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Published May 15, 2007 | Published + Supplemental Material
Journal Article Open

Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning

Abstract

Sonic hedgehog (Shh) acts as a morphogen to mediate the specification of distinct cell identities in the ventral neural tube through a Gli-mediated (Gli1-3) transcriptional network. Identifying Gli targets in a systematic fashion is central to the understanding of the action of Shh. We examined this issue in differentiating neural progenitors in mouse. An epitope-tagged Gli-activator protein was used to directly isolate cis-regulatory sequences by chromatin immunoprecipitation (ChIP). ChIP products were then used to screen custom genomic tiling arrays of putative Hedgehog (Hh) targets predicted from transcriptional profiling studies, surveying 50-150 kb of non-transcribed sequence for each candidate. In addition to identifying expected Gli-target sites, the data predicted a number of unreported direct targets of Shh action. Transgenic analysis of binding regions in Nkx2.2, Nkx2.1 (Titf1) and Rab34 established these as direct Hh targets. These data also facilitated the generation of an algorithm that improved in silico predictions of Hh target genes. Together, these approaches provide significant new insights into both tissue-specific and general transcriptional targets in a crucial Shh-mediated patterning process.

Additional Information

© 2007 Company of Biologists Ltd. Accepted 5 March 2007. We thank Curis for providing Hh-Ag, Alex Joyner and David Rowitch for providing constructs, Zhenjuan Wang (Harvard Genome Manipulation Facility) for pronuclear injections, Jennifer Couget (GGR) for expert guidance on performing microarrays, Julia Zeitlinger for helpful advice on optimizing ChIP, and Renate Hellmiss-Peralte for advice and assistance on figures. We are indebted to Leo Brizuela, Brett Chevalier and Mel Kronick at Agilent Technologies for supporting this project. We thank Dr James Briscoe for discussion of unpublished material. The Pax6 antibody (developed by the Kawakami Laboratory), Nkx2.2 and FoxA2 antibodies (developed by the Jessell laboratory) were obtained from the Developmental Studies Hybridoma Bank. We acknowledge support from the NIH-NINDS #R37 NS033642 (A.P.M.), NIH #R01 GM67250 and HG3903-01 (W.W. and H.J.),Helen Hay Whitney Foundation (S.V.) and the Charles King Trust Fellowship (S.V.).

Attached Files

Published - VOKdev07.pdf

Supplemental Material - 1.pdf

Supplemental Material - DEV001966FigS1.jpg

Supplemental Material - DEV001966FigS2.jpg

Supplemental Material - DEV001966FigS3.jpg

Supplemental Material - Supplemental_Movie.zip

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