Human Developmental Genetics  


  Annual Report

1. Characterisation of the mammalian sex determination pathway

How sex is determined has long been of major interest to developmental biologists. Although the Y chromosome linked gene SRYis known to initiate the testis-determining cascade the precise mechanism whereby this occurs and the downstream effectors of SRY action are unknown. The underlying etiology of most patients with anomalies of sex development is unexplained, suggesting that several sex-determining genes are unidentified. Interestingly, some of these patients carry duplications or deletions of chromosomal regions. To delimit these regions and identify the genes involved, we are performing ultra high resolution microarray comparative genome hybridisation (CGH) using both NimbleGen and Affymetrix platforms. These ongoing studies have already identified novel candidate sex-determining genes/loci. Using these technologies, we are analysing 300 cases of rare pathologies of sex-determination from our extensive DNA collections. In parallel, candidate genes that are determined by the CGH and other approaches are being screened for mutations in patients with anomalies of gonad development. A large series of mutations in several novel genes are currently being characterised using various functional assays.

This is funded in part by the ANR-Institut des Malades Rares, European Union FP7 project "EuroDSD" and by a research grant from the March of Dimes Foundation.

2. Genetic and epigenetic control of germ cell specification and differentiation. Our principal goal is to identify genetic and epigenetic factors that are necessary for germ cell development and maintenance. Much of our recent work involved extensive analyses of the human Y chromosome for mutations associated with male infertility. A growing body of evidence suggests that Y chromosome genes may be involved in epigenetic modification during germ cell specification and differentiation. We are determining epigenetic changes in mature sperm from patients using extensive microarray analysis and correlating this data with genetic anomalies. These include Y chromosome mutations that we have characterised in the large collection of patient material available through national and international collaborations with clinical centres.

In parallel, we are using murine embryonic stem (ES) cells as an ex-vivo model to understand the genetic/epigenetic factors involved in the process of specification of primordial germ cells (PGCs) and their differentiation into male germ cells. Stem cell factor (SCF) and its receptor, KIT, play a critical role during PGC proliferation, migration, survival and differentiation bothin vivoandin vitro.We have developed a novel genetically engineered ES cell system that we are using to study this critical signaling pathway (SCF/KIT) during the development of PGCs. Using a pharmacological approach KIT receptor is activated in KIT null ES cells at specific time points during their differentiation into the germ cell lineage. The resulting phenotypes are analysed based on the morphology and gene expression profile using real-time PCR. The global methylation status of the cells at different stages of germ cell differentiation from ES cells is being assayed using a murine whole genome microarray platform. This strategy is envisaged to provide important clues about the role of genetic/epigenetic factors involved and their interaction with each other during the specification of PGCs and their subsequent differentiation into male germ cells.

Keywords: sex determination, germ cell development, embryonic stem cells, infertility


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Activity Reports 2009 - Institut Pasteur
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