Rôles des facteurs de transcriptions SIM1, OTP et POU3F2 dans le développement de l'hypothalamus antérieur

The transcription factors SIM1, OTP and POU3F2 interact together to orchestrate the complex development of the hypothalamus, a region of the brain containing several small, circumscribed populations of neurons, including the paraventricular nucleus (PVN). This nucleus is an important integrating center, and the haploinsufficiency of the transcription factor Sim1, essential for the development of PVN, leads to overeating in both mice and humans. Different mice have been genetically engineered to help us find other essential genes that participate in this transcriptional cascade. Firstly, the anterior part of the hypothalamus was collected from mice embryos (E12.5) which overexpressed the Pou3f2 gene under the OTP7 promotor. The transcriptional analysis was compared to wt embryos from the same litter to see the different upregulated and downregulated genes. These genes were chosen according to their relevance to the development of the anterior hypothalamus. Three criteria were used to discriminate genes from one another: 1) an increase of more than 1.5, 2) a minimal expression in the embryo (E12.5) of at least 1000 reads and 3) the highest rank. This discriminatory method allowed us to identify the genes Lgi2, Fezf2, Sema3c, Six6, Sox14, Lmo4, Nwd2, Nkx2.1. To have a visuospatial idea of these affected genes, the validation of these results was done by in situ hybridization on E12.5 embryo hypothalamus. We have been able to see an overexpression in the PVN domain for the Lgi2, Fezf2, Sema3c, Lmo4 and Pou3f2 markers. The reduction of expression for Sox14 and Nkx2.1 markers were visible in the basal domain of the hypothalamus, which suggest a non cell autonomous effect of Pou3f2 being overexpressed. The reduction of Six6 couldn’t be consistently visible with repetition.  Secondly, a redundant role of OTP and SIM1 seems to occur in the development of the hypothalamus. We created a KO line of the Otp gene by deleting the second exon with CrispR-cas9 and characterized it. We then compared it to the Sim1+/tlz+ line that we already generated in the lab.  We were able to confirm our mutation model by seeing a reduction in the expression of crucial markers such as OT, OTP, AVP and TRH.  Thirdly, we crossed Otp+/- with Sim1+/tlz+ mice to obtain four different genotypes: wt, Otp+/-, Sim1+/tlz+ and Otp+/- Sim1+/tlz+. Since a redundant aspect has been observed for SIM1 and OTP transcription factors, we were wondering if the obesity phenotype would be worsened by carrying both mutation or not. These mice were weighted every week from 5-week-old up to 6 months old. Food intake has also been measured since the obesity has been reported to be caused by hyperphagia in Sim1 mutant mice. The male mice carrying the double mutation (Otp+/-Sim1+/tlz+) showed a more important weight gain than only Sim1+/tlz+ or Otp+/- mutants, but it was not the case for the female mice. The mice carrying the Otp mutation still got a more important weight gain than the wt mice (females and males). More mice would be necessary to determine if this weight gain is caused by hyperphagia only or if unbalance energy cost is part of the cause.

Keyphrases: Sim1, Otp, POU3F2, Hypothalamus, ocytocine, CRISPR-Cas9, transcription factors, paraventricular nucleus (PVN), hyperphagia, transcriptional cascade, in situ hybridization, promotor, mouse embryos, development