DEV001966 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables 1-9 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Generation of epitope-tagged Gli1 knock-in construct. (A) The construct is driven by the native ROSA26 promoter in an ‘off’ state, requiring Cre-mediated excision (LoxP sites depicted as triangles) for transcriptional activation. (B) Targeted Gli1 ES cells stained with FLAG and YFP antibodies showing the construct in an ‘off’ state in the absence of Cre activity (ES cells contain a tamoxifen inducible Cre) and in an ‘on’ state when cells are treated in parallel with 4-hydroxytamoxifen. (C) A constitutively active Gli-producing cell line was derived by excision of the stop cassette from the parent construct in B. Gli1 expression is predominately nuclear upon treatment with Leptomycin B. (D) Western blot showing the specificity and size of the GliFLAG protein, lacZ expression driven by the second Rosa26 allele serves as a loading control. pA, polyadenylation site; sa, splice acceptor; IRES, internal ribosomal entry site. Scale bar: 20 μm.

• Supplemental Figure S2 -

  Fig. S2. Gli position weight matrices. (A) Position weight matrix for Gli present in TRANSFAC and (B) matrix derived from first-tier ChIP sites using de novo motif analysis.

• Supplemental Figure S3 -

  Fig. S3. Expression of tier 1 ChIP target genes in Hh mutants. In situ analysis of tier 1 genes was performed using wild-type (WT), Hh gain-of-function (Ptch1^lacZ/lacZ) and loss-of-function (Smo^−/−) embryos. Compared with WT, Ptch1^lacZ/lacZ embryos contained increased levels of signaling and Smo^−/− embryos contained reduced or absent levels of expression, indicative of positively regulated targets. Note that panels B,C,F, and I exhibit neurally restricted expression patterns, whereas panels A,E,G, and H are expressed in multiple Hh-responsive domains. By contrast, Rab34 (D) is expressed more broadly. While it functions as a positive target, some residual expression remains in the Smo^−/− embryo (D′′).

• (Download) Animation_Launcher.html -

  Movie 1. BioTapestry model for Shh-driven transcriptional network underlying ventral neuronal specification. In this series of dynamic images, depicted in standard BioTapestry nomenclature (Longabaugh et al., 2005), neuronal specification is depicted as a sequential series of cell states. All genes expressed at that time are in black or colored font, whereas those genes not expressed are in gray font. Similarly, activation or repression at that time is depicted by colored lines, whereas inactive links (active in previous stages of specification) are depicted by gray lines. This diagram focuses explicitly on ventral cell specification; previous events in general neuronal specification are not depicted. 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). To view, click on Animation_Launcher.html to open up this directory off-line in your local web browser. A window will open with ‘Browse BioTapestry Network’ in the top-left corner. Click on this link and you will see an overall view of the network. Click on time points 1-6 to see a model for how specification progresses over time.