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Following activation, macrophages undergo extensive metabolic rewiring1,2. Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process3. Itaconate inhibits succinate dehydrogenase4,5, has electrophilic properties6 and is associated with a change in cytokine production4. Here, we compare the metabolic, electrophilic and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild-type and Irg1-/- macrophages, we show that neither dimethyl itaconate, 4-octyl itaconate nor 4-monoethyl itaconate are converted to intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only dimethyl itaconate and 4-octyl itaconate induce a strong electrophilic stress response, in contrast to itaconate and 4-monoethyl itaconate. This correlates with their immunosuppressive phenotype: dimethyl itaconate and 4-octyl itaconate inhibited IκBζ and pro-interleukin (IL)-1β induction, as well as IL-6, IL-10 and interferon-β secretion, in an NRF2-independent manner. In contrast, itaconate treatment suppressed IL-1β secretion but not pro-IL-1β levels and, surprisingly, strongly enhanced lipopolysaccharide-induced interferon-β secretion. Consistently, Irg1-/- macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive, metabolite and highlights the importance of using unmodified itaconate in future studies.

International audience; Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow–derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody–drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. Significance: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.

Macrophages rely on tightly integrated metabolic rewiring to clear dying neighboring cells by efferocytosis during homeostasis and disease. Here we reveal that glutaminase-1-mediated glutaminolysis is critical to promote apoptotic cell clearance by macrophages during homeostasis in mice. In addition, impaired macrophage glutaminolysis exacerbates atherosclerosis, a condition during which, efficient apoptotic cell debris clearance is critical to limit disease progression. Glutaminase-1 expression strongly correlates with atherosclerotic plaque necrosis in patients with cardiovascular diseases. High-throughput transcriptional and metabolic profiling reveals that macrophage efferocytic capacity relies on a non-canonical transaminase pathway, independent from the traditional requirement of glutamate dehydrogenase to fuel ɑ-ketoglutarate-dependent immunometabolism. This pathway is necessary to meet the unique requirements of efferocytosis for cellular detoxification and high-energy cytoskeletal rearrangements. Thus, we uncover a role for non-canonical glutamine metabolism for efficient clearance of dying cells and maintenance of tissue homeostasis during health and disease in mouse and humans. Merlin et al. find that non-canonical glutamine transamination is required for macrophage efferocytosis in atherosclerotic plaques by sustaining redox buffering and fueling energy production for cytoskeletal rearrangements.

Tuberculosis (TB) is one of the leading causes of death due to a single infectious agent. The development of a TB vaccine that induces durable and effective immunity to Mycobacterium tuberculosis (Mtb) infection is urgently needed. Early and superior Mtb control can be induced in M. bovis Bacillus Calmette-Guérin (BCG)-vaccinated hosts when the innate immune response is targeted to generate effective vaccine-induced immunity. In the present study, we show that innate activation of DCs is critical for mucosal localization of clonally activated vaccine-induced CD4+ T cells in the lung and superior early Mtb control. In addition, our study reveals that Th1/Th17 cytokine axis play an important role in superior vaccine-induced immunity. Our studies also show that activation of the nuclear factor kappa-light-chain enhancer of activated B cell (NF-κβ) pathway in lung epithelial cells is critical for the mucosal localization of activated vaccine-induced CD4+ T cells for rapid Mtb control. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control.

Getting around the blood–brain barrier The meninges comprise three membranes that surround and protect the central nervous system (CNS). Recent studies have noted the existence of myeloid cells resident there, but little is known about their ontogeny and function, and whether other meningeal immune cell populations have important roles remains unclear (see the Perspective by Nguyen and Kubes). Cugurra et al. found in mice that a large proportion of continuously replenished myeloid cells in the dura mater are not blood derived, but rather transit from cranial bone marrow through specialized channels. In models of CNS injury and neuroinflammation, the authors demonstrated that these myeloid cells have an immunoregulatory phenotype compared with their more inflammatory blood-derived counterparts. Similarly, Brioschi et al. show that the meninges host B cells that are also derived from skull bone marrow, mature locally, and likely acquire a tolerogenic phenotype. They further found that the brains of aging mice are infiltrated by a second population of age-associated B cells, which come from the periphery and may differentiate into autoantibody-secreting plasma cells after encountering CNS antigens. Together, these two studies may inform future treatment of neurological diseases. Science , abf7844, abf9277, this issue p. eabf7844 , p. eabf9277 ; see also abj8183, p. 396

