Dissection du programme développemental du noyau paraventriculaire de l'hypothalamus

A cascade of transcription factors composed of SIM1, ARNT2, OTP, BRN2 and SIM2 is required for the differentiation of the five major cell types populating the paraventricular nucleus (PVN) of the hypothalamus, a critical integrator of several homeostatic processes that are required for the survival of vertebrates. Haploinsufficency of Sim1 also causes isolated hyperphagia in mice and humans. The goal of our study is to dissect the developmental program of the PVN using an integrative approach in order to identify new genes that could potentially be implicated in the regulation of homeostasis in adults.  First, we used a comparative approach to analyse the PVN transcriptome at different developmental stages in mice embryos in order to identity new genes implicated in PVN development. We compared gene expression in the anterior hypothalamus of E12.5 Sim1-/- and Sim1+/+ littermate embryos using a microarray approach. We identified 56 genes acting downstream of Sim1 including 5 transcription factors - Irx3, Sax1, Rxrg, Ror and Neurod6. We proposed a model for the development of the anterior hypothalamus. In this model, the genes expressed in the medial domain of the mantle layer characterise cells that will form the PVN and genes expressed in the lateral domain identify cells that will give rise to ventrolateral areas of the hypothalamus. We also showed that Sim1, like Otp, is implicated in the differentiation, migration and proliferation of the neurons populating the PVN. Furthermore, we have isolated by laser captured microdissection the PVN and ARC nucleus in wild type mice at E14.5 and compared their transcriptomes. This technique allowed us to identity 34 transcription factors specific to the PVN and 76 factors specific to the ARC. These genes represent potential regulators of  hypothalamic development.  Second, we identified 3 blocks of sequence in the 5’ region of Otp that are conserved between human, mouse and fish. We constructed a transgene which included a 7 Kb fragment encompassing these sequences followed by a reporter gene. The analysis of 4 mice strains showed that this transgene is specifically expressed in the prospective PVN. We have generated a second transgene in which the 7 Kb fragment is located upstream of the cDNA encoding Brn2 or Sim1 and Gfp. We obtained 4 mice strains in which the Brn2 and Gfp expression pattern is similar to the Otp expression pattern. These mice will be used to study PVN development and food intake. Also, to determine if Brn2 expression only – without Sim1 gene expression - allows the development of PVN cells, we are presently crossing these mice with Sim1 deficient mice. In conclusion, we have generated the first transgene that is specifically expressed in the PVN. This transgene constitutes a critical tool for dissecting the developmental program of the hypothalamus.  Third, we have characterised the development of the anterior hypothalamus of  chick embryos which represent an interesting model for loss and gain of function experiments during the development of this brain region. Interestingly, our proposed model for the development of the anterior hypothalamus seems to be conserved in chick embryos. As a matter of fact, it is possible to classify genes according to their medio-lateral expression patterns and the outcome of the regions that they are defining.   Finally, we believe that this integrative approach will allow us to identify and characterize factors implicated in PVN development. From a clinical point of view, these factors could potentially be associated with pathologies such as obesity or arterial hypertension.

Keyphrases: Sim1, Otp, Brn2, PVN, Hypothalamus, Transcription factor, Paraventricular nucleus, Promoter, Transgenesis, Chick embryo, Development