Hao Chang

Wt1 negatively regulates β-catenin signaling during testis development

β-Catenin, as an important effector of the canonical Wnt signaling pathway and as a regulator of cell adhesion, has been demonstrated to be involved in multiple developmental processes and tumorigenesis. β-Catenin expression was found mainly on the Sertoli cell membrane starting from embryonic day 15.5 in the developing testes. However, its potential role in Sertoli cells during testis formation has not been examined. To determine the function of β-catenin in Sertoli cells during testis formation, we either deleted β-catenin or expressed a constitutively active form ofβ -catenin in Sertoli cells. We found that deletion caused no detectable abnormalities. However, stabilization caused severe phenotypes, including testicular cord disruption, germ cell depletion and inhibition of Müllerian duct regression. β-Catenin stabilization caused changes in Sertoli cell identity and misregulation of inter-Sertoli cell contacts. As Wt1 conditional knockout in Sertoli cells causes similar phenotypes to our stabilized β-catenin mutants, we then investigated the relationship of Wt1 and β-catenin in Sertoli cells and found Wt1 inhibits β-catenin signaling in these cells during testis development. Wt1 deletion resulted in upregulation of β-catenin expression in Sertoli cells both in vitro and in vivo. Our study indicates that Sertoli cell expression of β-catenin is dispensable for testis development. However, the suppression of β-catenin signaling in these cells is essential for proper testis formation and Wt1 is a negative regulator of β-catenin signaling during this developmental process.

Sox9 in Testis Determination

Abstract: Sox9 is an Sry‐box‐containing gene that encodes a transcriptional activator. During mouse gonadogenesis, Sox9 is detected in the male gonad at 11.5 days postcoitus (dpc). At 12.5 dpc, testicular cords form, morphologically distinguishing the male gonad from the ovary. From this stage onwards, Sox9 expression is restricted to the Sertoli cell lineage and persists in the adult. Humans with heterozygous mutations in SOX9 develop a skeletal syndrome known as campomelic dysplasia. Furthermore, most XY SOX9 heterozygotes show variable male‐to‐female sex reversal, implicating SOX9 in testis development. Sox9 heterozygous knockout mice die at birth with a syndrome similar to that of human campomelic dysplasia. In contrast to humans, XY Sox9+/− mice form normal appearing testes. Germ‐line knockout of Sox9 using a conditional null allele provides a tool for generating Sox9−/− mice by simple genetic crosses. However, Sox9−/− mice die soon after 11.5 dpc because of cardiovascular defects. In vitro culture of the urogenital ridges of XY Sox9−/− results in gonads lacking testicular cords and Sertoli cell marker expression, but with the expression of ovarian‐specific markers. Therefore, Sox9 is essential for diverting an intrinsically ovarian program of organogenesis toward testis formation.

When whorls collide: the development of hair patterns in frizzled 6 mutant mice

Surface appendages such as bristles, feathers and hairs exhibit both long- and short-range order. In the frizzled 6 null (Fz6–/–) mouse the orientations of the earliest born hair follicles are uncorrelated, but over time the follicles reorient to create patterns that are characterized by a high degree of local order. By quantifying follicle orientations over time, in both living and fixed tissues, we define the time course of local hair follicle refinement and the resulting evolution of a montage of competing patterns in Fz6–/– skin. We observe an apparently local process that within one week can organize a field of many tens of thousands of follicles, generating long-range order that extends over distances of more than one centimeter. Physical systems that undergo an analogous ordering of vector components suggest potential mechanisms that might apply to the patterning of hair follicles and related biological structures.