Summary: Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

BackgroundUnderstanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment.MethodsWe used transgenic mouse models to determine the role ofHoxb9in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options.ResultsOur human ACC dataset analyses showed high expression ofHOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression ofHoxb9in the adrenal cortex of mice with activatedCtnnb1led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, includingCcne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor.ConclusionsThese studies showHoxb9can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

SUMMARY CD137 is a costimulatory receptor expressed on natural killer cells, T cells, and subsets of dendritic cells. An agonistic monoclonal antibody (mAb) against CD137 has been used to reduce tumor burden or reverse autoimmunity in animal models and clinical trials. Here, we show that mice treated with an agonistic anti-CD137 mAb have reduced numbers of germinal center (GC) B cells and follicular dendritic cells (FDCs) in lymphoid tissues, which impair antibody responses to multiple T-cell-dependent antigens, including infectious virus, viral proteins, and conjugated haptens. These effects are not due to enhanced apoptosis or impaired proliferation of B cells but instead correlate with changes in lymphoid follicle structure and GC B cell dispersal and are mediated by CD137 signaling in CD4+ and CD8+ T cells. Our experiments in mice suggest that agonistic anti-CD137 mAbs used in cancer and autoimmunity therapy may impair long-term antibody and B cell memory responses., In Brief Agonistic anti-CD137 antibodies are being tested in clinical trials for their ability to enhance anticancer immunity. Hong et al. demonstrate that agonistic anti-CD137 antibody treatment causes defects in acute germinal B cell responses after virus infection or T-cell-dependent vaccines that impact long-lasting humoral immunity., Graphical Abstract

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC–regulated gene expression. In AR-SV–driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. Significance: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies. See related commentary by Rasool et al., p. 1011. This article is highlighted in the In This Issue feature, p. 995

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 “tag-mutate-enrich” mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies., The mechanisms of PARP inhibitor (PARPi) resistance are poorly understood. Here the authors employ a CRISPR mutagenesis approach to identify PARP1 mutants causing PARPi resistance and find that PARP1 mutations are tolerated in BRCA1 mutated cells, suggesting alternative resistance mechanisms.

Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.

Changes in bulk transcriptional profiles of heterogeneous samples often reflect changes in proportions of individual cell types. Several robust techniques have been developed to dissect the composition of such mixed samples given transcriptional signatures of the pure components or their proportions. These approaches are insufficient, however, in situations when no information about individual mixture components is available. This problem is known as the complete deconvolution problem, where the composition is revealed without any a priori knowledge about cell types and their proportions. Here, we identify a previously unrecognized property of tissue-specific genes – their mutual linearity – and use it to reveal the structure of the topological space of mixed transcriptional profiles and provide a noise-robust approach to the complete deconvolution problem. Furthermore, our analysis reveals systematic bias of all deconvolution techniques due to differences in cell size or RNA-content, and we demonstrate how to address this bias at the experimental design level., Complete gene expression deconvolution remains a challenging problem. Here, the authors provide a solution based on the recognition that expression levels of cell type specific genes are mutually linear across mixtures and mutually linear gene clusters correspond to cell type-specific signatures.

FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. Significance: Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. Cancer Discov; 6(8); 838–51. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 803

The prostate is a male exocrine gland that secretes components of the seminal fluid. In men, prostate tumors are one of the most prevalent cancers. Studies on the development of the prostate have given a better understanding of the processes and genes that are important in the formation of this organ and have provided insights into the mechanisms of prostate tumorigenesis. These developmental studies have provided evidence that some of the genes and signaling pathways involved in development are reactivated or deregulated during prostate cancer. The prostate goes through a number of different stages during organogenesis, which include organ specification, epithelial budding, branching morphogenesis, canalization, and cytodifferentiation. During development, these processes are tightly regulated, many of which are controlled by the male hormone androgens. The majority of prostate tumors remain hormone regulated, and antiandrogen therapy is a first-line therapy, highlighting the important link between prostate organogenesis and cancer. In this review, we describe some of the data on genes that have important roles during prostate development that also have strong evidence linking them to prostate cancer.

