Gonadal Identity in the Absence of pro-Testis Factor SOX9 and pro-Ovary Factor beta-catenin
ABSTRACT: The main goal of our study is to identify the molecular events that determine the gonadal identity in mammals. Although testis and ovary arise from a common embryonic primordium, they represent outcomes of opposing fate determination. This decision to differentiate into a testis or an ovary hinges upon the balance between two antagonizing factors, pro-testis SOX9 and pro-ovary β-catenin. This microarray analysis led to the identification of the genes involved in the fate of XX and XY gonads in absence of SOX9 and beta-catenin We developed mouse genetic models that lack either Sox9, β-catenin, or both specifically in the somatic cells. All embryos used in this study resulted from the crossing between Ctnnb1f/f; Sox9f/f females with Sf1-cre+/Tg ; Ctnnb1+/f; Sox9+/f males. XX and XY fetal gonads were collected at embryonic day E14.5
Project description:Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development.
Project description:The transcription factors SRY and SOX9 and RSPO1/WNT4/?-Catenin signaling act as antagonistic pathways to drive testis and ovary development respectively, from a common gonadal primordium in mouse embryos. In this work, we took advantage of a double knockout mouse model to study gonadal development when Sox9 and Wnt4 are both mutated. We show that the XX gonad mutant for Wnt4 or for both Wnt4 and Sox9 develop as ovotestes, demonstrating that ectopic SOX9 function is not required for the partial female-to-male sex reversal caused by a Wnt4 mutation. Sox9 deletion in XY gonads leads to ovarian development accompanied by ectopic WNT/?-catenin signaling. In XY Sox9 mutant gonads, SRY-positive supporting precursors adopt a female-like identity and develop as pre-granulosa-like cells. This phenotype cannot be fully prevented by the deletion of Wnt4 or Rspo1, indicating that SOX9 is required for the early determination of the male supporting cell identity independently of repressing RSPO1/WNT4/?-Catenin signaling. However, in XY Sox9 Wnt4 double mutant gonads, pre-granulosa cells are not maintained, as they prematurely differentiate as mature granulosa cells and then trans-differentiate into Sertoli-like cells. Together, our results reveal the dynamics of the specific and independent actions of SOX9 and WNT4 during gonadal differentiation: SOX9 is essential in the testis for early specification of male-supporting cells whereas WNT4 functions in the ovary to maintain female-supporting cell identity and inhibit male-specific vascular and steroidogenic cell differentiation.
Project description:In mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/?-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal.
Project description:Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/?-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6-9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding ? catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced ?-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a ?-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding ?-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of ?-catenin signaling to oppose testis determination.
Project description:In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1(KO)Sox9(cKO) ()), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1(KO)Sox9(cKO) mice. Moreover, since testis development occurs in XY Rspo1(KO)Sox9(cKO) mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9(cKO) mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised.
Project description:We used two Sertoli-cell-specific Cre lines: Wt1-CreERT2 and Sox9-CreERT2, to induce Sox9 ablation on a Sox8 -/- background in the adult testis. Sox9/8 double KO testes undergo testis-to-ovary genetic reprogramming and Sertoli-to-granulosa cell transdifferentiation.
Project description:During mammalian sex determination, expression of the Y-linked gene Sry shifts the bipotential gonad toward a testicular fate by upregulating a feed-forward loop between FGF9 and SOX9 to establish SOX9 expression in somatic cells. We previously proposed that these signals are mutually antagonistic with counteracting signals in XX gonads and that a shift in the balance of these factors leads to either male or female development. Evidence in mice and humans suggests that the male pathway is opposed by the expression of two signals, WNT4 and R-SPONDIN-1 (RSPO1), that promote the ovarian fate and block testis development. Both of these ligands can activate the canonical Wnt signaling pathway. Duplication of the distal portion of chromosome 1p, which includes both WNT4 and RSPO1, overrides the male program and causes male-to-female sex reversal in XY patients. To determine whether activation of beta-catenin is sufficient to block the testis pathway, we have ectopically expressed a stabilized form of beta-catenin in the somatic cells of XY gonads. Our results show that activation of beta-catenin in otherwise normal XY mice effectively disrupts the male program and results in male-to-female sex-reversal. The identification of beta-catenin as a key pro-ovarian and anti-testis signaling molecule will further our understanding of the mechanisms controlling sex determination and the molecular mechanisms that lead to sex-reversal.
Project description:: Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differ-entiation, while in mammals, this mechanism has been supplanted by the testis determining SRY gene. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic anal-yses to investigate the precise mechanism through which oestrogen impacts these pathways to ac-tivate -catenin—a factor essential for ovarian development. We show that oestrogen can activate -catenin within 30 minutes, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to -catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.
Project description:We have previously established an in vivo requirement for GATA4 and FOG2 transcription factors in sexual differentiation. Fog2 null mouse fetuses or fetuses homozygous for a targeted mutation in Gata4 (Gata4(ki)), which cripples the GATA4-FOG2 interaction, exhibit a profound and early block in testis differentiation in both sexes. Others have shown that XX mice with the Ods transgenic insertion or the Wt1-Sox9 YAC transgene overexpress the testis differentiation gene, Sox9. Thus, these XX animals undergo dominant sex reversal by developing into phenotypically normal, but sterile, males. Now we have determined that Fog2 haploinsufficiency prevents (suppresses) this dominant sex reversal and Fog2+/-Wt1-Sox9 or Ods XX animals develop normally--as fertile females. The suppression of sex reversal in Fog2 heterozygous females results from approximately 50% downregulation of the expression from the transgene-associated allele of Sox9. The GATA4/FOG2-dependent sex reversal observed in the transgenic XX gonads has to rely on gene targets other than the Y chromosome-linked Sry gene. Importantly, Fog2 null or Gata4(ki/ki) embryos (either XX or XY) fail to express detectable levels of Sox9 despite carrying the Ods mutation or Wt1-Sox9 transgene. Fog2 haploinsufficiency leads to a decreased amount of SOX9-positive cells in XY gonads. We conclude that FOG2 is a limiting factor in the formation of a functional GATA4/FOG2 transcription complex that is required for Sox9 expression during gonadogenesis.
Project description:Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-?-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/?-catenin activity and reduced <i>Sox9</i> expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human <i>ZNRF3</i> variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.