Transient development of ovotestes in XX Sox9 transgenic mice

AbstractThe sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9Tg/+ gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3′ UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9–EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth

Myocardial-specific R-spondin3 drives proliferation of the coronary stems primarily through the Leucine Rich Repeat G Protein coupled receptor LGR4

Coronary artery anomalies are common congenital disorders with serious consequences in adult life. Coronary circulation begins when the coronary stems form connections between the aorta and the developing vascular plexus. We recently identified the WNT signaling modulator R-spondin 3 (Rspo3), as a crucial regulator of coronary stem proliferation. Using expression analysis and tissue-specific deletion we now demonstrate that Rspo3 is primarily produced by cardiomyocytes. Moreover, we have employed CRISPR/Cas9 technology to generate novel Lgr4-null alleles that showed a significant decrease in coronary stem proliferation and thus phenocopied the coronary artery defects seen in Rspo3 mutants. Interestingly, Lgr4 mutants displayed slightly hypomorphic right ventricles, an observation also made after myocardial specific deletion of Rspo3. These results shed new light on the role of Rspo3 in heart development and demonstrate that LGR4 is the principal Rspondin 3 receptor in the heart

XY Sox9 embryonic loss-of-function mouse mutants show complete sex reversal and produce partially fertile XY oocytes.

International audienceGonadal differentiation is the first step of mammalian sex determination. The expression of the Y chromosomal testis determining factor Sry leads to up-regulation of the transcription factor Sox9 which promotes testis differentiation. Previous studies showed that Sox9 deficiency induces expression of ovarian markers in XY mutant fetal gonads before they die. To better understand the genome-wide transcriptional profile underlying this process we compared samples from XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads in which Sox9 is ablated in Sertoli-precursor cells during early stages of gonad development to XX Sox9(flox/flox) ovaries and XY Sox9(flox/flox) testes at E13.5. We found a complex mRNA signature that indicates wide-spread transcriptional de-regulation and revealed for XY mutants at E13.5 an intermediate transcript profile between male and female gonads. However, XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads develop as ovaries containing XY developing follicles at P0 but less frequently so than in XX control ovaries. Furthermore, we studied the extent to which developing XY mutant ovaries are able to mediate adult fertility and observed that XY oocytes from XY mutant ovaries are competent for fertilization; however, two thirds of them fail to develop beyond two-cell stage embryos. Taken together, we found that XY Sf1:Cre(Tg/+); Sox9(flox/flox) females are capable of producing viable offspring albeit at a reduced level

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here we show in mice that the signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/β-catenin signalling and their genetic deletion leads to a rapid decline of nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation

WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad.

International audienceThe gonad arises from the thickening of the coelomic epithelium and then commits into the sex determination process. Testis differentiation is activated by the expression of the Y-linked gene Sry, which promotes cell proliferation and differentiation of Sertoli cells, the supporting cells of the testis. In absence of Sry (XX individuals), activation of WNT/CTNNB1 signalling, via the upregulation of Rspo1 and Wnt4, promotes ovarian differentiation. However, Rspo1 and Wnt4 are expressed in the early undifferentiated gonad of both sexes, and Axin2-lacZ, a reporter of canonical WNT/CTNNB1 signalling, is expressed in the coelomic region of the E11.5 gonadal primordium, suggesting a role of these factors in early gonadal development. Here, we show that simultaneous ablation of Rspo1 and Wnt4 impairs proliferation of the cells of the coelomic epithelium, reducing the number of progenitors of Sertoli cells in XY mutant gonads. As a consequence, in XY Wnt4(-/-); Rspo1(-/-) foetuses, this leads to the differentiation of a reduced number of Sertoli cells and the formation of a hypoplastic testis exhibiting few seminiferous tubules. Hence, this study identifies Rspo1 and Wnt4 as two new regulators of cell proliferation in the early gonad regardless of its sex, in addition to the specific role of these genes in ovarian differentiation

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals

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/β-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

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals

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/β-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

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

<div><p>In mammals, male sex determination is governed by SRY-dependent activation of <em>Sox9</em>, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated <em>Sry</em> and <em>Sox9</em> 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 <em>Rspo1</em> mutant mice correlates with <em>Sox9</em> 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 <em>Rspo1</em> and <em>Sox9</em> (referred to as XX <em>Rspo1^KOSox9^cKO</em>⁾, indicating that <em>Sry</em> and <em>Sox9</em> are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both <em>Sox8</em> and <em>Sox10</em>, suggesting that activation of <em>Sox</em> genes other than <em>Sox9</em> can induce male differentiation in <em>Rspo1^KOSox9^cKO</em> mice. Moreover, since testis development occurs in XY <em>Rspo1^KOSox9^cKO</em> mice, our data show that <em>Rspo1</em> is the main effector for male-to-female sex reversal in XY <em>Sox9^cKO</em> mice. Thus, <em>Rspo1</em> 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.</p> </div

Non-differentiated XY and XX <i>Rspo1^KOSox9^cKO</i> gonads at 13.5 d<i>pc</i>.

<p>Immunofluorescence of SOX9 (Sertoli cell marker, in red) and AMH (Sertoli cell marker green) (a–d), AMH (Sertoli cell marker, in green) and SRY (pre-Sertoli and Sertoli cell marker in red) (e–h) and SF1 (undifferentiated supporting cell, Sertoli and Leydig cell marker) (i–l). Counterstain is DAPI (in blue). Lack of SOX9 and AMH expression in XY (b) and XX (c) <i>Rspo1^KOSox9^cKO</i> gonads shows that Sertoli cell differentiation did not occur at 13.5 d<i>pc</i>. Note that the kidneys (K) are positive for SOX9. This is accompagnied with the maintenance of SRY expression in the XY <i>Rspo1^KOSox9^cKO</i> gonads (f) whereas SRY expression has ceased in XY controls (e). SF1 expression is maintained in absence of Sertoli cells differentiation in XY and XX <i>Rspo1^KOSox9^cKO</i> gonads (j and k respectively) (scale bar: 100 µm). Note that SF1 is also expressed in steroidogenic cells of the adrenals (A). XY (a, e, i) and XX (d, h, l) <i>Rspo1^+/−; Sox9^flox/flox</i> controls, XY (b, f, j) and XX (c, g, k) <i>Sox9^cKO Rspo1^KO</i> respectively.</p

Opposing function of SOX and RSPO1 signaling in the fate of the gonad.

<p>A- In XX gonads, RSPO1 activates WNT/beta-catenin signaling to promote ovarian differentiation. Ablation of <i>Rspo1</i> results in partial sex reversal with ovotestis development, which coincides with <i>Sox9</i> expression. However additional deletion of <i>Sox9</i> in the XX <i>Rspo1</i>^KO (i.e., <i>Rspo1^KOSox9^cKO</i>) still allows ovotestis formation, implying that <i>Sry</i> and <i>Sox9</i> are not required for testicular differentiation in female-to-male sex reversal. B- In XY gonads, whereas <i>Sox9</i> deletion triggers ovarian development, additional deletion of <i>Rspo1</i> in XY <i>Rspo1^KOSox9^cKO</i> gonads restores testis development. This is associated with the expression of other SOX genes like SOX 8 and SOX10, other masculinising factors.</p