Overactive Beta-Catenin Signaling Causes Testicular Sertoli Cell Tumor Development in the Mouse

Overactive WNT/beta-catenin signaling has been found in many forms of cancer in human patients. Mouse models with mutations in different components of the WNT/beta-catenin signaling pathway have been generated to mimic tumorigenesis in humans. Mice with mutations that result in overactive WNT/beta-catenin signaling developed tumors in some tissues, such as digestive tract, skin, and ovary, but they failed to develop tumors in other tissues, such as mammary gland, liver, kidney, and primordial germ cells. To investigate whether overactive beta-catenin signaling is capable of inducing Sertoli cell tumorigenesis in testes, we generated Ctnnb1tm1Mmt/+;Tg(AMH-cre)1Flor male mice that express a constitutively active form of beta-catenin specifically in Sertoli cells. No tumors were observed at 4 mo of age, but 70% of the mutant males developed Sertoli cell tumors at 8 mo of age. At 1 yr of age, more than 90% of the mutant males developed tumors. No instances of extratesticular spread of the tumors were found in the mutant mice. These studies show a causal link between overactive WNT/beta-catenin signaling and Sertoli cell tumor development and provide a novel mouse model for the study of Sertoli cell tumor biology.

Responses of hair follicle–associated structures to loss of planar cell polarity signaling

Significance In mammals, hair follicles reside in the skin together with a set of follicle-associated structures: sebaceous glands, nerve fibers, specialized sensory cells, and muscle fibers. In general, the follicle and its associated structures are precisely oriented with respect to the body axes. The present study shows that, in mice genetically engineered to lack the follicle orienting system, the follicle-associated structures—with the exception of the specialized sensory cells—acquire an orientation that matches that of the follicle. These experiments imply that hair follicles communicate local orienting information to most of their associated structures. The mammalian hair follicle unit consists of a central follicle and a series of associated structures: sebaceous glands, arrector pili muscles, Merkel cells, and sensory nerve endings. The architecture of this multicellular structure is highly polarized with respect to the body axes. Previous work has implicated Frizzled6 (Fz6)-mediated planar cell polarity (PCP) signaling in the initial specification of hair follicle orientation. Here we investigate the origin of polarity information among structures within the hair follicle unit. Merkel cell clusters appear to have direct access to Fz6-based polarity information, and they lose polarity in the absence of Fz6. By contrast, the other follicle-associated structures likely derive some or all of their polarity cues from hair follicles, and as a result, their orientations closely match that of their associated follicle. These experiments reveal the interplay between global and local sources of polarity information for coordinating the spatial arrangement of diverse multicellular structures. They also highlight the utility of mammalian skin as a system for quantitative analyses of biological polarity.

Frizzled Receptors in Development and Disease.

Frizzled proteins are the principal receptors for the Wnt family of ligands. They mediate canonical Wnt signaling together with Lrp5 and Lrp6 coreceptors. In conjunction with Celsr, Vangl, and a small number of additional membrane and membrane-associated proteins, they also play a central role in tissue polarity/planar cell polarity (PCP) signaling. Targeted mutations in 9 of the 10 mammalian Frizzled genes have revealed their roles in an extraordinarily diverse set of developmental and homeostatic processes, including morphogenetic movements responsible for palate, ventricular septum, ocular furrow, and neural tube closure; survival of thalamic neurons; bone formation; central nervous system (CNS) angiogenesis and blood-brain barrier formation and maintenance; and a wide variety of processes that orient subcellular, cellular, and multicellular structures relative to the body axes. The last group likely reflects the mammalian equivalent of tissue polarity/PCP signaling, as defined in Drosophila, and it includes CNS axon guidance, hair follicle and tongue papilla orientation, and inner ear sensory hair bundle orientation. Frizzled receptors are ubiquitous among multicellular animals and, with other signaling molecules, they very likely evolved to permit the development of the complex tissue architectures that provide multicellular animals with their enormous selective advantage.

Generation of Viable Male and Female Mice from Two Fathers

In sexual species, fertilization of oocytes produces individuals with alleles derived from both parents. Here we use pluripotent stem cells derived from somatic cells to combine the haploid genomes from two males to produce viable sons and daughters. Male (XY) mouse induced pluripotent stem cells (Father #1) were used to isolate subclones that had spontaneously lost the Y chromosome to become genetically female (XO). These male-derived XO stem cells were used to generate female chimeras that were bred with genetically distinct males (Father #2), yielding progeny possessing genetic information that was equally derived from both fathers. Thus, functional oocytes can be generated from male somatic cells after reprogramming and spontaneous sex reversal. These findings have novel implications for mammalian reproduction and assisted reproductive technology.

Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance

In mammals, a set of anatomically diverse polarity processes – including axon growth and guidance, hair follicle orientation, and stereociliary bundle orientation in inner ear sensory hair cells – appear to be mechanistically related, as judged by their dependence on vertebrate homologues of core tissue polarity/planar cell polarity (PCP) genes in Drosophila. To explore more deeply the mechanistic similarities between different polarity processes, we have determined the extent to which frizzled 3 (Fz3) can rescue the hair follicle and Merkel cell polarity defects in frizzled 6-null (Fz6−/−) mice, and, reciprocally, the extent to which Fz6 can rescue the axon growth and guidance defects in Fz3−/− mice. These experiments reveal full rescue of the Fz6−/− phenotype by Fz3 and partial rescue of the Fz3−/− phenotype by Fz6, implying that these two proteins are likely to act in a conserved manner in these two contexts. Stimulated by these observations, we searched for additional anatomical structures that exhibit macroscopic polarity and that might plausibly use Fz3 and/or Fz6 signaling. This search has revealed a hitherto unappreciated pattern of papillae on the dorsal surface of the tongue that depends, at least in part, on redundant signaling by Fz3 and Fz6. Taken together, these experiments provide compelling evidence for a close mechanistic relationship between multiple anatomically diverse polarity processes.

The spatio-temporal domains of Frizzled6 action in planar polarity control of hair follicle orientation

In mammals, hair follicles cover most of the body surface and exhibit precise and stereotyped orientations relative to the body axes. Follicle orientation is controlled by the planar cell polarity (PCP; or, more generally, tissue polarity) system, as determined by the follicle mis-orientation phenotypes observed in mice with PCP gene mutations. The present study uses conditional knockout alleles of the PCP genes Frizzled6 (Fz6), Vangl1, and Vangl2, together with a series of Cre drivers to interrogate the spatio-temporal domains of PCP gene action in the developing mouse epidermis required for follicle orientation. Fz6 is required starting between embryonic day (E)11.5 and E12.5. Eliminating Fz6 in either the anterior or the posterior halves of the embryo or in either the feet or the torso leads to follicle mis-orientation phenotypes that are limited to the territories associated with Fz6 loss, implying either that PCP signaling is required for communicating polarity information on a local but not a global scale, or that there are multiple independent sources of global polarity information. Eliminating Fz6 in most hair follicle cells or in the inter-follicular epidermis at E15.5 suggests that PCP signaling in developing follicles is not required to maintain their orientation. The asymmetric arrangement of Merkel cells around the base of each guard hair follicle dependents on Fz6 expression in the epidermis but not in differentiating Merkel cells. These experiments constrain current models of PCP signaling and the flow of polarity information in mammalian skin.

Identification of Astrotactin2 as a Genetic Modifier That Regulates the Global Orientation of Mammalian Hair Follicles

Planar cell polarity (PCP) signaling controls the global orientation of surface structures, such as hairs and bristles, in both vertebrates and invertebrates. In Frizzled6 -/- (Fz6 -/-) mice, hair follicle orientations on the head and back are nearly random at birth, but reorient during early postnatal development to eventually generate a nearly parallel anterior-to-posterior array. We report the identification of a naturally occurring exon 5 deletion in Astrotactin2 (Astn2) that acts as a recessive genetic modifier of the Fz6 -/- hair patterning phenotype. A genetically engineered Astn2 exon 5 deletion recapitulates the modifier phenotype. In Fz6 -/- ;Astn2 ex5del/del mice, hair orientation on the back is subtly biased from posterior-to-anterior, leading to a 180-degree orientation reversal in mature mice. These experiments suggest that Astn2, an endosomal membrane protein, modulates PCP signaling.