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First published online 18 April 2007
doi: 10.1242/dev.001966

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

Steven A. Vokes¹, Hongkai Ji^2,3, Scott McCuine⁴, Toyoaki Tenzen¹, Shane Giles⁴, Sheng Zhong^3,*, William J. R. Longabaugh⁵, Eric H. Davidson⁶, Wing H. Wong³ and Andrew P. McMahon^1,7,

¹ Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
² Department of Statistics, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
³ Department of Statistics, Stanford University, Sequoia Hall, 390 Serra Mall, Stanford, CA, 94305, USA.
⁴ Agilent Technologies, 245 First Street, Suite 105, Cambridge, MA 02142, USA.
⁵ Institute for Systems Biology, Seattle, WA, 98103, USA.
⁶ Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
⁷ Harvard Stem Cell Institute, 16 Divinity Avenue, Cambridge, MA 02138, USA.

View larger version (40K): [in this window] [in a new window]   Fig. 1. EBs containing Gli1FLAG exhibit an amplified response to Hh stimulation. (A-P) Immunostaining of EBs to detect neural progenitor types in mouse. While both control (A-H) and Gli1FLAG samples (I-P) respond to Hh signaling by activating Nkx6.1 expression (F,N), only the latter samples contain Nkx2.2+ and FoxA2+ cells on Hh-Ag treatment (O,P). (Q) Distinct neural progenitors demarcated by expression of progenitor-type-specific markers were counted and represented as a percentage of all DAPI-positive cells in three independent EBs. The error bars represent the standard deviation. Scale bar: 100 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Chromatin immunoprecipitation of neuralized murine EBs. (A-H) Selected positive targets showing the mean fold enrichment of Gli1FLAG ChIPs representing three biological replicates, each with four technical replicates. The plots show the mean fold enrichment of ChIPed sequence versus input. Approximately half of the peaks lie outside the proximal promoter region. Multiple peaks are numbered to correspond to the peaks listed in Table 1 and in Table S2 in the supplementary material; arrows indicate previously described Gli regulatory regions. Below each graph, the position of exons (rectangles) and the direction transcription (arrows) are shown relative to the ChIPed region. (I,J) De novo motif analysis recovers a consensus site (I) similar to a previously described Gli1 consensus sequence (J) (Transfac # M01037) (I).

View larger version (82K): [in this window] [in a new window]   Fig. 3. Transgenic validation of Gli-binding regions demonstrates Gli-dependent expression in Hh target tissue. Selected candidate enhancers (Ptch1 peak2 and Nkx2.2 peak1, Rab34 peak1 and Nkx2.1 peak1) were cloned into minimal promoter lacZ reporter construct (A) to generate transgenic embryos and assayed at embryonic day 10.5. (B-G,W-Z) The embryo in panel E is cleared in benzyl alcohol and benzyl benzoate; all other whole mounts are in 80% glycerol. Histological sections through the spinal cord (forelimb level) (H-M) and brain (N-S) of transgenic and control embryos. Forelimbs (T-V,A',B') show are dissected from the corresponding whole mount. Negative control embryos and sections are shown in B,H,N,T. When compared with PtchlacZ/+ embryos, Ptch1 peak2-LacZ transgenics express a subset of the normal domain ß-gal activity (compare C,I,O,U with D,J,P,V), lacking expression in the limb bud mesenchyme (U,V). In situ hybridizations of Nkx2.2 (E,K,Q), transgenic Nkx2.2-lacZ embryos (F,L,R) or transgenics with the Gli-binding site mutated (G,M,S). In situ hybridizations of Rab34 (W,A') and transgenic Rab34-lacZ embryos (X,B'). In situ hybridizations of Nkx2.1 (Y) and transgenic Nkx2.1-lacZ embryo (Z). The arrows in X and Z indicate the domain of expression within the ventral diencephalon. Unlike the other transgenics, Nkx2.1-lacZ is driven by only one copy of the enhancer. All limb specimens are oriented with anterior to the left and distal up. Scale bars: 200 µm in H-S; 1 mm in A-G,T-Z,A',B'.

View larger version (80K): [in this window] [in a new window]   Fig. 4. BioTapestry models of Hh-driven cis-regulatory networks in mouse. Transcriptional targets of the Hh pathway can be defined by a globally responsive signaling cassette (A), containing components that are either known (unbroken lines) or likely (broken lines) to be part of a negative feedback loop. (B) A model for a Shh-driven transcriptional network underlying ventral neuronal specification. In this diagram, depicted in standard BioTapestry nomenclature (Longabaugh et al., 2005), neuronal specification is depicted as a sequential series of cell states. All genes not expressed are in gray, whereas currently expressed genes are in black or other colors. Similarly, inactive links (active in previous stages of specification) are depicted in gray, whereas active activation or repression is depicted using lines of other colors. An animation of these events can be viewed in Movie 1 in the supplementary material. This diagram focuses explicitly on ventral cell specification; thus previous events in general neuronal specification are not shown. Validated Gli targets (all identified or confirmed in our study) are indicated by blue diamonds (ChIP peak), orange diamonds (transgenic validation) or green diamonds (mutation of binding site in transgenic embryos).

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