Mouse vaginal development with lateral enlargement at late embryonic stages and caudal elongation after birth.

Müllerian ducts give rise to the oviducts, uterus, cervix, and vagina. During female reproductive tract development in mice, the bilateral Müllerian duct epithelium grows caudally until reaching the urogenital sinus epithelium. This is followed by further caudal growth with the reduction of the urogenital sinus epithelium. Finally, the vaginal epithelium of adult mice is entirely derived from the Müllerian duct epithelium. Here, we explored the mechanisms underlying mouse vaginal development via cell proliferation, apoptosis, and lineage analyses. We found that at the late embryonic stages, apoptosis occurred at the attachment site of bilateral Müllerian duct epithelia below the cervix, resulting in bilateral lumen traffic. The Müllerian duct epithelium was enclosed by the urogenital sinus epithelium at their boundary region on embryonic day (E) 16.5, whereas the Müllerian duct epithelium encased the urogenital sinus epithelium at postnatal day (P) 0 through lateral enlargement. Lateral Müllerian duct enlargement was accompanied by focal ERK activation within the curved epithelial tips and the specific localization of mitotic nuclei on the luminal side of the Müllerian duct epithelial layer at E17.5. Descent of the Müllerian duct epithelium and shortening of the urogenital sinus epithelium occurred rapidly after birth, accompanied by cell proliferation in the Müllerian duct epithelium and its peripheral mesenchymal tissues as well as intense apoptosis in the urogenital sinus epithelium around their boundary region. Urogenital sinus epithelium was localized at the base of the vagina at P7. In conclusion, the mouse vagina develops laterally at the late embryonic stages and caudally after birth.

Ovotesticular disorders of sex development in FGF9 mouse models of human synostosis syndromes.

In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

Mouse fibroblast growth factor 9 N143T mutation leads to wide chondrogenic condensation of long bones.

Long bones of the appendicular skeleton are formed through endochondral ossification. Endochondral bone formation initiates with mesenchymal condensation, followed by the formation of a cartilage template which is replaced by bone. Fibroblast growth factor 9 (FGF9) regulates bone development. Fgf9-/- mice exhibit disproportionate shortening of proximal skeletal elements. Fgf9 missense mutations in mice and humans induce joint synostosis. Thus, FGF9 is critical for regulating bone length and joint formation. Conversely, mechanisms regulating bone width remain unclear. Here, we showed that the homozygous elbow knee synostosis (Eks) mutant mice harboring N143T mutation in Fgf9 have wide long bones at birth. We investigated the cellular and molecular mechanisms underlying the widened prospective humerus in Fgf9Eks/Eks embryos. Increased and expanded FGF signaling in concert with wider expression domain of Fgf receptor 3 (Fgfr3) during chondrogenic condensation of the humerus led to widened cartilage, which resulted in the formation of wider prospective humeri in neonatal Fgf9Eks/Eks mice. Increased and expanded FGF signaling during chondrogenic condensation led to increased density of chondrocytes of the humeri accompanied by increased proliferation of chondrocytes which express inappropriately higher levels of cyclin D1 in Fgf9Eks/Eks embryos. The results suggest that FGF9 regulates the width of prospective long bones by controlling the width of chondrogenic condensation.

Three muscle slings of the pelvic floor in women: an anatomic study.

The region anterior to the anal canal in women is composed of intertwined smooth and skeletal muscles. The present study aimed to clarify skeletal muscle morphology in the anterior region of the anal canal. The pelvic floor muscles of 28 pelvic halves from 16 female cadavers (mean age 79.75 years) were dissected from the inferior aspect to examine the perineal muscles, followed by midline transection and dissection from the inner surface to examine the pelvic outlet muscles. The bulbospongiosus muscle was found to be attached to the lateral surface of the external anal sphincter. The superficial transverse perineal muscle crossed superiorly to the bulbospongiosus and coursed medially toward its contralateral muscle bundle deep to the anterior portion of the external anal sphincter. The superficial transverse perineal muscle formed the middle sling. From the medial aspect, the anterior part of the levator ani was divided into anterior and posterior bundles to form the anterior and posterior slings, respectively. This study proposes that three muscular slings could be important in supporting the pelvic floor in women. In addition, this study shows that the anterior skeletal muscular wall of the anal canal is composed of the anterior muscle bundle of the levator ani, superficial transverse perineal, and proper external anal sphincter muscles.