We describe a screen for cellular response to drugs that makes use of haploid embryonic stem cells. We generated ten libraries of mutants with piggyBac gene trap transposon integrations, totalling approximately 100,000 mutant clones. Random barcode sequences were inserted into the transposon vector to allow the number of cells bearing each insertion to be measured by amplifying and sequencing the barcodes. These barcodes were associated with their integration sites by inverse PCR. We exposed these libraries to commonly used cancer drugs and profiled changes in barcode abundance by Ion Torrent sequencing in order to identify mutations that conferred sensitivity. Drugs tested included conventional chemotherapeutics as well as targeted inhibitors of topoisomerases, poly(ADP-ribose) polymerase (PARP), Hsp90 and WEE1.

Rationale: Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. Previously, we demonstrated that Wnt/β-catenin signaling upregulates VSMC proliferation in vitro. Objective: We examined this pathway in vivo and investigated the involvement of specific Wnt proteins in VSMC proliferation. Methods and Results: Left carotid arteries of TOPgal (β-catenin signaling reporter) transgenic mice were ligated to induce intimal thickening. β-Catenin signaling was induced in the media and intima at 3 and 28 days after ligation, respectively, and was associated with VSMC proliferation and cyclin D1 expression. In vitro, a Wnt agonist promoted mouse VSMC proliferation, whereas Wnt inhibitory factor (WIF)-1 retarded platelet-derived growth factor-BB (PDGF-BB)–induced VSMC proliferation. Microarray analysis and quantitative PCR detected a significant induction of Wnt2 and Wnt4 mRNA in PDGF-BB-treated (proliferating) VSMCs compared to quiescent VSMCs. Western blotting revealed this increase was only translated into protein for Wnt4. Specific silencing RNA knockdown of Wnt4, but not Wnt2, significantly reduced VSMC proliferation. Recombinant Wnt4, but not Wnt2, significantly increased VSMC proliferation by ≈2-fold and silencing RNA knockdown revealed this is via Frizzled 1. Immunohistochemistry showed that increased Wnt4 protein correlated with VSMC proliferation and cyclin D1 expression ( P P +/− compared to Wnt4 +/+ mice. Conclusions: This study demonstrates that Wnt/β-catenin signaling occurs in proliferating VSMCs during intimal thickening and indicates that this is a result of Wnt4 upregulation.

Developmental pathways have been shown to be important in the initiation and progression of cancer in various tissues. We showed that the transcription factor SOX9 is expressed in the epithelia of the mouse embryonic prostate and is required for proper prostate development. We have performed an in vivo investigation into the role of SOX9 in prostate cancer in mouse and human. Studies on Pten and Nkx3.1 mutant mice show that cells with an increased level of SOX9 appear within the epithelia at the early stages of prostate neoplasia and this high expression correlates with all stages of neoplastic progression. Using genetically modified mice we show that overexpression of SOX9 in prostate epithelia leads to an increase in cell proliferation without inducing hyperplasia. In mice that were heterozygous for the conditional mutant allele of Pten, overexpression of SOX9 gave rise to an earlier induction of high-grade prostate intraepithelial neoplasia. Consistent with this role, loss of Sox9 in prostate epithelia led to a decrease in proliferating cells in normal and in homozygous Pten mutant mice with prostate neoplasia. Analysis of a cohort of 880 human prostate cancer samples showed that SOX9 expression is associated with increasing Gleason grades and higher Ki67 staining. These studies identify SOX9 as part of a developmental pathway that is reactivated in prostate neoplasia where it is involved in regulating proliferation and suggests it can contribute to carcinogenesis in specific genetic contexts.

Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad and we showed previously that the role of WNT4 was to inhibit endothelial and steroidogenic cell migration into the developing ovary. Here we show that Wnt4 also has a function in the male gonad. We find that Sertoli cell differentiation is compromised in Wnt4 mutant testes and that this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 −/− gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development.

Wnt4−/− XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst), which encodes a TGFβ superfamily binding protein, is a downstream component of Wnt4 signaling. Fst inhibits formation of the XY-specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary. Developmental Dynamics 230:210–215, 2004.

