Genomewide Expression Profiling in the Zebrafish Embryo Identifies Target Genes Regulated by Hedgehog Signaling During Vertebrate Development

Abstract

Hedgehog proteins play critical roles in organizing the embryonic development of animals, largely through modulation of target gene expression. Little is currently known, however, about the kinds and numbers of genes whose expression is controlled, directly or indirectly, by Hedgehog activity. Using techniques to globally repress or activate Hedgehog signaling in zebrafish embryos followed by microarraybased expression profiling, we have discovered a cohort of genes whose expression responds significantly to loss or gain of Hedgehog function. We have confirmed the Hedgehog responsiveness of a representative set of these genes with whole-mount in situ hybridization as well as real time PCR. In addition, we show that the consensus Gli-binding motif is enriched within the putative regulatory elements of a sizeable proportion of genes that showed positive regulation in our assay, indicating that their expression is directly induced by Hedgehog. Finally, we provide evidence that the Hedgehog-dependent spatially restricted transcription of one such gene, nkx2.9, is indeed mediated by Gli1 through a single Gli recognition site located within an evolutionarily conserved enhancer fragment. Taken together, this study represents the first comprehensive survey of target genes regulated by the Hedgehog pathway during vertebrate development. Our data also demonstrate for the first time the functionality of the Gli-binding motif in the control of Hedgehog signaling-induced gene expression in the zebrafish embryo.

Cloning of zebrafish nkx6.2 and a comprehensive analysis of the conserved transcriptional response to Hedgehog/Gli signaling in the zebrafish neural tube

“…Expression of nkx2.9 was dramatically expanded in embryos with ectopic Hh signaling, consistent with its classification as Hh induced Class II gene (Fig. 2C, Xu et al, 2006). The expression of the nkx2.9 was absent in all of the Hh pathway mutants examined (Fig.…”

“…1). nkx2.9 was subsequently identified in a microarray screen as a Hh responsive gene (Xu et al, 2006), but its expression during zebrafish embryogenesis was not documented. Assignment of homology was further verified by analysis of gene expression (see Fig.…”

Dynamics of Sonic hedgehog signaling in the ventral spinal cord are controlled by intrinsic changes in source cells requiring Sulfatase 1

“…5M,N). Furthermore, 24 hpf sulf1MO-injected embryos also failed to upregulate nkx2.9, specifically expressed in p3 cells (Guner and Karlstrom, 2007;Xu et al, 2006), whereas pax7a expression in dorsal progenitors and arx, expressed in MFP cells (Norton et al, 2005), were unaffected (supplementary material Fig. S3).…”

Generation of intestinal surface: an absorbing tale

“…Since many Nkx and Fox genes are targets of Hh signaling (Xu et al, 2006), it is possible that these mesenchymal factors play key roles in murine villus formation by affecting mesenchymal cluster formation downstream of the Hh pathway. This might also be true in the chick, given the clear role for Hh signaling in villus development in both species.…”