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First published online 21 January 2009
doi: 10.1242/dev.024703

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

¹ Department of Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley CA 94720, USA.
² Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge, CB2 1QN, UK.
³ Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK.
⁴ Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁵ California Institute for Quantitative Biosciences, Berkeley, CA 94158, USA.
⁶ Department of Physiology, Development and Neuroscience, Anatomy Building, Downing Street, Cambridge CB2 3DY, UK.

^* Author for correspondence (e-mail: amacher{at}berkeley.edu)

Accepted 30 December 2008

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.

Key words: Enhancer prediction, Gene regulation, No tail/brachyury, Spadetail/tbx16

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