the sexually dimorphic development of the external genitalia, which is dependent on the action of steroid hormones. Several key genes, for example, RARs, RXRs, RALDH2, and CYP26, were dynamically expressed during GT development. The teratogenic dose of RA at 9.0 d.p.c. induced a drastic malformation of the urethral plate during GT formation, but did not show gross abnormalities in its outgrowth. In RA-treated embryos, Fgf8 expression was still detected in the distal GT regions. Possible regulatory roles of the FGF and RA signaling systems in external genitalia formation are discussed.

Unique functions of Sonic hedgehog signaling during external genitalia development

Coordinated growth and differentiation of external genitalia generates a proximodistally elongated structure suitable for copulation and efficient fertilization. The differentiation of external genitalia incorporates a unique process, i.e. the formation of the urethral plate and the urethral tube. Despite significant progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes for external genitalia. The sonic hedgehog (Shh) gene and its signaling genes have been found to be dynamically expressed during murine external genitalia development. Functional analysis by organ culture revealed that Shh could regulate mesenchymally expressed genes, patched 1 (Ptch1), bone morphogenetic protein 4 (Bmp4), Hoxd13 and fibroblast growth factor 10 (Fgf10), in the anlage: the genital tubercle (GT). Activities of Shh for both GT outgrowth and differentiation were also demonstrated. Shh(-/-) mice displayed complete GT agenesis, which is compatible with such observations. Furthermore, the regulation of apoptosis during GT formation was revealed for the first time. Increased cell death and reduced cell proliferation of the Shh(-/-) mice GT were shown. A search for alterations of Shh downstream gene expression identified a dramatic shift of Bmp4 gene expression from the mesenchyme to the epithelium of the Shh mutant before GT outgrowth. Regulation of mesenchymal Fgf10 gene expression by the epithelial Shh was indicated during late GT development. These results suggest a dual mode of Shh function, first by the regulation of initiating GT outgrowth, and second, by subsequent GT differentiation.

Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis.

We recently cloned the murine homologue of Cyp26B1, a novel retinoic acid (RA)-metabolizing enzyme and showed that its gene expression pattern is unique from that of Cyp26A1 during early embryogenesis. Here, we complete this comparative expression analysis from embryonic day (E) 12 to postnatal stages. Cyp26B1 expression was found in developing tendons and precartilaginous elements and in perichondrium by E14.5, while Cyp26A1 expression was restricted to extremities of rib and vertebral cartilage. Cyp26A1 and Cyp26B1 were expressed, in the distal epithelium and mesenchyme of the limbs and genital tubercle, respectively. High Cyp26B1 expression was found in craniofacial areas undergoing morphogenetic growth, whereas Cyp26A1 message was restricted to the mouth and dental epithelium. Cyp26A1 alone was expressed in the developing neural retina, while both genes were co-expressed in the retinal pigment epithelium. Cyp26B1 was specifically expressed in the developing hindbrain (pons, cerebellum) and forebrain (striatum, hippocampus), with forebrain expression persisting postnatally. In addition, Cyp26B1 was expressed at specific levels of the differentiating upper and lower thoracic spinal cord, adjacent to the cervical and lumbar regions that express the RA-synthesizing enzyme RALDH-2. In viscera, Cyp26B1 transcripts were detected in the developing lung, kidney, spleen, thymus and testis, whereas Cyp26A1 transcripts were found in the diaphragm and outer stomach mesenchyme. Cyp26B1 was also specifically expressed in dermis surrounding the developing hair follicles. Regulated RA metabolism may therefore be required in many developing systems.