DLGH1 is a negative regulator of T-lymphocyte proliferation

Discs large homolog 1 (DLGH1), a founding member of the membrane-associated guanylate kinase family of proteins containing PostSynaptic Density-95/Discs large/Zona Occludens-1 domains, is an ortholog of the Drosophila tumor suppressor gene Discs large. In the mammalian embryo, DLGH1 is essential for normal urogenital morphogenesis and the development of skeletal and epithelial structures. Recent reports also indicate that DLGH1 may be a critical mediator of signals triggered by the antigen receptor complex in T lymphocytes by functioning as a scaffold coordinating the activities of T-cell receptor (TCR) signaling proteins at the immune synapse. However, it remains unclear if DLGH1 functions to enhance or attenuate signals emanating from the TCR. Here, we used Dlgh1 gene-targeted mice to determine the requirement for DLGH1 in T-cell development and activation. Strikingly, while all major subsets of T cells appear to undergo normal thymic development in the absence of DLGH1, peripheral lymph node Dlgh1(-/-) T cells show a hyper-proliferative response to TCR-induced stimulation. These data indicate that, consistent with the known function of Discs large proteins as tumor suppressors and attenuators of cell division, in T lymphocytes, DLGH1 functions as a negative regulator of TCR-induced proliferative responses.     

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.     

Roles of p63 in the diethylstilbestrol-induced cervicovaginal adenosis

Women exposed to diethylstilbestrol (DES) in utero develop abnormalities, including cervicovaginal adenosis that can lead to cancer. We report that transient disruption of developmental signals by DES permanently changes expression of p63, thereby altering the developmental fate of Müllerian duct epithelium. The cell fate of Müllerian epithelium to be columnar (uterine) or squamous (cervicovaginal) is determined by mesenchymal induction during the perinatal period. Cervicovaginal mesenchyme induced p63 in Müllerian duct epithelium and subsequent squamous differentiation. In p63(-/-) mice, cervicovaginal epithelium differentiated into uterine epithelium. Thus, p63 is an identity switch for Müllerian duct epithelium to be cervicovaginal versus uterine. P63 was also essential for uterine squamous metaplasia induced by DES-exposure. DES-exposure from postnatal day 1 to 5 inhibited induction of p63 in cervicovaginal epithelium via epithelial ERalpha. The inhibitory effect of DES was transient, and most cervicovaginal epithelial cells recovered expression of p63 by 2 days after discontinuation of DES-treatment. However, some cervicovaginal epithelial cells failed to express p63, remained columnar and persisted into adulthood as adenosis.     

Gene expression change in the Müllerian duct of the mouse fetus exposed to diethylstilbestrol in utero

In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Müllerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Müllerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 microg/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were downregulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and downregulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Müllerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.     

Biosynthesis and secretion of fibronectin in the cultured mesenchymal cells of the developing chick müllerian duct

We have developed a method to separate and isolate the mesenchymal cells from the epithelial cells in the left Müllerian duct of the developing chick. We then cultured the mesenchymal cells in a serum-free medium. Through an enzyme-linked immunosorbent assay, we detected fibronectin synthesis and release into the medium at stages of Müllerian duct development. Our results demonstrate that the amount of fibronectin secreted by cultured cells gradually decreased in accordance with Müllerian duct differentiation. Similar observations found in the developing embryonic intestine indicate that the highest fibronectin synthesis occurs during early stages of development, when morphogenetic movement and mesenchymal-epithelial interaction are prominent features of embryonic organ differentiation and growth.     

Cellular mechanisms of Müllerian duct formation in the mouse

Regardless of their sex chromosome karyotype, amniotes develop two pairs of genital ducts, the Wolffian and Müllerian ducts. As the Müllerian duct forms, its growing tip is intimately associated with the Wolffian duct as it elongates to the urogenital sinus. Previous studies have shown that the presence of the Wolffian duct is required for the development and maintenance of the Müllerian duct. The Müllerian duct is known to form by invagination of the coelomic epithelium, but the mechanism for its elongation to the urogenital sinus remains to be defined. Using genetic fate mapping, we demonstrate that the Wolffian duct does not contribute cells to the Müllerian duct. Experimental embryological manipulations and molecular studies show that precursor cells at the caudal tip of the Müllerian duct proliferate to deposit a cord of cells along the length of the urogenital ridge. Furthermore, immunohistochemical analysis reveals that the cells of the developing Müllerian duct are mesoepithelial when deposited, and subsequently differentiate into an epithelial tube and eventually the female reproductive tract. Our studies define cellular and molecular mechanisms for Müllerian duct formation.     