Dax-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (NR0B1)] is an orphan nuclear receptor acting as a suppressor of Ad4 binding protein/steroidogenic factor 1 [Ad4BP/SF-1 (NR5A1)] and as an anti-Sry factor in the process of gonadal sex differentiation. The roles of these nuclear receptors in the differentiation of the gonads and the adrenal cortex have been established through studies of the mutant phenotype in both mice and humans. However, the mechanisms underlying transcriptional regulation of these genes remain largely unknown. Here, we examined the relationship between Dax-1 gene transcription and the Wnt4 pathway. Reporter gene analysis revealed that Dax-1 gene transcription was activated by β-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with Ad4BP/SF-1. Interaction between β-catenin and Ad4BP/SF-1 was observed using yeast two-hybrid and in vitro pull-down assays. The region of Ad4BP/SF-1 essential for this interaction consists of an acidic amino acid cluster, which resides in the first helix of the ligand-binding domain. Mutation of the amino acid cluster impaired transcriptional activation of Dax-1 as well as interaction of Ad4BP/SF-1 with β-catenin. These results were supported by in vivo observations using Wnt4 gene-disrupted mice, in which Dax-1 gene expression was decreased significantly in sexually differentiating female gonads. We thus conclude that Wnt4 signaling mediates the increased expression of Dax-1 as the ovary becomes sexually differentiated.

Gene expression and functional studies have indicated that the molecular programmes involved in prostate development are also active in prostate cancer. PTEN has been implicated in human prostate cancer and is frequently mutated in this disease. Here, using the Nkx3.1:Cre mouse strain and a genetic deletion approach, we investigate the role of Pten specifically in the developing mouse prostate epithelia. In contrast to its role in other developing organs, this gene is dispensable for the initial developmental processes such as budding and branching. However, as cytodifferentiation progresses, abnormal luminal cells fill the ductal lumens together with augmented epithelial proliferation. This phenotype resembles the hyperplasia seen in postnatal Pten deletion models that develop neoplasia at later stages. Consistent with this, gene expression analysis showed a number of genes affected that are shared with Pten mutant prostate cancer models, including a decrease in androgen receptor regulated genes. In depth analysis of the phenotype of these mice during development revealed that loss of Pten leads to the precocious differentiation of epithelial cells towards a luminal cell fate. This study provides novel insight into the role of Pten in prostate development as part of the process of coordinating the differentiation and proliferation of cell types in time and space to form a functional organ.

DAX1, which encodes an unusual member of the nuclear hormone-receptor superfamily, is a gene that may be responsible for a sex-reversal syndrome in humans, referred to as dosage-sensitive sex reversal, in which XY individuals carrying duplications of Xp21, part of the small arm of the X chromosome, develop as females. XY mice carrying extra copies of mouse Dax1 as a transgene show delayed testis development when the gene is expressed at high levels, but do not normally show sex reversal. Complete sex reversal occurs, however, when the transgene is tested against weak alleles of the sex-determining Y-chromosome gene Sry. These results show that DAX1 is largely, if not solely, responsible for dosage-sensitive sex reversal and provide a model for early events in mammalian sex determination, when precise levels and timing of gene expression are critical.

Mammalian sex determination occurs in the gonad of the developing embryo. This process is dependent on the Y-chromosome-encoded Sry gene that acts in the somatic cells of the genital ridge. The transient nature of Sry gene expression suggests that it acts as a switch from one cell fate to another. One of the roles of Sry is to initiate the differentiation of Sertoli cells, which are the first cell type of the testis to be formed. Two genes are thought to be important in Sertoli cell differentiation and function, Sox9, an Sry-related gene, and SF-1, a nuclear hormone receptor. Sox9 is expressed in Sertoli cells throughout development of the mouse embryo, and inactivating mutations in this gene in humans give rise to XY females. SF-1 is also expressed in Sertoli cells and is thought to activate the AMH gene--an early marker of these cells. DAX-1, an X-linked member of the nuclear hormone superfamily, is a candidate for a human condition in which duplication of regions of the X chromosome results in XY females. Expression of this gene during mouse development is associated with ovary development and is down-regulated in the differentiating testis. Mutations in DAX-1 in humans have shown that this gene is not necessary for testis development. The properties of the DAX-1 gene suggest that it is important in ovary determination and might therefore be antagonistic to the action of the Sry gene.

