Induction of WNT inhibitory factor 1 expression by Müllerian inhibiting substance/antiMullerian hormone in the Müllerian duct mesenchyme is linked to Müllerian duct regression.

A key event during mammalian sexual development is regression of the Müllerian ducts (MDs) in the bipotential urogenital ridges (UGRs) of fetal males, which is caused by the expression of Müllerian inhibiting substance (MIS) in the Sertoli cells of the differentiating testes. The paracrine signaling mechanisms involved in MD regression are not completely understood, particularly since the receptor for MIS, MISR2, is expressed in the mesenchyme surrounding the MD, but regression occurs in both the epithelium and mesenchyme. Microarray analysis comparing MIS signaling competent and Misr2 knockout embryonic UGRs was performed to identify secreted factors that might be important for MIS-mediated regression of the MD. A seven-fold increase in the expression of Wif1, an inhibitor of WNT/ß-catenin signaling, was observed in the Misr2-expressing UGRs. Whole mount in situ hybridization of Wif1 revealed a spatial and temporal pattern of expression consistent with Misr2 during the window of MD regression in the mesenchyme surrounding the MD epithelium that was absent in both female UGRs and UGRs knocked out for Misr2. Knockdown of Wif1 expression in male UGRs by Wif1-specific siRNAs beginning on embryonic day 13.5 resulted in MD retention in an organ culture assay, and exposure of female UGRs to added recombinant human MIS induced Wif1 expression in the MD mesenchyme. Knockdown of Wif1 led to increased expression of ß-catenin and its downstream targets TCF1/LEF1 in the MD mesenchyme and to decreased apoptosis, resulting in partial to complete retention of the MD. These results strongly suggest that WIF1 secretion by the MD mesenchyme plays a role in MD regression in fetal males.

Mesenchymal-to-epithelial transition contributes to endometrial regeneration following natural and artificial decidualization.

Despite being a histologically dynamic organ, mechanisms coordinating uterine regeneration during the menstrual/estrous cycle and following parturition are poorly understood. In the current study, we hypothesized that endometrial epithelial tissue regeneration is accomplished, in part, by mesenchymal-to-epithelial transition (MET). To test this hypothesis, fate mapping studies were completed using a double transgenic (Tg) reporter strain, Amhr2-Cre; Rosa26-Stop(fl/fl-EYFP) (i.e., flox-stop EYFP reporter). EYFP expression was observed in Müllerian duct mesenchyme-derived stroma and myometrium, but not epithelia in young and peripubertal double Tg female mice. However, mosaic EYFP expression was observed in epithelia of double Tg mice after parturition. To ensure the observed epithelial EYFP expression was not due to leaky Amhr2 promoter activity, resulting in aberrant Cre expression, transgenic mice expressing LacZ under the control of the Amhr2 promoter (Amhr2-LacZ) were used to monitor ß-galactosidase (ß-Gal) activity within the uterus. ß-Gal activity was not detected in luminal or glandular epithelia regardless of age, reproductive status, or degree of damage incurred within the uterus. Lastly, a unique population of transitional cells was identified that expressed the epithelial cell marker, pan-cytokeratin, and the stromal cell marker, vimentin. These cells localized predominantly to the regeneration zone in the mesometrial region of the endometrium. These findings suggest a previously unappreciated role for MET in endometrial regeneration and have important implications for proliferative diseases of the endometrium such as endometriosis.

Cell migration and activated PI3K/AKT-directed elongation in the developing rat Müllerian duct.

In vertebrates, the Müllerian duct elongates along the Wolffian duct, a mesonephric structure that is required for Müllerian duct formation. Recently, several genes required for initial Müllerian duct formation have been identified. However, the precise mechanism of Müllerian duct elongation remains to be elucidated. In this study, we investigated dynamic morphological changes in the elongating Müllerian duct in rat urogenital ridges in organ culture manipulated by microincision and/or chemical inhibitors. Mechanical division of the developing Müllerian duct showed that epithelial cells of the Müllerian duct actively migrate along the anterior-posterior axis independent of the proliferative expansion of the anterior portion of the duct. We found that the PI3K/AKT signaling pathway is activated in the Müllerian duct epithelium and is required for elongation of the tip of the duct; however, migration of Müllerian duct epithelial cells proximal to the tip remains intact when PI3K/AKT is inactivated. Although much is known about the molecular and cellular mechanisms leading to Müllerian duct regression, the present findings provide a fuller understanding of the mechanisms contributing to Müllerian duct formation and to the general process of early tubulogenesis.

A mesenchymal perspective of Müllerian duct differentiation and regression in Amhr2-lacZ mice.

