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Fig. 5 Repressor activity of Pnx was required for the formation of posterior neurons. Schematic representation of Pnx constructs. Ten picograms of pnx, 5 pg of EnRpnx or 20 pg of VP16pnx RNA were co-injected with 50 pg of β-galactosidase RNA into one blastomere of two- to four-cell stage zebrafish embryos. The embryos were fixed at the three-somite stage, and the expression of β-galactosidase was stained by X-gal. (B-E) Overexpression of pnx in posterior neuroectoderm increased the numbers of cells expressing ngn1 (B) and elavl3 (C), whereas the overexpression of pnx in anterior neuroectoderm inhibited their expression (indicated by an arrowhead in B). Dorsal (B,C) and dorsolateral (D,E) views. (D,E) ngn1-expressing cells were increased in the right side (injected side, E) of the embryos, in which pnx and β-galactosidase RNA were localized, compared with the control side (left side, D). Expression of EnR-Pnx also elicited an increased expression of ngn1 (right side, F) and elavl3 (G). By contrast, expression of VP16-Pnx strongly inhibited the expression of ngn1 (left side, H) and elavl3 (right side, I). Highly magnified views of the pnx RNA (J,K) and VP16pnx RNA (L,M). The borderlines between the injected (lower) and the non-injected (upper) sides are indicated by broken lines. (N,O) Overexpression of pnx slightly expanded neuroectoderm expressing sox19 (N), but expression of VP16pnx did not significantly affect the formation of neural plate (O). The left sides are injected sides.