Coexistence of Dense and Sparse Areas in the Longitudinal Smooth Muscle of the Anal Canal: Anatomical and Histological Analyses Inspired by Magnetic Resonance Images.

Magnetic resonance images of the anal canal show small, circular, low-intensity areas arranged in a row and a high-intensity area surrounding them internally and externally in the longitudinal muscle layer that cannot be explained by current anatomical findings. The purpose of this study was to elucidate the detailed structure of the longitudinal smooth muscle of the anal canal and to interpret the magnetic resonance image of the longitudinal muscle. Specimens for macroscopic anatomy and histology were obtained from six and seven cadavers, respectively. The histological nature of the longitudinal muscle was examined by staining serial transverse and coronal sections of the lateral wall of the anal canal with Masson's trichrome stain and using immunohistochemistry for smooth and skeletal muscle fibers. Dense and sparse areas of smooth muscle fibers coexisted in the longitudinal muscle layer. The dense areas formed columnar muscle bundles approximately 1.0-1.5 mm in diameter, and they continued from the longitudinal muscle bundles of the rectum. The columnar muscle bundles of the longitudinal anal muscle were internally and externally surrounded by sparsely arranged smooth muscle fibers that ran longitudinally. The coexistence of dense and sparse areas of smooth muscle fibers suggests that the structure of the smooth muscle is optimized for its function. This histological nature is probably reflected in the magnetic resonance image of the longitudinal muscle as the coexistence of low- and high-intensity areas. Clin. Anat. 33:619-626, 2020. © 2019 Wiley Periodicals, Inc.

Various significant connections of the male pelvic floor muscles with special reference to the anal and urethral sphincter muscles.

The male pelvic floor is a complex structure formed by several muscles. The levator ani muscle and the perineal muscles are important components of the pelvic floor. The perineal muscles comprise the external anal sphincter, bulbospongiosus, superficial transverse perineal muscles, and ischiocavernosus. Although the connections of the muscles of the pelvic floor have been reported recently, the anatomical details of each muscle remain unclear. In this study, we examined the male pelvic floor to clarify the connection between the muscles related to function. Fifteen male pelvises were used for microscopic dissection, and three male pelvises were used for histological examination. On the lateral aspect, the perineal muscles were connected to each other. Bundles of the levator ani muscle extended to connect to the perineal muscles. In addition, the extended muscle bundle from the levator ani muscle and the perineal muscles surround the external urethral sphincter. On the medial aspect, the levator ani muscle and the external anal sphincter form the anterior and posterior muscular slings of the anal canal. The connection between the perineal muscles and levator ani muscle indicates a possible close relationship between the functions of the urethra and anus.

Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy.

The anatomy of the rectourethralis muscle is essential for performing radical prostatectomy and proctectomy. The rectourethralis muscle is known to continue to the rectal wall posteriorly and to the membranous urethra anteriorly. However, the lateral extent of the rectourethralis muscle remains unclear. This study aimed to verify the hypothesis that the rectourethralis muscle laterally extends and directly adheres to the levator ani. Eight male cadavers were used for macroscopic dissection, and three male cadavers were used for immunohistological analysis using anti-smooth muscle and anti-skeletal muscle antibodies. The rectourethralis muscle laterally extended smooth muscle fibers both superoposteriorly and inferoanteriorly toward the levator ani. The smooth muscle fibers sandwiched the levator ani superoanteriorly and inferoanteriorly. A few smooth muscle fibers of the rectourethralis muscle inserted into the levator ani. This study clarified the spatial distribution of the rectourethralis muscle and its detailed positional relationship with the levator ani. The findings are valuable especially to urologists and anorectal surgeons for dissecting an optimal layer around the urethra and the rectum, and for avoiding rectal or urethral injuries during surgery. Clin. Anat. 31:1167-1176, 2018. © 2018 Wiley Periodicals, Inc.

