A BINDING-SITE FOR GLI PROTEINS IS ESSENTIAL FOR HNF-3-BETA FLOOR PLATE ENHANCER ACTIVITY IN TRANSGENICS AND CAN RESPOND TO SHH IN-VITRO

The floor plate plays important roles in ventral pattern formation and axonal guidance within the neural tube of vertebrate embryos, A criti cal event for floor plate development is the induction of a winged hel ix transcription factor, Hepatocyte Nuclear Factor-3 beta (HNF-3 beta) . The enhancer for floor plate expression of HNF-3 beta is located 3' of the transcription unit and consists of multiple elements, HNF-3 bet a induction depends on the notochord-derived signal, Sonic hedgehog (S hh). Genetic analysis in Drosophila has led to the identification of g enes involved in the Hh signalling pathway, and cubitus interruptus (c i), encoding a protein with five zinc finger motifs, was placed downst ream, In the present work, we test the involvement of Gli proteins, th e mouse homologues of Ci, in activation of the floor plate enhancer of HNF-3 beta. Transgenic analysis shows that a Gli-binding site is requ ired for the activity of the minimal floor plate enhancer of HNF-3 bet a in vivo. Three Gli genes are differentially expressed in the develop ing neural tube, Gli expression is restricted to the ventral part, whi le Gli2 and Gli3 are expressed throughout the neural tube and dorsally , respectively, Strong Gli and Gli2, and weak Gli3 expressions transie ntly overlap with HNF-3 beta at the time of its induction, Consistent with ventrally localized expression, Gli expression can be up-regulate d by Shh in a cell line, Finally, the Gli-binding site acts as a Shh r esponsive element, and human GLI, but not GLI3, can activate this bind ing site in tissue culture, Taken together, these findings suggest tha t Gli, and probably also Gli2, are good candidates for transcriptional activators of the HNF-3 beta floor plate enhancer, and the binding si te for Gli proteins is a key element for response to Shh signalling, T hese results also support the idea that Gli/Ci are evolutionary conser ved transcription factors in the Hedgehog signalling pathway.