Mutations of the orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, cause complex endocrine phenotypes that include impaired adrenal development and hypogonadotrophic hypogonadism. These similar phenotypes suggest that SF-1 and DAX-1 act in the same pathway(s) of endocrine development. To explore this model, we now compare directly their sites of expression. In mouse embryos, SF-1 expression in the urogenital ridge and brain either preceded or coincided with Dax-1 expression, with coordinate expression thereafter in the adrenal cortex, testis, ovary, hypothalamus, and anterior pituitary. The striking colocalization of SF-1 and Dax-1 supports the model that they are intimately linked in a common pathway of endocrine development. The slightly earlier onset of SF-1 expression and its ability to bind specifically to a conserved sequence in the Dax-1 5'-flanking region suggested that SF-1 may activate Dax-1 expression. However, promoter activity of Dax-1 5'-flanking sequences did not require this potential SF-1-responsive element, and Dax-1 expression was unimpaired in knockout mice lacking SF-1, establishing that SF-1 is not required for Dax-1 gene expression in these settings. Although the precise mechanisms remain to be established and may be multifactorial, our results strongly suggest that these two orphan nuclear receptors interact in a common pathway of endocrine development.

Heterozygous mutations in SOX9 lead to a human dwarfism syndrome, Campomelic dysplasia. Consistent with a role in sex determination, we find that Sox9 expression closely follows differentiation of Sertoli cells in the mouse testis, in experimental sex reversal when fetal ovaries are grafted to adult kidneys and in the chick where there is no evidence for a Sry gene. Our results imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor, perhaps in all vertebrates.

Prostate cancer is a major cause of male death in the Western world, but few frequent genetic alterations that drive prostate cancer initiation and progression have been identified. β-Catenin is essential for many developmental processes and has been implicated in tumorigenesis in many tissues, including prostate cancer. However, expression studies on human prostate cancer samples are unclear on the role this protein plays in this disease. We have used in vivo genetic studies in the embryo and adult to extend our understanding of the role of β-Catenin in the normal and neoplastic prostate. Our gene deletion analysis revealed that prostate epithelial β-Catenin is required for embryonic prostate growth and branching but is dispensable in the normal adult organ. During development, β-Catenin controls the number of progenitors in the epithelial buds and regulates a discrete network of genes, including c-Myc and Nkx3.1. Deletion of β-Catenin in a Pten deleted model of castration-resistant prostate cancer demonstrated it is dispensable for disease progression in this setting. Complementary overexpression experiments, through in vivo protein stabilization, showed that β-Catenin promotes the formation of squamous epithelia during prostate development, even in the absence of androgens. β-Catenin overexpression in combination with Pten loss was able to drive progression to invasive carcinoma together with squamous metaplasia. These studies demonstrate that β-Catenin is essential for prostate development and that an inherent property of high levels of this protein in prostate epithelia is to drive squamous fate differentiation. In addition, they show that β-Catenin overexpression can promote invasive prostate cancer in a clinically relevant model of this disease. These data provide novel information on cancer progression pathways that give rise to lethal prostate disease in humans., Author Summary Prostate cancer is a major cause of male death in the Western world, but few genes involved in this disease have been identified. We have undertaken an in-depth in vivo analysis in the prostate of the β-Catenin protein, which has been shown to be important in many processes during embryogenesis and has been implicated in tumorigenesis. Our studies demonstrate that β-Catenin is essential for prostate development but is dispensable in the normal adult organ. Analysis of a mouse model of a frequently mutated human prostate tumour suppressor, Pten loss, revealed that β-Catenin is not required for neoplastic formation in this model, even in castrated conditions. However, increased β-Catenin levels can cooperate with Pten loss to promote the progression of aggressive invasive prostate cancer together with squamous metaplasia. These data uncover the role of β-Catenin in the prostate and provide new insights on how pathways interact to drive human prostate cancer.