A mutation in transcription factor MAFB causes Focal Segmental Glomerulosclerosis with Duane Retraction Syndrome.

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease in children and adults. Genetic factors significantly contribute to early-onset FSGS, but the etiologies of most adult cases remain unknown. Genetic studies of monogenic syndromic FSGS exhibiting extra-renal manifestations have uncovered an unexpected biological role for genes in the development of both podocytes and other cellular lineages. To help define these roles, we studied two unrelated families with FSGS associated with Duane Retraction Syndrome, characterized by impaired horizontal eye movement due to cranial nerve malformation. All four affected individuals developed FSGS and Duane Retraction Syndrome in their first to second decade of life, manifested as restricted abduction together with globe retraction and narrowed palpebral fissure on attempted adduction. Hypoplasia of the abducens nerves and hearing impairment occurred in severely affected individuals. Genetic analyses revealed that affected individuals harbor a rare heterozygous substitution (p.Leu239Pro) in MAFB, a leucine zipper transcription factor. Luciferase assays with cultured monocytes indicated that the substitution significantly reduced transactivation of the F4/80 promoter, the known MAFB recognition element. Additionally, immunohistochemistry indicated reduced MAFB expression in the podocytes of patients. Structural modeling suggested that the p.Leu239Pro substitution in the DNA-binding domain possibly interferes with the stability of the adjacent zinc finger. Lastly, podocytes in neonatal mice with p.Leu239Pro displayed impaired differentiation. Thus, MAFB mutations impair development and/or maintenance of podocytes, abducens neurons and the inner ear. The interactions between MAFB and regulatory elements in these developing organs are likely highly specific based on spatiotemporal requirements.

Morphology of the region anterior to the anal canal in males: visualization of the anterior bundle of the longitudinal muscle by transanal ultrasonography.

PURPOSE: The anterior bundle of the longitudinal muscle in the region anterior to the anal canal in males has been described, although the anatomical details remain unclear. The present study was undertaken to clarify the precise morphology of the anterior bundle of the longitudinal muscle and its relationship to the surrounding structures, and to visualize the anterior bundle of the longitudinal muscle via transanal ultrasonography. METHODS: Histological examination was carried out using seven male cadavers; an additional three male cadavers were used for transanal ultrasonography, and the ultrasonography images were compared with the actual sagittal sections. In addition, transanal ultrasonography images of 50 male patients at Tokatsu-Tsujinaka Hospital were studied. RESULTS: The region anterior to the anal canal consisted of smooth muscles and skeletal muscles. The anterior bundle of the longitudinal muscle was situated between the bulbospongiosus and the external anal sphincter, and consisted of smooth muscle. The bundle was identified in the transanal ultrasonography of cadavers by comparison with the actual sections. Transanal ultrasonography images of living bodies showed the anterior bundle of the longitudinal muscle as a hypoechoic layer of approximately 17.7 mm in length. CONCLUSIONS: The detailed anatomical findings of the anterior bundle of the longitudinal muscle suggested "an alternate arrangement of smooth muscles and skeletal muscles" in the region anterior to the anal canal and facilitated the visualization of the anterior bundle of the longitudinal muscle in transanal ultrasonography.

Tissue-specific roles of FGF signaling in external genitalia development.

