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Figure 2. Increased Six1 alters otic gene expression. Transcripts encoding wild-type Six1 (Six1WT, either 150 or 400 pg), activating Six1 (VP16, 100 pg) or repressive Six1 (EnR, 100 pg) were microinjected into blastomeres that contribute to otic structures on one side of embryos containing endogenous levels of Six1. (A) The effects on otic vesicle gene expression (blue) were compared between embryos injected with Six1WT mRNA (+Six1WT) versus those injected with either Six1VP16 (+Six1VP16) or Six1EnR (+Six1EnR) mRNAs. If Six1 acts as a repressor (top row), then additional Six1WT should reduce gene expression, Six1VP16 should either cause no change or increase it, and Six1EnR should also reduce it. If Six1 acts as an activator (bottom row), then additional Six1WT should increase gene expression, Six1VP16 should also increase it, and Six1EnR should cause either no change or reduce it. (B–P) Gene expression was assayed by ISH for eya2 (B,C), prdm1 (E,F), spry1 (H,I), tspan13 (K,L) and pa2g4 (N,O). The control, uninjected side of each embryo is on the left and the mRNA-injected side of the same embryo is on the right. Otic gene expression on the control side is indicated by black arrows, and that on the mRNA-injected side by red arrows. In C and F, the width of the otic vesicle is indicated by a black (control) or red (injected) bar. Frequencies of the effects (blue, decreased expression; yellow, increased expression; grey, no change) are indicated by bar graphs (D,G,J,M,P). For eya2 (D) and prdm1 (G), the frequencies for Six1VP16 were significantly different from both Six1WT-150 and SixWT-400 (*, p < 0.0001). For tspan13 (M), the frequencies for Six1VP16 were significantly different from Six1WT-150 (*, p < 0.05), but not from SixWT-400. For pa2g4 (P), the frequencies for Six1VP16 were significantly different from SixWT-400 (*, p < 0.0001), and the frequencies for Six1EnR were significantly different from SixWT-150 (*, p < 0.01). White numbers inside each bar denotes the number of embryos analyzed. |