Paracrine-mediated apoptosis in reproductive tract development

In mammalian development, the signaling pathways that couple extracellular death signals with the apoptotic machinery are still poorly understood. We chose to examine Müllerian duct regression in the developing reproductive tract as a possible model of apoptosis during morphogenesis. The TGFbeta-like hormone, Müllerian inhibiting substance (MIS), initiates regression of the Müllerian duct or female reproductive tract anlagen; this event is essential for proper male sexual differentiation and occurs between embryonic days (E) 14 and 17 in the rat. Here, we show that apoptosis occurs during Müllerian duct regression in male embryos beginning at E15. Female Müllerian ducts exposed to MIS also exhibited prominent apoptosis within 13 h, which was blocked by a caspase inhibitor. In both males and females the MIS type-II receptor is expressed exclusively in the mesenchymal cell layer surrounding the duct, whereas apoptotic cells localize to the epithelium. In addition, tissue recombination experiments provide evidence that MIS does not act directly on the epithelium to induce apoptosis. Based on these data, we suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions.     

Wilms' tumor protein Wt1 is an activator of the anti-Müllerian hormone receptor gene Amhr2

The Wilms' tumor protein Wt1 plays an essential role in mammalian urogenital development. WT1 mutations in humans lead to a variety of disorders, including Wilms' tumor, a pediatric kidney cancer, as well as Frasier and Denys-Drash syndromes. Phenotypic anomalies in Denys-Drash syndrome include pseudohermaphroditism and sex reversal in extreme cases. We have used cDNA microarray analyses on Wt1 knockout mice to identify Wt1-dependent genes involved in sexual development. The gene most dramatically affected by Wt1 inactivation was Amhr2, encoding the anti-Müllerian hormone (Amh) receptor 2. Amhr2 is an essential factor for the regression of the Müllerian duct in males, and mutations in AMHR2 lead to the persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism. Here we show that Wt1 and Amhr2 are coexpressed during urogenital development and that the Wt1 protein binds to the promoter region of the Amhr2 gene. Inactivation and overexpression of Wt1 in cell lines was followed by immediate changes of Amhr2 expression. The identification of Amhr2 as a Wt1 target provides new insights into the role of Wt1 in sexual differentiation and indicates, in addition to its function in early gonad development and sex determination, a novel function for Wt1, namely, in Müllerian duct regression.     

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.     

Involvement of a matrix metalloproteinase in MIS-induced cell death during urogenital development

Programmed cell death of the Müllerian duct eliminates the primitive female reproductive tract during normal male sexual differentiation. Müllerian inhibiting substance (MIS or AMH) triggers regression by propagating a BMP-like signaling pathway in the Müllerian mesenchyme that culminates in apoptosis of the Müllerian duct epithelium. Presently, the paracrine signal(s) used in this developmental event are undefined. We have identified a member of the matrix metalloproteinase gene family, Mmp2, as one of the first candidate target genes downstream of the MIS cascade to function as a paracrine death factor in Müllerian duct regression. Consistent with a role in regression, Mmp2 expression was significantly elevated in male but not female Müllerian duct mesenchyme. Furthermore, this sexually dimorphic expression of Mmp2 was extinguished in mice lacking the MIS ligand, suggesting strongly that Mmp2 expression is regulated by MIS signaling. Using rat organ genital ridge organ cultures, we found that inhibition of MMP2 activity prevented MIS-induced regression, whereas activation of MMP2 promoted ligand-independent Müllerian duct regression. Finally, MMP2 antisense experiments resulted in partial blockage of Müllerian duct regression. Based on our findings, we propose that similar to other developmental programs where selective elimination or remodeling of tissues occurs, localized induction of extracellular proteinases is critical for normal male urogenital development.     

Studies on sex-organ development. Prenatal effect of oestrogenic hormone on tubular-gland cell morphogenesis and ovalbumin-gene expression in the chick Müllerian duct.

