Wnt4 action in gonadal development and sex determination

Wnt4 is a growth factor involved in multiple developmental processes such as the formation of the kidney, adrenal, mammary gland, pituitary and the female reproductive system. During mammalian embryogenesis, Wnt4 is expressed in the gonads of both sexes before sex determination events take place and is subsequently down-regulated in the male gonad. Inactivation of the Wnt4 gene in mice has revealed that it is involved at several steps of female reproductive development. Wnt4 is implicated in Müllerian duct regression, the formation of sex-specific vasculature, the inhibition of steroidogenesis and in sex-specific cell migration events. A mouse model of sex-reversal has partially unravelled the molecular pathways in which Wnt4 operates during the development of the female reproductive system. However, the specific molecular mechanism of action of Wnt4 during gonadal development remains unknown. This and downstream signaling pathways involved in Wnt4 action during female gonad development are reviewed and models of Wnt4 action are proposed for Müllerian duct formation, sex-specific vasculature development, and sex determination events. Further identification of critical downstream effectors of the Wnt4 signaling pathway in mouse models and in patients with sex-reversal conditions could help in understanding sex-reversal pathologies in humans.     

Role of steroidogenic factor 1 and aromatase in temperature-dependent sex determination in the red-eared slider turtle.

Red-eared slider turtles are genetically bipotential for sex determination. In this species, as in many other reptiles, incubation temperature of the egg determines gonadal sex. At higher incubation temperatures females are produced and increasing temperature appears to increase estrogen production in the embryonic brain. Treatment of eggs incubating at a male-producing temperature with exogenous estrogen causes ovaries to form. At a female-biased incubation temperature, prevention of estrogen biosynthesis or administration of nonaromatizable androgens results in the development of testes. In mammals, steroidogenic factor 1 (SF-1) regulates most genes required for estrogen biosynthesis, including aromatase. In both mammals and red-eared sliders, SF-1 is differentially expressed in males and females during gonadogenesis. We have examined both SF-1 gene expression and aromatase activity in embryos incubating at different temperatures and after manipulation to change the course of gonadal development. Our findings indicate a central role for SF-1 in enacting the effect of estrogen. Estrogen treatment directly or indirectly downregulates SF-1 and, ultimately, causes development of females. The inhibition of estrogen results in upregulation of SF-1 and male hatchlings. Thus, SF-1 may lie at the center of one molecular crossroad in male versus female differentiation of the red-eared slider.     

Expression of the SMADIP1 gene during early human development

There are four members of the platelet-derived growth factor (PDGF) family; PDGF-A, PDGF-B, PDGF-C and PDGF-D. Their biological effects are mediated via two tyrosine kinase receptors, PDGFR-alpha and PDGFR-beta, and PDGF-mediated signaling is critical for development of many organ systems. Analysis in adult tissues showed that PDGF-C was mainly expressed in kidney, testis, liver, heart and brain. During development, PDGF-C expression was widespread and dynamic, and found in somites and their derivatives, in kidney, lung, brain, and in several other tissues, particularly at sites of developing epidermal openings. PDGF-C may therefore have unique functions during tissue development and maintenance.     

A Boolean network model of human gonadal sex determination

Background

Gonadal sex determination (GSD) in humans is a complex biological process that takes place in early stages of embryonic development when the bipotential gonadal primordium (BGP) differentiates towards testes or ovaries. This decision is directed by one of two distinct pathways embedded in a GSD network activated in a population of coelomic epithelial cells, the Sertoli progenitor cells (SPC) and the granulosa progenitor cells (GPC). In males, the pathway is activated when the Sex-Determining Region Y (SRY) gene starts to be expressed, whereas in females the WNT4/ β-catenin pathway promotes the differentiation of the GPCs towards ovaries. The interactions and dynamics of the elements that constitute the GSD network are poorly understood, thus our group is interested in inferring the general architecture of this network as well as modeling the dynamic behavior of a set of genes associated to this process under wild-type and mutant conditions.

Methods

We reconstructed the regulatory network of GSD with a set of genes directly associated with the process of differentiation from SPC and GPC towards Sertoli and granulosa cells, respectively. These genes are experimentally well-characterized and the effects of their deficiency have been clinically reported. We modeled this GSD network as a synchronous Boolean network model (BNM) and characterized its attractors under wild-type and mutant conditions.

Results

Three attractors with a clear biological meaning were found; one of them corresponding to the currently known gene expression pattern of Sertoli cells, the second correlating to the granulosa cells and, the third resembling a disgenetic gonad.

