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Fig. 7. Otx2 and Lim1 promote hhex transcription downstream of Sia/Twn. (A) GR-sia RNA (30 pg) + gfp RNA (200 pg) was injected into ventral–vegetal cells and explants were isolated at blastula stage based on GFP fluorescence. Explants were cultured with or without CHX for 30 min, and then DEX was added to activate the GR-Sia as indicated. In situ hybridization at stage 11 shows that chordin, otx2, and lim1, but not hhex are directly induced by GR-Sia in CHX treated explants. This experiment was repeated 3 independent times with identical results and > 10 explants for each condition. (B) A model showing two mechanisms by which Sia/Twn could promote hhex transcription. (C) Induction of hhex by Sia/Twn is partially mediated by Otx2 and Lim1. In situ hybridization of hhex, gsc, otx2, and lim1 in gastrulae injected as follows: Sia/Twn-MOs (20 ng each, dorsal 4-cell), sia RNA (50 pg, ventral–vegetal 8-cell), Sia/Twn-MOs (20 ng each, dorsal 4-cell) + otx2 and lim1 RNA (50 pg each dorsal–vegetal 8-cell), otx2 and lim1 RNA (50 pg each ventral–vegetal 8-cell), Otx2-MO + Lim-MO1 (20 ng each, dorsal 4-cell) or Gsc-MO (60 ng, dorsal 4-cell). (D) WT or ∆HD mutant −0.65 Kb hhex:luc reporters were injected into either C1 dorsal (green) or C4 ventral cells (red), with or without the indicated mRNAs (pg). Histograms show the average normalized luciferase activity and standard deviation from a single injection experiment performed in biological triplicate. A representative example from 3 independent experiments is shown. *p < 0.05 in Student T-test when compared to injection of reporter alone and ns = no statistical difference. |