The effects of diethylstilboestrol on morphogenesis and cyto-differentiation of the chick-embryo left Müllerian duct were examined. Embryos were treated at different stages of development with maximal-responsive doses of diethylstilboestrol over a 5-day interval. The shell gland and magnum regions of the Müllerian duct were then assayed for growth and histological morphogenesis. The results were correlated with diethylstilboestrol-induced ovalbumin-gene expression as measured by ovalbumin-mRNA (mRNAov) accumulation and the relative rate of ovalbumin synthesis. Treatment of the embryo from day 10 to day 15 of incubation induces morphogenesis of tubular-gland cells in the Müllerian-duct magnum. Although these cells constitute 10% of the total cell population and contain an average of 8000 molecules of mRNAov per cell, ovalbumin synthesis is only 0.85% of total magnum protein synthesis. The Müllerian-duct magnum of embryos treated from day 13 to day 18 of incubation contains about 30% tubular-gland cells, which have accumulated an average of 7000 molecules of mRNAov per cell, but ovalbumin synthesis is only 3.25% of total magnum protein synthesis. The Müllerian-duct magnum of embryos treated from day 16 to day 21 of incubation contains about 50% tubular-gland cells, which have accumulated an average of 6500 mRNAov molecules per cell, and ovalbumin synthesis is 10% of total magnum protein synthesis. Oestrogen responsiveness develops simultaneously in the Müllerian-duct magnum and shell-gland regions. Compared with the rate of diethylstilboestrol-induced oviduct growth, the relative rate of diethylstilboestrol-induced Müllerian-duct growth increases with embryonic age, from 20-fold lower in the 10-day embryo to only 3-fold lower in the 16-day embryo. All results are discussed in comparison with the responses to oestrogen of the immature chick oviduct, and in terms of the ontogeny of hormone-competent epithelial and stromal components of the Müllerian duct. It is concluded that the development of oestrogenic competence in the embryonic Müllerian duct is a multiphasic phenomenon. A dramatic increase in hormone responsiveness in the Müllerian duct occurs between days 10 and 16 of development, and a less dramatic final maturation of oestrogen responsiveness occurs between day 16 of development and 1 week after hatching.     

Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system

The vertebrate urogenital system forms due to inductive interactions between the Wolffian duct, its derivative the ureteric bud, and their adjacent mesenchymes. These establish epithelial primordia within the mesonephric (embryonic) and metanephric (adult) kidneys and the Müllerian duct, the anlage of much of the female reproductive tract. We show that Wnt9b is expressed in the inductive epithelia and is essential for the development of mesonephric and metanephric tubules and caudal extension of the Müllerian duct. Wnt9b is required for the earliest inductive response in metanephric mesenchyme. Further, Wnt9b-expressing cells can functionally substitute for the ureteric bud in these interactions. Wnt9b acts upstream of another Wnt, Wnt4, in this process, and our data implicate canonical Wnt signaling as one of the major pathways in the organization of the mammalian urogenital system. Together these findings suggest that Wnt9b is a common organizing signal regulating diverse components of the mammalian urogenital system.     

Human müllerian inhibitory factor messenger ribonucleic acid is hormonally regulated in the fetal testis and in adult granulosa cells

The regression of the Müllerian ducts (the embryologic precursor of uterus, vagina, and Fallopian tubes) in the male fetus is caused by Müllerian inhibitory factor (MIF), a glycoprotein produced by fetal Sertoli cells. Although this Müllerian duct involution is complete before midgestation, the amount of MIF mRNA did not vary among 25 human fetal testis samples from 13 to 25.8 weeks of gestation. In cultured 20-week human testis cells, cAMP increased MIF mRNA 8.3-fold, but the human gonadotropins FSH and CG had no effect. In cultured adult human granulosa cells, CG and cAMP increased MIF mRNA accumulation to 430% and 890%, respectively, but FSH had no effect. The expression and hormonal regulation of MIF mRNA in midgestation testes and in adult granulosa cells indicate that MIF has physiological roles in the human gonad other than Müllerian duct regression.     

Establishment and characterization of clonal cell lines from the vagina of p53-deficient young mice

Clonal cell lines have been established from vagina of prepubertal female p53(-/-) mice. Because the mouse vagina has a dual origin (the cranial three-fifths derived from the Müllerian duct and the caudal two-fifths derived from the urogenital sinus), both parts were separately subjected to cloning. Sixteen epithelial and two fibroblastic cell lines were established from the cranial three-fifths (Müllerian vagina group), and four epithelial and three fibroblastic cell lines were established from the caudal two-fifths (sinus vagina group). They were maintained in Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 containing 10% fetal calf serum and 17 beta-estradiol at 10(-8) M. Two cell lines (one epithelial and one fibroblastic) were examined using soft agar assay, but no colonies were formed. The doubling time of the cell lines was approximately 24 h, and all of them divided more than 200 times without crisis, suggesting that they were immortalized. All epithelial cell lines expressed cytokeratin 8. However, the epithelial cell lines expressed cytokeratin 14 and cytokeratin 10 when exposed to medium containing different concentrations of Ca(2+). Fibroblastic cell lines expressed vimentin. All epithelial and fibroblastic cell lines expressed estrogen receptor-alpha protein. This is the first successful establishment of clonal cell lines from the normal mouse vagina, and these lines may provide good models in vitro of the vagina for the study of the mechanism of estrogen action.     

