A mesodermal gene regulatory network directed by zebrafish No tail

Using chromatin immunoprecipitation combined with microarrays we have identified targets of No tail (Ntl), a zebrafish Brachyury ortholog that plays a central role in mesoderm formation. We show that Ntl regulates a downstream network of other transcription factors and identify an in vivo Ntl binding site that resembles the consensus T-box binding site (TBS) previously identified by in vitro studies. We show floating head (flh) is a direct transcriptional target of Ntl in the notochord and that TBS in the flh upstream region are required for Ntl directed expression. Using this genome-scale data we have assembled a Gene Regulatory Network that describes mesoderm formation and patterning in the early zebrafish embryo and which predicts a role for Ntl in somite segmentation. Keywords: ChIP-chip Genomic array design. Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). We have previously described the ChIP-chip technique in zebrafish and the design of promoter microarrays which cover a 2kb region around the transcription start sites (TSSs) of 11,512 zebrafish genes based on Zv4 (Wardle et al, 2006. Genome Biology 7:R71). In order to capture additional regulatory information further from the basal promoter region we also designed an expanded set of 60mer probes that cover 9kb upstream and 3kb downstream of the TSS of these genes using the same parameters as for the 2kb microarrays. We also incorporated several sets of control probes, both positive and negative. On each array there are 753 negative probes designed against zebrafish gene desert regions and Arabidopsis thaliana genes. In addition we included duplicates of promoter probes for several genes that we anticipated may be bound by factors of interest for this and other studies (wnt11, flh, vent, msgn1, myod, fgf8, pcdh8) and these probes are arrayed on both slides slide. We also included in this design 502 intensity control probes. Finally there are 2,256 controls added by Agilent. The final design contained 378,002 probes, including 365,537 experimental probes divided between nine microarray slides: slides 1-8 contained 40,616 experimental probes and slide 9 contained 40, 609. Further information on design and manufacture of the microarrays can be found on the Agilent Technologies website (www.agilent.com).