Conclusions

The BNM of GSD that we present summarizes the experimental data on the pathways for Sertoli and granulosa establishment and sheds light on the overall behavior of a population of cells that differentiate within the developing gonad. With this model we propose a set of regulatory interactions needed to activate either the SRY or the WNT4/ β-catenin pathway as well as their downstream targets, which are critical for further sex differentiation. In addition, we observed a pattern of altered regulatory interactions and their dynamics that lead to some disorders of sex development (DSD).

     

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.     

Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor alpha and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.     

Expression profiles for six zebrafish genes during gonadal sex differentiation

Background  

The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation.     

早期胚胎的发育选择:性别决定

性别决定是一个复杂的发育调控过程, 早期胚胎发育过程中, 雌雄二者必居其一的发育选择是胚胎性腺形成必须的发育决定。文章综述了动物性别决定的遗传系统、性腺发生、性别决定关键基因及其作用机制, 从分子进化的角度分析了性染色体与性别决定形成机制, 提示性别决定基因在进化中总是趋向异配性染色体。     

Expression of ASK1 during chick and early mouse development

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.     

Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation

Alternative splicing of Wt1 results in the insertion or omission of the three amino acids KTS between zinc fingers 3 and 4. In vitro experiments suggest distinct molecular functions for + and -KTS isoforms. We have generated mouse strains in which specific isoforms have been removed. Heterozygous mice with a reduction of +KTS levels develop glomerulosclerosis and represent a model for Frasier syndrome. Homozygous mutants of both strains die after birth due to kidney defects. Strikingly, mice lacking +KTS isoforms show a complete XY sex reversal due to a dramatic reduction of Sry expression levels. Our data demonstrate distinct functions for the two splice variants and place the +KTS variants as important regulators for Sry in the sex determination pathway.     

Expression of the RSK2 gene during early human development

The 90 kDa ribosomal S6 serine/threonine kinase 2 gene (RSK2, U08316) has been recently identified as a disease-causing gene in an X-linked disorder, the Coffin-Lowry Syndrome (MIM 303600) characterized by severe mental retardation, facial dysmorphisms and progressive skeletal malformations. To investigate its possible role in cerebral cortex development, we performed RNA in situ hybridization at three stages of human development: day 32 (Carnegie 15), 9 weeks (Carnegie 23) and 13 weeks. RSK2 expression is detected in the embryonic anterior and posterior telencephalon (hippocampus anlagen), mesencephalon, rhombencephalon and cerebellum. RSK2 gene expression is also observed in dorsal root ganglia, cranial nerve ganglia, and sensory epithelium of the inner ear, liver, lung and jaw anlagen. This pattern of expression may be involved in cognitive impairment and facial dysmorphisms found in Coffin-Lowry Syndrome.     

Expression of steroidogenic factors 1 and 2 in normal human pancreas

Orphan nuclear receptor steroidogenic factor-1 (SF-1) is crucial for development and function of steroidogenic organs. The steroidogenic factor-2 (SF-2) is an essential factor involved in cholesterol transfer and activation of promoters of steroidogenic enzymes CYP11A1, CYP17 and Steroidogenic Acute Regulatory Protein (StAR). We have previously demonstrated steroidogenic activity in pancreatic tissue. The aim of this study was to investigate the presence of SF-1 and SF-2 in human pancreas. Total RNA was extracted from normal male (five) and female (five) samples, obtained from the organs donor program. RT-PCR approach was used to analyze the expression of SF-1 and SF-2. Immunohistochemical analysis was performed for SF-1. The bands of expression were present in both male and female samples, although differential expression was observed. For both factors, the signal detected was more evident in males than in females. A similar pattern was present in the immunohistochemical study. Normal human pancreas expresses SF-1 and SF-2 factors similarly to ovary and adrenals. A distinctive characteristic is the sexually dimorphic expression of these factors. Our data provide evidence suggesting that the pancreas achieves steroidogenic activity supporting the presence of gender- and location-related differences in the expression of these steroidogenic factors.     

Expression of nerve growth factor receptor during human peripheral nerve development

The expression of NGF receptors on human Schwann cells during development and myelination and in culture was analyzed using a murine monoclonal antibody to human NGF receptor. Nonmyelinated femoral nerves from 13- to 14-week fetuses stained strongly for NGF receptor, whereas tissues from later stages of development showed a decrease in the staining intensity. These changes correlated with the initiation of myelination (17-19 weeks), as observed by phase-contrast and electron microscopy, and the reactivity with monoclonal antibody 4C5, a marker of mature Schwann cells. In adult nerves, only the perineurium and few endoneurial cells were stained with anti-NGF receptor antibody. Cultured human fetal Schwann cells were positive for NGF receptor by immunofluorescence irregardless of donor age or length of time in culture. The decreased staining of NGF receptor with nerve maturation may reflect a dependence of antigen expression on Schwann cell differentiation and/or neuron-Schwann cell interaction.