A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro

Hiroshi Sasaki ) 2 Chi-chung Hui 1 Masato Nakafuku 0 Hisato Kondoh 2 0 Division of Signal Transduction and Metabolic Regulation in Animal Cells, Graduate School of Biological Sciences, Nara Advanced Institute of Science and Technology , Ikoma, Nara 630-01 , Japan 1 Program in Developmental Biology and Division of Endocrinology, Research Institute, The Hospital for Sick Children, 555 University Avenue , Toronto, Ontario M5G 1X8 , Canada 2 Laboratory of Developmental Biology, Institute for Molecular and Cellular Biology, Osaka University , 1-3 Yamada-oka, Suita, Osaka 565 , Japan activity in transgenics and can respond to Shh in vitro SUMMARY The floor plate plays important roles in ventral pattern formation and axonal guidance within the neural tube of vertebrate embryos. A critical event for floor plate development is the induction of a winged helix transcription factor, Hepatocyte Nuclear Factor-3b (HNF-3b ). The enhancer for floor plate expression of HNF-3b is located 3 of the transcription unit and consists of multiple elements. HNF-3b induction depends on the notochord-derived signal, Sonic hedgehog (Shh). Genetic analysis in Drosophila has led to the identification of genes involved in the Hh signalling pathway, and cubitus interruptus (ci), encoding a protein with five zinc finger motifs, was placed downstream. In the present work, we test the involvement of Gli proteins, the mouse homologues of Ci, in activation of the floor plate enhancer of HNF-3b . Transgenic analysis shows that a Gli-binding site is required for the activity of the minimal floor plate enhancer of HNF-3b in vivo. Three Gli genes are differentially expressed in the developing neural tube. Gli expression is restricted to the ventral part, The floor plate is a group of specialized cells located in the ventral midline of the vertebrate neural tube. It plays important roles in neural tube development as a source of signalling molecules for dorsoventral patterning and axonal guidance (for reviews, see Jessell and Dodd, 1992; Dodd and Jessell, 1993). A floor plate-derived signalling molecule, Sonic hedgehog (Shh), which is a vertebrate homologue of Drosophila Hedgehog (Hh), induces differentiation of ventral type neurons (Ericson et al., 1995; Marti et al., 1995; Roelink et al., 1995). Another floor plate-derived signalling molecule, netrin-1, works as both a chemoattractant and a chemorepellent of axons, depending on neuronal cell types (Kennedy et al., 1994; Colamarino and Tessier-Lavigne, 1995). Differentiation of the floor plate is induced by a signal derived from the notochord or by Shh. High concentrations of Shh-N peptide or contact with the notochord are required for while Gli2 and Gli3 are expressed throughout the neural tube and dorsally, respectively. Strong Gli and Gli2, and weak Gli3 expressions transiently overlap with HNF-3b at the time of its induction. Consistent with ventrally localized expression, Gli expression can be up-regulated by Shh in a cell line. Finally, the Gli-binding site acts as a Shh responsive element, and human GLI, but not GLI3, can activate this binding site in tissue culture. Taken together, these findings suggest that Gli, and probably also Gli2, are good candidates for transcriptional activators of the HNF-3b floor plate enhancer, and the binding site for Gli proteins is a key element for response to Shh signalling. These results also support the idea that Gli/Ci are evolutionary conserved transcription factors in the Hedgehog signalling pathway. floor plate induction in neural explants (Marti et al., 1995; Roelink et al., 1995). A critical event of floor plate development is the expression of a winged-helix transcription factor, HNF-3b (Ang et al., 1993; Ruiz i Altaba et al., 1993; Sasaki and Hogan, 1993, 1994). Ectopic expression of HNF-3b in the neural tube induces ectopic expression of series of floor plate marker genes and prevents dorsal/lateral maker expression, indicating that HNF-3b acts as a regulator of floor plate development (Sasaki and Hogan, 1994; Ruiz i Altaba et al., 1995a). The HNF-3b expression in the floor plate is induced by a signal derived from the notochord or by Shh, and its induction does not require de novo protein synthesis (Roelink et al., 1995; Ruiz i Altaba et al., 1995b; Chiang et al., 1996). This makes HNF-3b a candidate for a direct target of Shh signalling. Previously, we have identified cis-regulatory regions of the mouse HNF-3b gene in transgenic mouse embryos. Enhancers for floor plate expression and node/notochord expression were Fig. 1. Schematic presentation of the previous knowledge of mouse HNF-3b floor plate enhancer. The transcription unit and its direction are indicated by boxes and an arrow, respectively. Minimal floor plate enhancer activity requires combination of a 1.5 kb A fragment and a 400 bp HaeIII fragment. Analysis using internal deletion series within the 400 bp fragment identified multiple regions involved in regulation. The regions del-3 and del-4 are required for anterior floor plate gene expression, while regions del-5 and del-6 are essential for the enhancer activity (Sasaki and Hogan, 1996). identified in separate regions 3 and 5 of the transcription unit, respectively. Deletion analysis of the minimal floor plate enhancer identified multiple elements required for its activity (Fig. 1) (Sasaki and Hogan, 1996). The present study was initiated by finding a Gli-protein-binding site within one of these essential elements. GLI is a gene that was originally identified as an amplified gene in a human glioblastoma (Kinzler et al., 1987). It encodes a nuclear protein, containing five zinc finger motifs, which binds to DNA in a sequence-specific manner (Kinzler et al., 1988; Kinzler and Vogelstein, 1990). In humans, there are three GLI genes (GLI, GLI2, GLI3) (Ruppert et al., 1988), which have closely related zinc fingers, and their mouse counterparts have also been identified (Gli, Gli2, Gli3) (Walterhouse et al., 1993; Hui et al., 1994). The Drosophila homologue of Gli genes is cubitus interruptus (ci), which is involved in the Hedgehog (Hh) signalling pathway (Orenic et al., 1990). According to genetic analysis, the Hh pathway also includes transmembrane Hh receptor Patched (Ptc), a seven-transmembrane protein Smoothened (Smo), a serine-threonine kinase Fused (Fu), cAMP-dependent protein kinase A (PKA) and a gene costal-2 (cos-2) (Alcedo et al., 1996; Chen and Struhl, 1996; van den Heuvel and Ingham, 1996; for reviews, see Forbes et al., 1993; Perrimon, 1995). Among them, Ci was proposed to be a transcription factor that works at the most downstream point of the pathway (Forbes et al., 1993; Domnguez et al., 1996). In fact, recent experiments showed that Ci acts as a transcriptional activator and that it activates one of the target genes, ptc, through GLI-binding sites (Alexandre et al., 1996). At least a part of the Hh signalling pathway seems to be conserved between Drosophila and vertebra (...truncated)