Ntla and Tbx16 directly regulate dlc expression (A)–(H) at 65% epiboly ntla^-/- and spt^-/- mutants have reduced dorsal-lateral expression of dlc, which is more pronounced in ntla^-/-; spt^-/- double mutants (asterisks). The vegetal-animal width of the expression is also reduced in mutant embryos. (I)–(L) At bud stage, dlc expression in ntla^-/- mutants is similar to wild-type embryos, although somite shape is sometimes disrupted. Expression is much reduced in spt^-/- mutants and absent in ntla^-/-; spt^-/- double mutants. (M)–(P) At 12 somite stage, dlc expression is absent from the posterior in ntla^-/- mutants, but remains in the somites. In spt^-/- mutants, expanded expression is seen in the tailbud, while expression is absent in ntla^-/-; spt^-/- double mutants. (Q)–(T) Normal expression of dlc is seen in embryos injected with Ntla-GR or Tbx16-GR and treated with cycloheximide (CHD). Addition of dexamethasone (DEX) together with cycloheximide, results in expanded dlc expression at the margin, indicating dlc is directly regulated by Ntla and Tbx16. (A)–(D) show animal views, dorsal to the left; (E) - ((H) and (Q) - (T) show dorsal views, animal to the top. (I) - (P) show dorsal views, anterior to the top.

dlc CRM drives expression of reporter in tailbud, PSM and somites. (A) Confocal images of transient transgenics at 5, 8, 12 and 18 somite stages (5s, 8s, 12s, 18s) show mCherry expression (red; under the control of the dlc CRM) overlaps with GFP (green; under the control of a ubiquitous promoter) in the somites (arrows). Conversely, although strong GFP expression is seen in the nervous system, including the eye, mCherry expression is not seen here. (B) mCherry protein is detected in the somites by immunohistochemistry in 12 somite stage embryos (arrows). (C) Sites of mCherry expression detected by immunohistochemistry at 12ss (examples in (B)) were recorded and used to produce a representative composite of mCherry expression from 80 embryos. Black dots represent mCherry-expressing cells in the somites, PSM, tailbud or notochord; red dots represent mCherry expressing cells outside these tissues. 44% of embryos which expressed GFP also expressed mCherry in the somites, PSM, tailbud and/or notochord at 12ss. (D) Co-localisation of mCherry protein driven by the dlc CRM in the talibud (red) and endogenous dlc mRNA at 12 somite stage (green) shows overlapping expression. Left image is a dorsal view, right image is a lateral view. (E) Co-localisation of dlc mRNA (green) with ntla or tbx16 mRNA (red) at 12 somite stage shows overlapping expression in the tailbud. Lateral views are shown. (F) ChIP-PCR on 12-somite stage embryos using primers that amplify a region within the dlc CRM (see Fig. 1A and Table S1) show that dlc is bound by Ntla (red bars) and Tbx16 (blue bars) at this stage. For comparison, enrichment of binding around a negative region 2.2 kb upstream of dcn (see Table S1 for primer sequence) is shown. (G) Ntla-GR and Tbx16-GR activate luciferase expression through the dlc CRM in the presence of dexamethasone and cycloheximide at 12ss, indicating regulation by these factors is direct.