Διερεύνηση του ρόλου μεταγραφικών παραγόντων και σηματοδοτικών μηχανισμών στην ανάπτυξη του κεντρικού νευρικού συστήματος

Abstract

HOX genes play a fundamental role in patterning the developing hindbrain and the posterior nervous system as well as in the generation of diverse neuronal subtypes. Their precise role is difficult to address because of functional redundancy of these genes. Hoxb1 is expressed in a single compartment of the developing hindbrain (rhombomere 4) and it is necessary for correct patterning of neural precursors and it is also implicated in the generation of neural crest cells in this region. Furthermore, acting in concert with signalling protein Shh, it is responsible for the generation of branchiomotor neurons and suppression of serotonergic neurons in that region. In this study, we attempted to clarify the role of Hoxb1 in the development with loss and gain of function experiments in mouse and chick embryos. One of our goals was to make transgenic animals that allow the inducible ectopic expression of Hoxb1. We first generated targeted mouse embryonic stem (ES) cells carrying an inducible Hoxb1 transgene. Positive clones were injected into blastocysts of C57BL / 6 mice to generate mouse chimeras, which were then interbred with wild type mice 129sv. The agouti offsprings positive for the transgene were crossed with transgenic En1-cre mice to activate the Hoxb1 transgene starting from embryonic day 9.5 in the posterior midbrain and rhombomere 1. We analyzed double transgenic embryos at different developmental stages. In situ RNA Hybridization, immunofluorescence with Hoxb1 antibody and histological analysis led us to conclude that the resulting low ectopic expression of Hoxb1 in this experimental setup was not sufficient to cause phenotypic changes. To elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos and investigated the effect on the expression of the LIM/ homeobox containing genes Lhx2, 5 and 9. In mouse ES derived neural stem cells where Hoxb1 was induced Lhx2, 5 and 9 were downregulated (Gouti et al., 2008). Additionally, bioinformatic analysis has shown that these genes contain in their sequence Hoxb1 binding sites, which indeed are conserved between species. Our goal was to investigate whether Hoxb1 regulates the expression of Lhx2, 5 and 9 in the neural tube during development. We first studied the expression of Lhx5 in wild-type mouse embryos and in embryos that lack functional Hoxb1 protein. RNA in situ hybridization and immunofluorescence experiments showed that Hoxb1 repressed Lhx5 expression. Then Hoxb1 was ectopically expressed in the neural tube of chick embryos via electroporation and expression of Lhx 5 and 9 was analyzed one or two days later. Indeed, the ectopic expression of Hoxb1 along the neural tube in chick embryos lead to reduced expression of both Lhx5 and Lhx9 as shown by RNA in situ hybridization and immunofluorescence for GFP experiments. Lhx5 and Lhx9 are expressed broadly in the developing neural tube in specific subdomains. Our findings that Hoxb1 can repress their expression suggest that Hox genes may act as upstream regulators in shaping their expression domains and thus participate in neuronal subtype specification.