Identification of direct T-box target genes in the developing zebrafish mesoderm

The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. Here, we survey the genome for downstream target genes of the Spt and Ntl T-box transcription factors. We find evidence for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, we searched for target regulatory sequence via a combination of binding motif searches and comparative genomics. We identified regulatory elements for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, we provide evidence that both are directly regulated by T-box transcription factors. We also find that deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process. Keywords: Morpholino Knockdown We measured transcript levels in zebrafish embryos at 75% epiboly. We compared levels in spadetail (spt) and/or no tail (ntl) morpholino injected embryos to those in control embryos mock injected with Danieau solution plus 0.25% phenol red. Three biological replicates are included for each treatment.