The germ layer origin of mouse vaginal epithelium restricts its responsiveness to mesenchymal inductors: uterine induction

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the lower fused Müllerian ducts. While previously it has been reported that neonatal vaginal epithelium can be induced to differentiate as uterus, which normally develops from the middle portion of the Müllerian ducts, it has not been determined whether this ability is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test if germ layer origin influences the ability of vaginal epithelium to undergo uterine differentiation, we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with uterine mesenchyme, and grown them for 4 weeks in female mice. Mesoderm-derived Müllerian vaginal epithelium in combination with uterine mesenchyme formed the simple columnar epithelium typical of uterus. Similar results were obtained with neonatal cervical epithelium, another mesodermal Müllerian duct derivative. On the other hand, sinus vaginal epithelium combined with uterine mesenchyme formed small cysts lined by a stratified squamous vaginal-like epithelium. This epithelium never showed evidence of cycling between the cornified and mucified states as is typically seen in vaginal epithelium combined with vaginal stroma. These results indicate that the ability of epithelium to form uterus is limited to mesoderm-derived epithelia and suggest that endoderm-derived sinus vaginal epithelium cannot undergo the typical differentiative modifications in response to the hormonal fluctuations of the estrous cycle when associated with uterine stroma.     

Androgens and the development of the vagina

Today it is generally held that the vagina develops from sinovaginal bulbs and that the lower third of the definitive vagina is derived from the urogenital sinus. Here we show that the entire vagina arises by downward growth of Wolffian and Müllerian ducts, that the sinovaginal bulbs are in fact the caudal ends of the Wolffian ducts, and that vaginal development is under negative control of androgens. We designed a genetic experiment in which the androgen receptor defect in the Tfm mouse was used to examine the effects of androgens. Vaginal development was studied by 3D reconstruction in androgen-treated female embryos and in complete androgen-insensitive littermates. In androgen-treated females, descent of the genital ducts was inhibited, and a vagina formed in androgen-insensitive Tfm embryos as it does in normal females. By immmunohistochemical localization of the androgen receptor in normal mouse embryos, we demonstrated that the androgen receptor was expressed in Wolffian duct and urogenital sinus-derived structures, and was entirely absent in the Müllerian duct derivatives. We conclude that the Wolffian ducts are instrumental in conveying the negative control by androgens on vaginal development. The results are discussed under evolutionary aspects at the transition from marsupial to eutherian mammals.     

Essential roles of mesenchyme-derived beta-catenin in mouse Müllerian duct morphogenesis

Members of the Wnt family of genes such as Wnt4, Wnt5a, and Wnt7a have been implicated in the formation and morphogenesis of the Müllerian duct into various parts of the female reproductive tract. These WNT ligands elicit their action via either the canonical WNT/beta-catenin or the non-canonical WNT/calcium pathway and could possibly function redundantly in Müllerian duct differentiation. By using the Müllerian duct-specific anti-Müllerian hormone receptor 2 cre (Amhr2-cre) mouse line, we established a conditional knockout model that removed beta-catenin specifically in the mesenchyme of the Müllerian duct. At birth, loss of beta-catenin in the Müllerian duct mesenchyme disrupted the normal coiling of the oviduct in the knockout embryo, resembling the phenotype of the Wnt7a knockout. The overall development of the female reproductive tract was stunted at birth with a decrease in proliferation in the mesenchyme and epithelium. We also discovered that Wnt5a and Wnt7a expression remained normal, excluding the possibility that the phenotypes resulted from a loss of these WNT ligands. We examined the expression of Frizzled (Fzd), the receptors for WNT, and found that Fzd1 is one receptor present in the Müllerian duct mesenchyme and could be the putative receptor for beta-catenin activation in the Müllerian duct. In summary, our findings suggest that mesenchymal beta-catenin is a downstream effector of Wnt7a that mediates the patterning of the oviduct and proper differentiation of the uterus.     

Marsupial anti-Mullerian hormone gene structure,regulatory elements,and expression

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.