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Fig. 1. Hairy2 has DNA-binding dependent and independent activities. (A) Whole-mount in situ hybridization analysis of embryos injected with Hairy2-GR mRNA (500 pg) or Hairy2DBM-GR mRNA (500 pg) and hybridized with the indicated probes. SoxD is ectopically induced and Msx1 and Pax3 expanded by Hairy2-GR but not by Hairy2DBM-GR. In contrast, both Hairy2-GR and Hairy2DBM-GR increase Sox10. Frequency of embryos with the indicated phenotype: a, 89% ectopic, n = 19; b, 100% normal, n = 26; c, 59% expanded, n = 27; d, 65% expanded, n = 32; e, 93% normal, n = 29; f, 72% normal, n = 28; g, 68% expanded, n = 25; h, 57% expanded, n = 22). Lateral views of tailbud embryos (a,b,g,h) with control and injected sides or dorsal views of neurula embryos (c–f) with the injected side on the right, anterior to bottom, are shown. LacZ was used as lineage tracer. (B) Both Hairy2-GR and Hairy2DBM-GR overexpression (500 pg mRNA each) increases the number of pH3 positive cells (arrows) (a, 75%, n = 24; b, 47% n = 30). Neurula embryos viewed from the dorsal side, with the injected side on the right are shown. Rhodamine dextran was used as a lineage tracer. (C) Injection of Hairy2-GR mRNA (500 pg), but not Hairy2DBM-GR mRNA (500 pg), rescues Bax (50 pg mRNA) mediated embryonic lethality. The external aspect of the embryos at early gastrula stage was monitored for the presence in the injected area of depigmented enlarged dying cells which are expulsed from the embryo and trapped in between the surface of the embryo and the vitelline membrane. Examples of embryos classified as developing normally (a) or showing a low (b) or a high number of dying cells (c) is shown. The percentage of embryos developing normally or showing a mild or severe phenotype observed in each condition is shown in (c) (−, Hairy2-GR mRNA injected minus DEX). The number of embryos examined is indicated. Embryos were injected at the four cell-stages in the animal pole of one blastomere. Rhodamine dextran was used to identify the injected area (not shown). |