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding beta-galactosidase (beta-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express beta-gal activity in an Amhr2-specific pattern. In the gonads, beta-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. beta-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, beta-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males beta-gal expression encircled the duct. beta-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal beta-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.

Sexual dimorphism of G-protein subunit Gng13 expression in the cortical region of the developing mouse ovary.

In our search for genes required for the development and function of mouse gonads, we identified Gng13 (guanine nucleotide binding protein 13, gamma), a gene with an embryonic expression pattern highly restricted to the ovary. Based on reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, Gng13 is expressed in both XX and XY gonads at embryonic day (E) 11.5, but becomes up-regulated in the XX gonad by E12.5. Expression is retained after treatment with busulfan, a chemical known to eliminate germ cells, pointing to the soma as a site of Gng13 transcription. In situ hybridization of embryonic ovarian tissue sections further localized the expression to the cortex of the developing XX gonad. Gng13 expression in the adult is also highly restricted. Northern blot analyses and Genomic Institute of the Novartis Research Foundation expression profiling of adult tissues detected very high expression in the cerebrum and cerebellum, in addition to, a weaker signal in the ovary. Gng13 belongs to a well-known family of signal transduction molecules with functions in many aspects of development and organ physiology. Here, we report that, in the developing mouse embryo, expression of Gng13 mRNA is highly restricted to the cortex of the XX gonad during sexual differentiation, suggesting a role for this gene during ovarian development.

Expression analysis and evolutionary conservation of the mouse germ cell-specific D6Mm5e gene.

During our search for genes required for gonadal development and function in the mouse, we identified D6Mm5e (DNA segment, Chr 6. Miriam Meisler 5, expressed), a gene with an expression pattern highly restricted to the embryonic ovary and the postnatal testis. Based on RT-PCR, Northern blot, and in situ hybridization analyses, we show that D6Mm5e is expressed in the germ cells of the female embryo upon their initial entry into meiosis, and in male germ cells during the last stages of spermatogenesis. Two transcripts are detected in the gonads resulting from the alternative splicing of exon 8. This splicing event does not introduce a frame shift, and creates an mRNA product that uses the same stop codon as the longer transcript. Although D6Mm5e does not belong to any known protein family and does not contain any known protein signature motifs, the high level of evolutionary conservation and the cellular and temporal expression suggest that D6Mm5e may have a role in male and female gametogenesis. Here we report the gonad-restricted mRNA expression profile of D6Mm5e in the mouse, and the evolutionary conservation of its amino acid sequence.

Müllerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Müllerian duct regression.

Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stage- and tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor (MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-to-mesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by differentially regulating their expression.

Conditional deletion of beta-catenin in the mesenchyme of the developing mouse uterus results in a switch to adipogenesis in the myometrium.

Precise cell fate decisions during differentiation of uterine tissues from the embryonic Müllerian duct are critical for normal fertility. Wnt-7a, a member of the Wnt family of secreted signaling molecules that can signal through a canonical beta-catenin pathway, is necessary for the correct differentiation of both anterior/posterior and radial axes of the uterus. In order to investigate the role of beta-catenin directly in mouse uterine development, we have generated mice that are deficient in beta-catenin expression in the embryonic Müllerian duct. We have found that conditional deletion of beta-catenin in the Müllerian duct mesenchyme before postnatal differentiation of the uterine layers results in a phenotype that is distinct from the phenotype observed by deletion of Wnt-7a. Shortly after birth, the uteri of the conditional mutants appear smaller and less organized. The uteri of adult conditional beta-catenin mutants are grossly deficient in smooth muscle of the myometrium, which has been replaced by adipose, a phenotype resembling human lipoleiomyoma. We also show that the adipocytes in the uteri of mice conditionally deleted for beta-catenin are derived from Müllerian inhibiting substance type II receptor-expressing cells suggesting that they share a common origin with the uterine smooth muscle cells. These results describe the first molecular evidence linking disruption of beta-catenin expression in mesenchymal cells with a switch from myogenesis to adipogenesis in vivo.

Genomic structure and expression analysis of the mouse testis-specific ribbon protein (Trib) gene.

During our analyses of genes required for the development and function of the mouse gonads, we identified a novel testis-specific mRNA, transcribed from a gene that we have named testis-specific ribbon protein (Trib). In the mouse, Trib is located on chromosome 15, overlapping with and transcribed in the opposite orientation of the meiosis specific gene Smc1beta. The deduced amino acid sequence of testis ribbon (TRIB) protein is highly conserved between human, mouse, and rat and contains the ribbon motifs found in the largely uncharacterized microtubule ribbon protein ribbon43a (RIB43A). We show by Northern blot analyses and reverse transcription-polymerase chain reaction (RT-PCR) that Trib mRNA is specifically expressed in the adult testis. In situ hybridization indicates that Trib is expressed solely in germ cells during the leptotene-pachytene stages of spermatogenesis. The high level of evolutionary conservation and the cellular and temporal expression suggest that Trib may be required for mouse spermatogenesis and male fertility. Here, we describe the genomic structure and expression profile of mouse Trib and compare its homology with other ribbon proteins.

