Cerebellar nuclei excitatory neurons regulate developmental scaling of presynaptic Purkinje cell number and organ growth.

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.

Lhx1 is required in Müllerian duct epithelium for uterine development.

The female reproductive tract organs of mammals, including the oviducts, uterus, cervix and upper vagina, are derived from the Müllerian ducts, a pair of epithelial tubes that form within the mesonephroi. The Müllerian ducts form in a rostral to caudal manner, guided by and dependent on the Wolffian ducts that have already formed. Experimental embryological studies indicate that caudal elongation of the Müllerian duct towards the urogenital sinus occurs in part by proliferation at the ductal tip. The molecular mechanisms that regulate the elongation of the Müllerian duct are currently unclear. Lhx1 encodes a LIM-homeodomain transcription factor that is essential for male and female reproductive tract development. Lhx1 is expressed in both the Wolffian and Müllerian ducts. Wolffian duct-specific knockout of Lhx1 results in degeneration of the Wolffian duct and consequently the non-cell-autonomous loss of the Müllerian duct. To determine the role of Lhx1 specifically in the Müllerian duct epithelium, we performed a Müllerian duct-specific knockout study using Wnt7a-Cre mice. Loss of Lhx1 in the Müllerian duct epithelium led to a block in Müllerian duct elongation and uterine hypoplasia characterized by loss of the entire endometrium (luminal and glandular epithelium and stroma) and inner circular but not the outer longitudinal muscle layer. Time-lapse imaging and molecular analyses indicate that Lhx1 acts cell autonomously to maintain ductal progenitor cells for Müllerian duct elongation. These studies identify LHX1 as the first transcription factor that is essential in the Müllerian duct epithelial progenitor cells for female reproductive tract development. Furthermore, these genetic studies demonstrate the requirement of epithelial-mesenchymal interactions for uterine tissue compartment differentiation.

STAT3 regulates uterine epithelial remodeling and epithelial-stromal crosstalk during implantation.

Embryo implantation is regulated by a variety of endometrial factors, including cytokines, growth factors, and transcription factors. Earlier studies identified the leukemia inhibitory factor (LIF), a cytokine produced by uterine glands, as an essential regulator of implantation. LIF, acting via its cell surface receptor, activates the signal transducer and activator of transcription 3 (STAT3) in the uterine epithelial cells. However, the precise mechanism via which activated STAT3 promotes uterine function during implantation remains unknown. To identify the molecular pathways regulated by STAT3, we created SW(d/d) mice in which Stat3 gene is conditionally inactivated in uterine epithelium. The SW(d/d) mice are infertile due to a lack of embryo attachment to the uterine luminal epithelium and consequent implantation failure. Gene expression profiling of uterine epithelial cells of SW(d/d) mice revealed dysregulated expression of specific components of junctional complexes, including E-cadherin, α- and ß-catenin, and several claudins, which critically regulate epithelial junctional integrity and embryo attachment. In addition, uteri of SW(d/d) mice exhibited markedly reduced stromal proliferation and differentiation, indicating that epithelial STAT3 controls stromal function via a paracrine mechanism. The stromal defect arose from a drastic reduction in the production of several members of the epidermal growth factor family in luminal epithelium of SW(d/d) uteri and the resulting lack of activation of epidermal growth factor receptor signaling and mitotic activity in the stromal cells. Collectively, our results uncovered an intricate molecular network operating downstream of STAT3 that regulates uterine epithelial junctional reorganization, and stromal proliferation, and differentiation, which are critical determinants of successful implantation.

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium.

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5' sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.

Critical tumor suppressor function mediated by epithelial Mig-6 in endometrial cancer.

Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal-epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6(f/f)) mice were crossed to Wnt7a-Cre mice (Wnt7a(cre+)Mig-6(f/f)). Wnt7a(cre+)Mig-6(f/f) mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7a(cre+)Mig-6(f/f) mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7a(cre+)Mig-6(f/f) mice was prevented by progesterone treatment, whereas this treatment had no effect in PR(cre/+)Mig-6(f/f) mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer.

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

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

Stromal-to-epithelial transition during postpartum endometrial regeneration.