Adrenal and gonadal steroids are essential for life and reproduction. The orphan nuclear receptor SF1 (NR5A1) has been shown to regulate the expression of enzymes involved in steroid production in vitro. However, the in vivo role of this transcription factor in steroidogenesis has not been elucidated. In this study, we have generated steroidogenic-specific Cre-expressing mice to lineage mark and delete Sf1 in differentiated steroid-producing cells of the testis, the ovary and the adrenal gland. Our data show that SF1 is a regulator of the expression of steroidogenic genes in all three organs. In addition, Sf1 deletion leads to a radical change in cell morphology and loss of identity. Surprisingly, sexual development and reproduction in mutant animals were not compromised owing, in part, to the presence of a small proportion of SF1-positive cells. In contrast to the testis and ovary, the mutant adult adrenal gland showed a lack of Sf1-deleted cells and our studies suggest that steroidogenic adrenal cells during foetal stages require Sf1 to give rise to the adult adrenal population. This study is the first to show the in vivo requirements of SF1 in steroidogenesis and provides novel data on the cellular consequences of the loss of this protein specifically within steroid-producing cells.

International audience; Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4⁻/⁻ NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.

Sry is expressed at higher levels in the adult testis, where no function has been determined, than in the genital ridge, its critical site of action. cDNA and 5' RACE clones isolated from testis or from Sry-transfected cell lines have an unusual structure, with 3' sequences located in a 5' position. RNAase protection assays and reverse transcription polymerase chain reactions confirmed that these unusual RNA molecules represent the most abundant transcript in testis. Furthermore, oligonucleotide hybridization and RNAase H digestion proved that these Sry RNA molecules are circular. Similar transcripts were detected in the testes of mice with Mus musculus musculus, Mus musculus domesticus, and Mus spretus Sry genes. The circular RNA is found in the cytoplasm but is not substantially bound to polysomes. We suggest that the circles arise from normal splicing processes as a consequence of the unusual genomic structure surrounding the Sry locus in the mouse.

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches., Author Summary In Western countries, prostate cancer is the most common male cancer and the second biggest cause of cancer-related deaths in men. Men with a familial history of either breast or ovarian cancer have an elevated predisposition to prostate cancer, suggesting there is a genetic element to this disease. Indeed, the inheritance of a mutated form of the breast cancer susceptibility gene BRCA2 has been linked to the development of prostate cancer, although the precise role that BRCA2 dysfunction plays in the development of prostate cancer is unclear. To address this, we have generated an animal model in which the mouse Brca2 gene is specifically deleted in the adult prostate. These mice develop precancerous prostate lesions, which progress in severity and incidence with the loss-of-function of an additional tumour suppressor, Trp53. Importantly, blocking male steroidal hormone production by castration leads to partial regression of the prostate lesions, however cells continue to proliferate after androgen withdrawal. This suggests human BRCA2 mutant prostate tumours, like the majority of prostate cancers, will respond to hormone therapy, but will relapse, as frequently occurs in this disease. In summary, our model suggests that BRCA2 acts as a tumour suppressor in the prostate and provides a pre-invasive model to test novel therapeutics.

International audience

It has been proposed that the mammalian adrenal cortex and gonad are derived from the same primordium present during early urogenital development. Molecular pathways involved in the differentiation of the adrenal cortex from the adrenogonadal primordium (AGP) have yet to be determined. Here we show in mice that the transcription co-factor Cited2 is required for the specification of the adrenal cortex from the AGP. We present genetic and molecular evidence demonstrating that Cited2 interacts with the transcription factor Wt1 to stimulate expression of the nuclear hormone receptor Sf-1 (Nr5a1) in the AGP prior to the separation between gonad and adrenal cortex. We show a direct correlation between the expression levels of Sf-1 in the AGP and the defects in adrenal development found in mice with different Cited2 and Wt1 mutant backgrounds. Analysis of embryos heterozygous for mutations in both Sf-1 and Cited2 confirmed that these genes act in the same pathway during adrenal development. Our studies reveal a regulatory mechanism in which Cited2 acts as a Wt1 co-factor to increase, at a critical time in embryogenesis, the levels of the essential transcription factor Sf-1 in the AGP above the threshold required to determine adrenal development. These results highlight the importance of transcription factor dosage in organogenesis and the role of transcription co-factors such as Cited2 in determining the levels of these factors.

Germ cell sex determination is directed by the gonad and is characterized by a difference in the timing of entry into meiosis. Recent data show that retinoic acid signalling is responsible for the induction of germ cell meiosis in the developing ovary. In the fetal testis, this process is inhibited by a retinoic acid metabolizing enzyme.