BACKGROUND: The developmental processes of the genital tubercle (GT), the anlage of the external genitalia, possess several developmental aspects, including GT outgrowth, urethral tube formation, and epithelial differentiation of the urethra. The GT comprises the mesenchyme derived from the lateral mesoderm, ectodermal epithelium, and endodermal epithelium (embryonic urethral epithelium). The three tissue layers develop the GT coordinately. RESULTS: Around the initial stage of GT outgrowth (E11.5), FGF signaling was detected in the mesenchyme of the GT. FGF signaling was detected in the three tissue layers of the GT around the early stage of urethral formation (E13.5). Subsequently, FGF signaling was predominantly detected in the urethral epithelium (E14.5). Tissue-specific roles of FGF signaling in GT development were revealed by conditional Fgfr gene knockout approaches. Mesenchymal FGF signaling in the early-stage GT is required for its outgrowth. Ectodermal FGF signaling in the GT is required for the differentiation of the ectoderm and urethral epithelium at their junction to form the proper urethral tube. Endodermal FGF signaling in the GT is required for the stratification and cell adhesive characteristics of the urethral epithelium. CONCLUSIONS: The current study suggests that spatiotemporally regulated FGF signaling plays tissue-specific roles in multiple processes of external genitalia development.

Region-specific regulation of cell proliferation by FGF receptor signaling during the Wolffian duct development.

The Wolffian duct (WD) is a primordium of the male reproductive tract and kidney collecting duct system. Fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family, are essential for kidney development. Although the functions of FGFR signaling in kidney morphogenesis have been analyzed, their function in WD development has not been comprehensively investigated. Here, we demonstrate that Fgfr2 is the major Fgfr gene expressed throughout the WD epithelia and that it is essential for the maintenance of the WD, specifically in the caudal part of the WD. Hoxb7-Cre mediated inactivation of Fgfr2 in the mouse WD epithelia resulted in the regression of the caudal part of the WD and abnormal male reproductive tract development. Cell proliferation and expression of the downstream target genes of RTK signaling (Etv4 and Etv5) were decreased in the caudal part of the WD epithelia in the mutant embryos. Cranial (rostral) WD formation and ureteric budding were not affected. Ret, Etv4, and Etv5 expression were sustained in the ureteric bud of the mutant embryos. Taken together, these data suggest region-specific requirements for FGFR2 signaling in the developing caudal WD epithelia.

Essential roles of epithelial bone morphogenetic protein signaling during prostatic development.

Prostate is a male sex-accessory organ. The prostatic epithelia consist primarily of basal and luminal cells that differentiate from embryonic urogenital sinus epithelia. Prostate tumors are believed to originate in the basal and luminal cells. However, factors that promote normal epithelial differentiation have not been well elucidated, particularly for bone morphogenetic protein (Bmp) signaling. This study shows that Bmp signaling prominently increases during prostatic differentiation in the luminal epithelia, which is monitored by the expression of phosphorylated Smad1/5/8. To elucidate the mechanism of epithelial differentiation and the function of Bmp signaling during prostatic development, conditional male mutant mouse analysis for the epithelial-specific Bmp receptor 1a (Bmpr1a) was performed. We demonstrate that Bmp signaling is indispensable for luminal cell maturation, which regulates basal cell proliferation. Expression of the prostatic epithelial regulatory gene Nkx3.1 was significantly reduced in the Bmpr1a mutants. These results indicate that Bmp signaling is a key factor for prostatic epithelial differentiation, possibly by controlling the prostatic regulatory gene Nkx3.1.

Dynamic intersection of the longitudinal muscle and external anal sphincter in the layered structure of the anal canal posterior wall.

