Transcriptomic analysis reveal novel genes with sexually dimorphic expression in the zebrafish gonad and brain

Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs), 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and ‘rest-of-body’ from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel) that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4) has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar roles in gonadogenesis in zebrafish and other vertebrates, our array may even provide information on genetic disorders affecting gonadal phenotypes and fertility in mammals.Fellowships from Temasek Life Science Laboratory Internal research grants from Temasek Life Science LaboratoryWeb of Scienc

Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes

Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a “male-like” gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario

Polygenic Sex Determination System in Zebrafish

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Gonad differentiation in zebrafish is regulated by the canonical Wnt signalling pathway

Zebrafish males undergo a ‘‘juvenile ovary-to-testis’’ gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Mu¨ llerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testislike profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a ‘‘pro-female’’ pathway that regulates gonad differentiation in zebrafish.Web of Scienc

A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY g

Transcriptomic Analyses Reveal Novel Genes with Sexually Dimorphic Expression in the Zebrafish Gonad and Brain

Background Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. Methodology/Principal Findings We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs), 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and ‘rest-of-body’ from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel) that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4) has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. Conclusions/Significance We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar roles in gonadogenesis in zebrafish and other vertebrates, our array may even provide information on genetic disorders affecting gonadal phenotypes and fertility in mammals

Regulation of SOX9 in mammalian sex determination and differentiation

© 2015 Dr. Rajini SreenivasanMammalian testis development is initiated by the key sex-determining gene, SRY, which together with SF-1, activates SOX9 expression. Activation occurs through the testis-specific enhancer of SOX9 core element, TESCO. Human mutations in these genes or their regulatory regions can disrupt testicular development, leading to disorders of sex development (DSDs) which are congenital conditions resulting in atypical chromosomal, gonadal or anatomic sex. Around 70% of 46,XY DSDs remain unexplained genetically. This thesis investigates several aspects of SOX9 regulation in male development. Firstly, the evolution of a novel sex-determining mechanism with altered Sox9 regulation in unusual mammals lacking SRY is described. Secondly, genotype-phenotype correlation was discovered between mutant SF-1 activity on TESCO and DSD severity. Thirdly, the identification and characterisation of novel testicular and genital tubercle SOX9 enhancers mutated in DSDs are described. The sex determining mechanism of mammals Ellobius lutescens and Ellobius tancrei that lack Sry and a Y chromosome has remained elusive. A 14 bp deletion in TESCO was discovered in these species, resulting in the loss of a highly conserved SOX/TCF binding site. Introduction of the 14 bp deletion into mouse TESCO led to elevated TESCO activity in in vitro reporter assays. Increased TESCO activity may therefore form the basis for the loss of Sry from this species, since consequent increased SOX9 levels would render Sry dispensable for testis development. Mutations in SF-1 are associated with highly variable reproductive phenotypes in 46,XY DSD. The mechanism underlying the broad spectrum of phenotypes and the precise mechanism of SF1 action that fails in DSDs are unknown. Twenty SF-1 mutants identified in 46,XY DSD patients were examined for their ability to activate TESCO in transactivation assays in vitro. Fifteen of the twenty mutants showed reduced SF-1 activation on TESCO, eleven with abnormal subcellular localisation. Fourteen SF-1 mutants were predicted in silico to alter DNA, ligand or cofactor interactions. A positive genotype-phenotype correlation was observed with the extent of reduction in TESCO activation being proportional to the severity of patient phenotypes. These data implicate aberrant SF1-mediated transcriptional regulation of SOX9 in 46,XY DSDs. The lack of TESCO mutations in 46,XY DSD patients prompted a search for additional testicular enhancers that may be functioning in humans. Array comparative genomic hybridisation analysis of isolated DSD patients revealed a 78 kb minimal sex determining region (RevSex) far upstream of SOX9 that was duplicated in 46,XX and deleted in 46,XY DSDs. It was postulated that RevSex contains a gonadal enhancer and the most highly conserved sub-region within RevSex, called SR4, was subjected to further analysis. However, SR4 was neither responsive to sex determining factors in vitro nor active in the gonads of transgenic mice, suggesting that SR4 may not be functioning as a testicular enhancer. SR4 transgenic mice showed reporter activity in the genital tubercle, the primordium of the penis and clitoris, a previously unreported domain of Sox9 expression. SOX9 protein was detected in the genital tubercle, notably in the urethral plate epithelium, preputial glands, ventral surface ectoderm and corpus cavernosa. SR4 may therefore function as a Sox9 genital tubercle enhancer, mutations of which could possibly lead to hypospadias, a birth defect seen in the DSD patients in the RevSex study. However, conditional ablation of Sox9 in the genital tubercle using Shh-Cre/+;Sox9flox/floxmice revealed no genital tubercle abnormalities, possibly due to compensation by similar Sox factors. SR4 activity and the observed SOX9 expression pattern suggest that SR4 may be functioning as an enhancer that drives Sox9 expression in the genital tubercle. These findings suggest a novel role for Sox9 in the direct regulation of external genitalia development. The work described in this thesis on the genetic regulation of SOX9 has shed light on the molecular mechanisms that control mammalian sex determination and differentiation. This research has enabled a better understanding of the mechanism of evolutionary adaptation and speciation, as well as the molecular aetiology of DSDs

Effects of high water temperature during early development on the zebrafish gonadal transcriptome

17th International Congress of Comparative Endocrinology (ICCE), 15-19 July 2013, Barcelona, SpainFish exhibit a wide range of reproduction systems and sex depends on the interplay between genetic and environmental influences. Zebrafish is broadly used in many research areas and although it is clearly an undifferentiated gonochorist, details of its sex determining mechanism are still puzzling researchers. Recent studies argue for a polygenic mechanism with secondary effects from temperature. In fish, including zebrafish, high temperature can bias the final sex ratio towards males. However, the molecular mechanisms that underlie the heat-induced sex reversal is still poorly understood. We show the effects caused by high temperature during the period from 7 to 32 days post fertilization, which includes the sex differentiation period. First, we describe that heat response is family-specific since the masculinizing effects of high temperature vary among broods from different families. Second, we study the transcriptomic responses of gonads in adults and juveniles by using a genomewide microarray. The analysis revealed that temperature is able to modulate the gonadal transcriptome resulting in novel transcriptomic profiles. These profiles were found in both juvenile and adult fish. Then we validated the results by using qPCR array for more than forty genes. A subset of key genes involved in the reproduction system were identified and their pattern was studied. Finally, we identify endocrine pathways that represent a short-term response to high temperature, a long-term response or both. Overall, we provide further evidence that zebrafish sex determination and differentiation is consistent with a polygenic mechanism with temperature influences. We characterize novel heat-induced transcriptomes and identify markers that could be used for early detection of temperature-based masculinization. This is relevant for understanding the possible effects of elevated water temperature on a climate change scenarioPeer reviewe