Sex differentiation in mammals occurs in three steps. The first is the establishment of chromosomal sex at fertilization, followed by the differentiation of the gonad into an ovary or testis, and finally the establishment of the phenotypic sex of the embryo and adult, which is regulated by the gonad. Disruption of any of these stages gives rise to sexual ambiguities that include 46,XY pure gonadal dysgenesis, 46,XX true hermaphroditism, and variable degrees of intersexuality. In this review, we focus on the development of the mammalian gonad from a bipotential primordium that differentiates into either an ovary or a testis. We describe the recent increase in our knowledge of the genetic defects that directly affect gonadal development, sex determination, and sex differentiation, with emphasis on the comparison of genetic studies in mice with studies of naturally occurring mutations in humans.

The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. Here we show that the role of WNT4 in gonad development is to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. We show that WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad,preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis did not inhibit coelomic vessel formation but vascular pattern was affected. Leydig cell differentiation was not affected in these transgenic animals and our data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development.

We tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion of Sry from the Y and subsequent insertion of an Sry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain.

The nuclear hormone receptor DAX1 has been implicated in mammalian gonad development and sex determination. The expression of the gene in the gonad follows a dynamic pattern in time and place in the embryo and the adult. We have undertaken the first in vivo study of the regulation of Dax1 expression. Using a transgenic mouse approach we have identified a novel 500-bp region 4 kb upstream of the mouse Dax1 start codon that is essential for LacZ reporter gene expression in the embryonic gonad. Within this region, a highly conserved steroidogenic factor 1 (SF1) consensus-binding site is necessary to direct LacZ expression to the embryonic gonad implicating SF1 in the regulation of Dax1 in the developing gonad. Consistent with this, Dax1 is expressed at much reduced levels in gonads of embryos that are deficient in SF1. In addition, our results show that SF1 consensus-binding sites close to the start of Dax1 transcription are important in regulating levels of expression in the developing gonad. These studies have identified the critical in vivo regulatory region for expression of Dax1 in the early gonad and provide novel information on how a specific enhancer element acts in different cell types at different stages of development.

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

Our knowledge of mammalian sex determination is based on two main areas of study. First, the characterization of the biological events that determine the sexual development of the individual, including patterns of gene expression, and second, the study of genetic mutations in humans and mice that lead to abnormal sexual phenotypes. In the search for molecular components of this process, the identification of genes in loci involved in human disease has been especially fruitful. Serendipity has also played a hand, where in several cases targeted mutations in mouse genes, being studied for other reasons, have led to unexpected sex reversal phenotypes. The collection of molecular candidates implicated in sex determination is now quite extensive. We are not able to fit all of these into simple pathways, where one gene acts on the next and so on in a linear fashion, as seems possible in the invertebrate model organisms, Caenorhabditis elegans and Drosophila. In part this is due to gaps in our knowledge, as we are clearly missing several key components, but it is looking increasingly likely that the system is much better described as a network of factors. In fact, the story so far is like some partly recovered script for a play. Thus some gene products are main characters with roles at several different stages, some act as a chorus, in a combinatorial fashion with others, whereas a few play a critical role in one scene and then disappear. The mechanism presumably evolved to be delicately poised to respond to the initial trigger to be male or female and then to amplify this decision while avoiding development of intersex phenotypes. It is therefore likely to be a system full of back-ups and functional redundancy. The complexity may also follow from the relatively late embryonic stage at which the decision is reached. This means that events occurring in one cell lineage have to be coordinated with others in the context of a developing organ and eventually the whole organism. We review here the story that is beginning to emerge, focusing mostly on the set of transcription factors that appear to play important leading and supporting roles. In mammals, the genetic sex of the embryo is established at fertilization with the inheritance of an X or Y chromosome from the father. However, the sex-determining process is set in motion only during the period of organogenesis when the gonads develop. The Y chromosome, through the testis-determining gene Sry, acts dominantly to trigger differentiation of testes from the indifferent gonads (or genital ridges) that would otherwise develop as ovaries (Gubbay et al. 1990; Sinclair et al. 1990). Sry is the only gene needed from the Y chromosome to establish male development, as shown by transgenic experiments where XX mice carrying the Sry gene develop as males (Koopman et al. 1991). Conversely, mutations in Sry can lead to relatively normal female development of XY mice and humans (Gubbay et al. 1992; Hawkins et al. 1992). Once the gonads begin to differentiate as testes or ovaries, they secrete factors, notably anti-Mullerian hormone (AMH, otherwise known as Mullerian-inhibiting substance, or MIS) and testosterone from the testes, which determine the sexual development of the rest of the embryo. There are many steps in this process that, when affected, will give rise to different degrees of sex reversal. The challenge has been to correlate the phenotype of affected individuals with the appropriate step and translate this into a molecular mechanism.