PURPOSE: The minute details of the structure of the anal canal are still not well understood. The complex structural configuration of the muscles, ligaments and raphes remains unclarified. This study was undertaken to determine the precise structure of the posterior part of the anal canal and to facilitate an understanding of previous studies. METHODS: For macroscopic examination, 14 right pelvic halves from 14 Japanese cadavers were used. Observation and dissection were performed from the median plane. In the histological examination, six left pelvic halves were used. The sections of the posterior parts of the anal canal were stained with hematoxylin and eosin, Elastic van Gieson, anti-smooth actin antibody and anti-skeletal myosin antibody. RESULTS: We identified the following muscles arranged from the internal side to the external side: internal anal sphincter, longitudinal muscle (LM), external anal sphincter (EAS) and levator ani muscle (LAM). Two different types of conformation of the posterior part of the anal canal were found, each bearing a different shape of EAS. In both types, LM penetrated the inferior part of EAS. After penetrating EAS, some fibers of LM ran posterosuperiorly and attached to the "the posterior fibers" which reach the dorsal side of the coccyx. CONCLUSIONS: We defined and labeled the connective tissues between the anal canal and coccyx on the basis of their relative position to LAM. Based on a comparison of the two types of the posterior part of the anal canal, we propose that there are two phases due to constriction and relaxation of LM.

Epithelial Bmp (Bone morphogenetic protein) signaling for bulbourethral gland development: a mouse model for congenital cystic dilation.

The bulbourethral gland (BUG) is a male-specific organ, which secretes part of the semen fluid. As the BUG is located in the deep pelvic floor, its developmental process is still unclear. Bone morphogenetic protein (Bmp) signaling plays pivotal roles in various organs. However, the function of Bmp signaling for BUG development is still unclear. The present study aimed to elucidate the role of Bmp signaling in the development of the BUG. We observed the prominent nuclear accumulation of phosphorylated (p) SMAD1/5/8, the downstream molecules of Bmp signaling, during BUG epithelial development. These results suggest that Bmp signaling contributes to BUG development. Bmp receptor1a (Bmpr1a) is known as the major type 1 signal transducer in some organogeneses. To analyze the Bmp signaling function for BUG development, we examined epithelial cell-specific Bmpr1a gene conditional mutant mice utilizing the tamoxifen-inducible Cre recombinase system. We observed cystic dilation and epithelial hyperplasia of the BUG in the Bmpr1a conditional knockout mice. The mutant cystic BUG specimens also showed inflammatory lesions. These BUG abnormalities resembled some of the BUG malformations observed in human congenital syndromes. The current study suggests that Bmp signaling possesses an essential role in BUG development and homeostasis. This would be the first report showing that the mutation of the Bmpr1a gene in the BUG epithelia phenocopied some abnormalities of human congenital syndromes affecting the BUG duct.

Dosage-dependent hedgehog signals integrated with Wnt/beta-catenin signaling regulate external genitalia formation as an appendicular program.

Embryonic appendicular structures, such as the limb buds and the developing external genitalia, are suitable models with which to analyze the reciprocal interactions of growth factors in the regulation of outgrowth. Although several studies have evaluated the individual functions of different growth factors in appendicular growth, the coordinated function and integration of input from multiple signaling cascades is poorly understood. We demonstrate that a novel signaling cascade governs formation of the embryonic external genitalia [genital tubercle (GT)]. We show that the dosage of Shh signal is tightly associated with subsequent levels of Wnt/beta-catenin activity and the extent of external genitalia outgrowth. In Shh-null mouse embryos, both expression of Wnt ligands and Wnt/beta-catenin signaling activity are downregulated. beta-catenin gain-of-function mutation rescues defective GT outgrowth and Fgf8 expression in Shh-null embryos. These data indicate that Wnt/beta-catenin signaling in the distal urethral epithelium acts downstream of Shh signaling during GT outgrowth. The current data also suggest that Wnt/beta-catenin regulates Fgf8 expression via Lef/Tcf binding sites in a 3' conserved enhancer. Fgf8 induces phosphorylation of Erk1/2 and cell proliferation in the GT mesenchyme in vitro, yet Fgf4/8 compound-mutant phenotypes indicate dispensable functions of Fgf4/8 and the possibility of redundancy among multiple Fgfs in GT development. Our results provide new insights into the integration of growth factor signaling in the appendicular developmental programs that regulate external genitalia development.

FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion.

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9(Eks)) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.