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Fig. 2. Regulation of hhex expression by Nodal and Wnt/β-catenin signaling. (A) Nodal and Wnt signaling are required for hhex expression. In situ hybridization of hhex and gfp in bisected −6.0Kb:hhex:gfp transgenic gastrulae injected with β-catenin-MO (20 ng), ∆Ntcf3 RNA (500 pg), stabilized pt-β-catenin RNA (250 pg), xnr1 RNA (50 pg), and/or cer-s RNA (750 pg). The number of embryos exhibiting the depicted phenotype is indicated. Transgenic F1 males are heterozygous, and approximately 50% of the embryos are expected to express gfp. (B) The schematic indicates the cells injected with the hhex:luc reporter (300 pg) plus pRL-TK:Renilla (25 pg). Co-injection with xnr1 RNA (1–500 pg) and/or β-catenin RNA (20–200 pg) resulted in a dose-dependent activation the hhex:luc reporter in the ectoderm. The histogram shows the average normalized luciferase activity and standard deviation from a single injection experiment performed in biological triplicate. A representative example of 4 independent experiments is shown. (C) hhex is a direct Nodal-target and an indirect Wnt-target. Blastula stage animal cap tissue was cultured either untreated, with Activin or with BIO. Some explants were incubated in CHX for 30 min prior to and during culture to block protein synthesis. At stage 11, the animal caps were assayed by in situ for hhex, gfp, cerb, and xnr3. This experiment was repeated 3 times, with identical results and a total of 12–15 caps per treatment. A representative example is shown. |
