Function of the homeobox gene Rax in the early hypothalamic development of the mouse

ORQUERA, DP; NASIF, S; RUBINSTEIN, M; DE SOUZA, FS

Chascomús, Bs As

Taller; II Taller de Biología Celular y del Desarrollo; 2014

Rax (also Rx) is a homeodomain-containing transcription factor (TF) expressed in the developing retina and ventral forebrain of all vertebrates. Rax-deficient mice and zebrafish lack eye structures, but the function of the gene in the hypothalamus has not been studied in detail. By in situ hybridisation, we observed that Rax is expressed in the anterior hypothalamic region during early development, corresponding to embryonic days (E) E8.5 to E9.5, but it becomes increasingly restricted to more posterior levels of the hypothalamus as development progresses. To study the role of Rax in hypothalamic patterning, we employed mutant mice carrying a loxP-flanked Rax exon 2 and an inducible Cre recombinase system which allowed us to eliminate its function at different embryonic stages. In this way we observed that the elimination of Rax function over many time points, ranging from embryonic days (E) 7.5 to E10.5, causes underdevelopment of the retina and of the infundibulum, a region of the posterior hypothalamus that gives rise to the posterior lobe of the pituitary. Interestingly, the anterior hypothalamus was severely affected only when Rax function was eliminated between E7.5 and E8.0, causing a striking thinning of the neuroepithelium. Analysis of molecular markers indicate that differentiated neurons expressing proopiomelanocortin (Pomc), tyrosine hydroxylase (TH) and the TFs orthopedia (Otp) and Six6 are either absent or severely reduced in these mutant embryos, indicating that Rax is essential for the formation of the anterior hypothalamus before E8.5. This contrasts with molecular markers of the posterior hypothalamus, which are expressed normally or, in the cases of  Otx2, Tbx3 and Rax itself, become more broadly expressed, invading the anterior hypothalamus of mutant embryos. Some of these phenotypes are reminiscent of those observed when the gene encoding the secreted factor Sonic hedgehog (Shh) are eliminated from the hypothalamus. Remarkably, we determined that the elimination of Rax function before E8.0 completely supressed Shh expression in the anterior hypothalamus. Indeed, double immunohistochemistry reveals that Rax and Shh are coexpressed at the anteroventral  forebrain at late headfold stages (E8.5), indicating that Rax has the potential of controlling Shh at early development. In conclusion, we show that Rax is essential for anterior hypothalamic specification and patterning, likely by its action on Shh expression.