Genetic studies of the AMH/MIS signaling pathway for Müllerian duct regression.

Anti-Müllerian hormone (AMH)/Müllerian-inhibiting substance (MIS) is a member of the transforming growth factor-beta (TGF-beta) superfamily. Like other TGF-beta family members, AMH is likely to signal through two transmembrane serine/threonine kinase receptors. Whereas the AMH type II receptor has been clearly defined, only recently has there been evidence about the identity of the AMH type I receptor for Müllerian duct regression in vivo. We generated a new cre mouse line expressing the recombinase in AMH target cells. This line was then used to conditionally inactivate the Bmpr1a gene in the Müllerian duct, resulting in males with a uterus. Thus, Bmpr1a plays an essential role in the process of Müllerian duct regression. To investigate the role of Bmpr1a in granulosa cells, we took advantage of transgenic mice overexpressing human AMH. Surprisingly, these transgenic females that were also conditionally mutant for Bmpr1a in the Müllerian duct had no uterus. These results suggest that when AMH is overexpressed, other TGF-beta family type I receptors can potentially transduce AMH signals.

Requirement of Bmpr1a for Müllerian duct regression during male sexual development.

Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation. In males, the fetal testis produces the transforming growth factor beta (TGF-beta) family member anti-Müllerian hormone (Amh, also known as Müllerian-inhibiting substance (Mis)), which causes regression of the Müllerian ducts, the primordia of the oviducts, uterus and upper vagina. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri. The type I receptor and Smad proteins that are required in vivo for Müllerian duct regression have not yet been identified. Here we show that targeted disruption of the widely expressed type I bone morphogenetic protein (BMP) receptor Bmpr1a (also known as Alk3) in the mesenchymal cells of the Müllerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Müllerian ducts. Because Bmpr1a is evolutionarily conserved, these findings indicate that a component of the BMP signaling pathway has been co-opted during evolution for male sexual development in amniotes.

Genetic studies of MIS signalling in sexual development.

The Müllerian ducts are composed of an epithelium and surrounding mesenchyme that have the potential to differentiate into female reproductive organs, including the oviducts, uterus and upper vagina. In eutherian mammals, Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) secreted by the fetal testis causes the regression of the Müllerian ducts to prevent the differentiation of female reproductive organs in males. MIS signalling in the Müllerian duct is mediated by the MIS type II receptor (MISRII) that is expressed in the mesenchyme surrounding the epithelium. MIS signalling alters the Müllerian duct mesenchyme, leading to the elimination of the ductal epithelium. Loss of MIS signalling, by mutation of MIS or MISRII, leads to the differentiation of female reproductive organs in males that can cause cryptorchidism and infertility. We have exploited the mouse MisrII locus to express heterologous genes in the cellular target of MIS signalling, the Müllerian duct mesenchyme. This approach can be used with conditional genetic strategies to identify factors that are required for the regression of the female genital duct system.

Iris hypoplasia in mice that lack the alternatively spliced Pax6(5a) isoform.

PAX6 is an evolutionarily conserved transcription factor that plays a critical role in vertebrate and invertebrate eye formation. Heterozygous null mutations in the PAX6 gene result in aniridia in humans and a distinct small eye syndrome in rodents. Vertebrates primarily express two alternatively spliced isoforms of Pax6 that differ by the presence or absence of exon 5a (e5A) that encodes an additional 14 aa residues within the paired domain. The e5a-containing isoform, PAX6(5a), is specific to and conserved in vertebrates. To determine the role of PAX6(5a), we have generated mice that lack e5a of the Pax6 gene. Unlike Pax6 null mice that exhibit anopthalmia with central nervous system defects and lethality, 5a isoform-null mice have iris hypoplasia and defects in the cornea, lens, and retina. Although invertebrates have structures that respond to light intensity and act to restrict light exposure of the eyes, a significant and distinct feature of the vertebrate eye is its ability to regulate the amount of incoming light through contractile pupils. This feature of the eye not only allows vertebrates to see in various light conditions but also enhances image resolution. The requirement of the 5a isoform in iris formation suggests that the evolution of this isoform contributed to advanced features of the vertebrate eye.