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Fig. 5. Dorsal expansion of Shh/Foxa2 expression and Grg4-mediated suppression of Otx trans-activating ability. (A-J) In situ hybridisation of E10.5 and E12.5 En1^cre/+ and En1^cre/+; Otx2^flox/flox embryos with Otx2 (A,F), Otx1 (B,G), Grg4 (C,H), Shh (D,I) and Foxa2 (E,J) probes. (K,L) RNAse protection assays showing the trans-activating ability of Otx1, Otx2 and Otx2-Q₅₀ alone or in combination with Grg4 expressing plasmid on the multimerised (4x) bts (K) and np (L) target sequence. (M-O) Otx1 (M) and Otx2 (N,O) mutant molecules carrying the amino acid deletions () indicated are assayed alone or in combination with the Grg4-expressing plasmid. (P,Q) Co-immunoprecipitation assays between Grg4-Flag and Otx1 or Otx2 (P) and between Grg4-Flag and Otx2 mutant proteins not responding to Grg4-corepression (Q) show that, when co-transfected with the Grg4-Flag expressing vector, the Flag antibody co-immunoprecipitates Grg4-Flag and Otx1 or Otx2 (P), while it fails to co-immunoprecipitate Grg4-Flag and Otx2 mutant proteins (Q). (R) Comparison between the Otx1 and Otx2 amino acid domain (bold) responding to Grg4 and those known for other transcription factors reveals conservative substitutions (grey box) or identity (white box). The ß-act indicates that a similar amount of total RNA is analysed for each transfection. Numbers indicate the DNA amount in micrograms transfected for each plasmid. ABB, alar-basal boundary; I, input; IP, immunoprecipitation.

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