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Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

ABSTRACT: Despite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29).RNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue.This study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.

SUBMITTER: Ayers KL

PROVIDER: S-EPMC4574023 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

Ayers Katie L KL Lambeth Luke S LS Davidson Nadia M NM Sinclair Andrew H AH Oshlack Alicia A Smith Craig A CA

BMC genomics 20150916

<h4>Background</h4>Despite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29).<h4>Results</h4>RNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal ...[more]

PMID: 26377738

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Project description:BackgroundThe American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD.ResultsOur characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted.ConclusionsGonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

Project description:Gonadal sex differentiation – testis versus ovary formation – is a fundamental process required for reproduction and evolution. Reflecting this importance, the embryonic gonads of vertebrate species comprise the same key cell types; germ cells, supporting cells and interstitial steroidogenic cells. Remarkably, the genetic triggers for gonadal sex differentiation vary across species (the SRY gene in mammals, DMRT1 in birds and some turtles, temperature in many reptiles, AMH and various other genes in fishes). Despite this variation, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using the chicken embryo as a model and considering the entire gonad. We sampled over 30,000 cells across several developmental stages, prior, during and after the onset of gonadal sex differentiation. The data provide several new insights into cell lineage specification during vertebrate gonadogenesis. Combining lineage tracing with single cell transcriptomics, the data show that somatic supporting cells of the embryonic chicken gonad do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, the early somatic precursors cells of the gonads in both sexes derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. In particular, PAX2 marks immigrating mesenchymal cells that give rise to the supporting cell lineage. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes, and derivation of embryonic steroidogenic cells from a differentiated supporting cell lineage. We provide significantly improved resolution of gonadal cell types and identify several new gonadal marker genes. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.

Project description:To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish.

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Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

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Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

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