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, 2008). Using Ntla and Tbx16 position weight matrices (PWMs; Garnett et al., 2009?and?Morley et al., 2009) this analysis detects the four TBS upstream of zebrafish dlc and predicts the clustering to be significant, suggesting it is a CRM (pvalQuinapyramine the Ntla PWM identifies TBS3 (red bar) and Tbx16 PWM identifies TBS4 (blue bar). We then used this software to inspect the upstream region of dlc orthologues in four other teleost fish species (fugu, tetraodon, medaka and stickleback) and in two tetrapods (Xenopus and human). This found significant clustering of T-box binding sites (pvalthis website be important in the regulation of dlc orthologues in these other species, although these sites are not within highly conserved non-coding elements (HCNEs; Frazer et al., 2004; data not shown). Medaka dll3 upstream sequence contains several TBS, but they did not form clusters that reached the significance threshold ( Fig. 1C), however such clusters of TBS can be found in the long introns of dll3 (data not shown). These results show that Ntla and Tbx16 bind upstream of dlc in zebrafish during gastrulation and suggest that T-domain factors may also bind upstream of dlc orthologues in other species. We next selleck screening library asked if Ntla and Tbx16 regulate expression of dlc in zebrafish embryos by analysing its expression in mutant embryos ( Fig. 2 and Table S2). In wild type embryos at 65% epiboly, dlc expression is seen in the ventral-lateral margin but is excluded from the dorsal margin. In ntla?/? and spt?/? single mutant embryos expression is down-regulated on the dorsal side ( Fig. 2A�CC, E�CG), and is further down-regulated in ntla?/?; spt?/? double mutants ( Fig. 2D, H). The width of the marginal expression in the vegetal-animal direction is also reduced in mutant embryos at this stage even though a normal germ ring forms ( Halpern et al., 1993?and?Kimmel et al., 1989 and data not shown). At bud stage wild type dlc expression is seen in the tailbud and in stripes in the anterior paraxial mesoderm ( Fig. 2I). In bud stage ntla?/? mutant embryos, dlc is expressed in a pattern similar to wild type embryos, although the shape of the forming somites is often disrupted ( Fig. 2J). In spt?/? mutant embryos, dlc expression is seen in the tailbud and in a few cells adjacent to the midline which may correspond to the clusters of cells observed in the paraxial region of the trunk in these embryos ( Fig. 2K; Kimmel et al., 1989). Expression is absent from ntla?/?; spt?/? double mutants in the trunk and in the tailbud ( Fig. 2L).