Duplications of a chromosome Xp21 locus DSS (dosage sensitive sex reversal) are associated with male to female sex reversal. An unusual member of the nuclear hormone receptor superfamily, DAX1, maps to the DSS critical region and is responsible for X-linked adrenal hypoplasia congenita. Here we describe the isolation of the mouse Dax1 gene and its pattern of expression during development. Expression was detected in the first stages of gonadal and adrenal differentiation and in the developing hypothalamus. Moreover, Dax1 expression is down-regulated coincident with overt differentiation in the testis, but persists in the developing ovary. Comparison of the predicted protein products of the human and mouse genes show that specific domains are evolving rapidly. Our results suggest a basis for adrenal insufficiency and hypogonadotropic hypogonadism in males affected by adrenal hypoplasia congenita and are consistent with a role for DAX1 in gonadal sex determination.

The mammalian prostate arises from the urogenital sinus and few factors have been identified to be important in the early stages of prostate development. In this study we show that the transcription factor Sox9 is expressed in the epithelia of all mouse prostatic lobes from the initial stages of their development. We used a conditional approach with mice expressing Cre recombinase under the control of Nkx3.1 regulatory sequences to delete Sox9 from the developing prostate. Mice with a prostate specific deletion of Sox9 showed a lack of ventral prostate development and abnormal anterior prostate differentiation. Analysis of these mutant animals revealed an early loss of expression of genes specific to the prostate epithelia such as Nkx3.1 and Shh and a marked reduction in proliferation in the ventral prostate but not in other lobes. Fgf signalling, through the MAPK pathway, has been shown to be important in prostate development and a lobe specific phenotype was reported for a prostate specific Fgfr2 mutant mouse model. Here we show that the levels of Fgfr2 and Sprouty2, a downstream target of Fgf signalling, were severely reduced in the ventral prostate of Sox9 mutant animals but not in other lobes. Prostate organ culture studies with a Mek inhibitor, U0126, and a Fgf receptor inhibitor, SU5402, indicate that the timing of expression of Cre in the mutant animals could account for the lobe specific phenotype in the Sox9 and Fgfr2 mutants. These studies imply that Sox9 is required for the early differentiation of the prostate bud epithelia.

Steroidogenic cells of the adrenal and gonad are thought to be derived from a common primordium that divides into separate tissues during embryogenesis. In this paper, we show that cells with mixed adrenal and Leydig cell properties are found dispersed in the insterstitium of the embryonic and adult mouse testis. They express the adrenal markers Cyp11b1 and Cyp21 and respond to ACTH. Consistent with these properties, we show that the embryonic testis produces the adrenal steroid corticosterone. These cells also express Cyp17 and respond to hCG stimulation but do not express the Leydig specific marker Insl3 showing that they are a population of steroidogenic cells distinct from Leydig cells. Based on their properties, we refer to these cells as adrenal-like cells of the testis and propose that they are the mouse equivalent of the precursors of human adrenal rests, tumors found primarily in male patients with congenital adrenal hyperplasia. Organ culture studies show that ACTH-responsive cells are present at the gonad/mesonephros border and seem to migrate into the XY but not the XX gonad during development. Consistent with this, using transgenic Cyp11a1 reporter mice, we definitively show that steroidogenic cells can migrate from the mesonephros into the XY gonad. We also show that the region between the mesonephros and the gonad harbors steroidogenic cell precursors that are repressed by the presence of the mesonephros. We propose that this region is the source of the adrenal-like cells that migrate into the testis as it develops and are activated when Leydig cells differentiate. These studies reveal the complex nature of steroidogenic cell differentiation during urogenital development.

Sex-determining genes have been identified in flies, worms and mammals but not, until recently, in non-mammalian vertebrates. Now, a gene has been isolated from the Y chromosome of the teleost fish medaka that is functionally comparable to the mammalian testis-determining gene, Sry.