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Voies de signalisation de l’acide rétinoïque dans l’ontogenèse et la différenciation des spermatogonies souches – RAPSSODI

Submission summary

RAPSSODI focuses on the mechanisms through which retinoic acid (RA) controls the fate of male germ stem cells (spermatogonia) in vivo. RA, the natural active vitamin A metabolite, acts through activation of nuclear receptors (RAR alpha, beta and gamma), that bind to target gene promoters, usually in the form of heterodimers with rexinoid receptors (RXR). As RAR are expressed in every tissue, control of RA action in the organism involves local syntheses and degradations. Synthesis is catalysed by 3 isotypes of retinaldehyde dehydrogenases (RALDH) and degradation by 3 isotypes of cytochrome hydroxylases of the CYP26 family. RA exerts a wide range of effects on developing and adult tissues by regulating cell proliferation, differentiation and death, and most interestingly, by determining the fate of certain stem cell populations. In addition, a body of research supports the view of RA-derivatives as preventive and therapeutic agents against certain carcinomas. To gain insights into in vivo functions of RA, we use a spatio-temporally-controlled somatic mutation strategy that allows distinguishing cell-autonomous from paracrine functions of RA. As a model, we choose the seminiferous epithelium (SE) of the testis composed of germ cells and of somatic, supporting, Sertoli cells, and surrounded by peritubular myoid cells as: (1) this tissue is the most sensitive to RA-deficiency; (2) it contains a well-characterized stem cell population; (3) Cre-expressing mouse lines exist allowing efficient gene ablation in different cell-types composing and surrounding the SE. RA is indispensable to stem spermatogonia differentiation. On the basis of our own findings, we hypothesized that RA produced by RALDHs in pre-pubertal Sertoli cells acts in a paracrine manner on pre-meiotic germ cells. These cells respond to RA by expressing the Stra8 gene, thereby triggering the first round of meiotic divisions. Then RA, now produced by meiotic spermatocytes, is delivered to spermatogonia which differentiate and ultimately enter meiosis to perpetuate the spermatogenic cycle. The catabolic barrier generated by CYP26s in peritubular myoid cells likely insulates the SE from circulating RA to prevent untimely initiation of spermatogonia differentiation. Because spermatogonia do not express RXR, we have proposed that RARgamma (the sole RAR detected in these cells) mediates the RA signal through a mechanism that does not implicate canonical RAR/RXR units of transcriptional activation, as it was already proven to be true for RARalpha in Sertoli cells. RAPSSODI combines innovative approaches (somatic mutagenesis, high-throughput DNA sequencing, generation of mouse/cell lines expressing tagged proteins, mass spectrometry) to test for these scenarios and identify downstream effectors of RA in a physiological context. Firstly, we propose to determine, through somatic mutagenesis, the contribution of RA synthesizing and degrading enzymes to SE homeostasis in order to define the cell-types involved and the timing in setting up RA signalling in the SE, as well as to decipher RA-dependent cell interactions that are mandatory for differentiation of spermatogonia. In parallel, we will identify, through comparative analysis of RNA populations, RA-controlled genes in Sertoli cells and in spermatogonia. This will allow elucidation of the RA-dependent network that control spermatogonia differentiation and, beyond, of molecular mechanisms underlying the ability of RA to promote differentiation of other stem cell populations in vivo. We will also test, through somatic mutagenesis, whether RARgamma acts independently of RXR in spermatogonia, and identify, with the help of a mouse line expressing a tagged-RARgamma, its non canonical mechanism of action, whose knowledge is expected to be extremely helpful to explore novel therapeutic strategies. Lastly, we propose to identify the effectors of the RA-controlled molecular cascade orchestrating the meiotic response. Understanding how the RA signal commits the stem cell progeny towards meiosis rather than toward mitosis will open a new field of investigation on a stem cell population with therapeutic potential. RAPSSODI partners are the only, worldwide, that can perform this approach combining both genetic and molecular tools, as (1) we already have in our possession mutant mouse lines that cover every aspect of the RA signalling pathways, (2) we master and have access to a large number of recent, high throughput, molecular techniques, two indispensable conditions for the successful completion the project.

Project coordination

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


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Beginning and duration of the scientific project: - 0 Months

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