Endometrium is the inner lining of the uterus which is composed of epithelial and stromal tissue compartments enclosed by the two smooth muscle layers of the myometrium. In women, much of the endometrium is shed and regenerated each month during the menstrual cycle. Endometrial regeneration also occurs after parturition. The cellular mechanisms that regulate endometrial regeneration are still poorly understood. Using genetic fate-mapping in the mouse, we found that the epithelial compartment of the endometrium maintains its epithelial identity during the estrous cycle and postpartum regeneration. However, whereas the stromal compartment maintains its identity during homeostatic cycling, after parturition a subset of stromal cells differentiates into epithelium that is subsequently maintained. These findings identify potential progenitor cells within the endometrial stromal compartment that produce long-term epithelial tissue during postpartum endometrial regeneration.

The engrailed homeobox genes are required in multiple cell lineages to coordinate sequential formation of fissures and growth of the cerebellum.

The layered cortex of the cerebellum is folded along the anterior-posterior axis into lobules separated by fissures, allowing the large number of cells needed for advanced cerebellar functions to be packed into a small volume. During development, the cerebellum begins as a smooth ovoid structure with two progenitor zones, the ventricular zone and upper rhombic lip, which give rise to distinct cell types in the mature cerebellum. Initially, the cerebellar primordium is divided into five cardinal lobes, which are subsequently further subdivided by fissures. The cellular processes and genes that regulate the formation of a normal pattern of fissures are poorly understood. The engrailed genes (En1 and En2) are expressed in all cerebellar cell types and are critical for regulating formation of specific fissures. However, the cerebellar cell types that En1 and En2 act in to control growth and/or patterning of fissures has not been determined. We conditionally eliminated En2 or En1 and En2 either in both progenitor zones and their descendents or in the two complementary sets of cells derived from each progenitor zone. En2 was found to be required only transiently in the progenitor zones and their immediate descendents to regulate formation of three fissures and for general growth of the cerebellum. In contrast, En1 and En2 have overlapping functions in the cells derived from each progenitor zone in regulating formation of additional fissures and for extensive cerebellar growth. Furthermore, En1/2 function in ventricular zone-derived cells plays a more significant role in determining the timing of initiation and positioning of fissures, whereas in upper rhombic lip-derived cells the genes are more important in regulating cerebellar growth. Our studies reveal the complex manner in which the En genes control cerebellar growth and foliation in distinct cell types.

Spatially restricted and developmentally dynamic expression of engrailed genes in multiple cerebellar cell types.

The cerebellum is a highly organized structure partitioned into lobules along the anterior-posterior (A-P) axis and into striped molecular domains along the medial-lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P) 21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCß4 and EPHA4.

Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses.

Female fertility requires estrogen to specifically stimulate estrogen receptor α (ERα)-dependent growth of the uterine epithelium in adult mice, while immature females show proliferation in both stroma and epithelium. To address the relative roles of ERα in mediating estrogen action in uterine epithelium versus stroma, a uterine epithelial-specific ERα knockout (UtEpiαERKO) mouse line was generated by crossing Esr mice with Wnt7a-Cre mice. Expression of Wnt7a directed Cre activity generated selective deletion of ERα in uterine epithelium, and female UtEpiαERKO are infertile. Herein, we demonstrate that 17ß-estradiol (E(2))-induced uterine epithelial proliferation was independent of uterine epithelial ERα because DNA synthesis and up-regulation of mitogenic mediators were sustained in UtEpiαERKO uteri after E(2) treatment. IGF-1 treatment resulted in ligand-independent ER activation in both wild-type (WT) and UtEpiαERKO and mimicked the E(2) stimulatory effect on DNA synthesis in uterine epithelium. Uterine epithelial ERα was necessary to induce lactoferrin, an E(2)-regulated secretory protein selectively synthesized in the uterine epithelium. However, loss of uterine epithelial ERα did not alter the E(2)-dependent progesterone receptor (PR) down-regulation in epithelium. Strikingly, the uterine epithelium of UtEpiαERKO had robust evidence of apoptosis after 3 d of E(2) treatment. Therefore, we surmise that estrogen induced uterine hyperplasia involves a dispensable role for uterine epithelial ERα in the proliferative response, but ERα is required subsequent to proliferation to prevent uterine epithelial apoptosis assuring the full uterine epithelial response, illustrating the differential cellular roles for ERα in uterine tissue and its contribution during pregnancy.

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.

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.