XB-IMG-148313
Xenbase Image ID: 148313
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Fig. 1.
Identification of bcar3 as a target of Gata2 in ectoderm during gastrulation. (A) Schematic of strategy for microarray analysis and target gene identification. Gata2 MO (40 ng), Fog RNA (500 pg) or Gata2 RNA (500 pg) was injected into both cells of two-cell embryos and ectoderm was explanted when embryos reached stage 10. When sibling embryos reached stage 12, RNA was isolated from ectoderm for microarray analysis. Genes showing upregulation in Gata2 MO and Fog RNA injected embryos, and downregulation in Gata2 RNA injected embryos were identified as putative negative targets of Gata2. Genes showing downregulation in Gata2 MO and Fog RNA injected embryos, and upregulation in Gata2 RNA injected embryos were identified as putative positive targets of Gata2. (B) Table showing fold change in bcar3 expression relative to uninjected controls detected by microarray analysis and schematic diagram of Bcar3 structure, SH2: Src homology 2 domain, S/P-rich: Serine/Proline-rich domain, GEF: Guanine nucleotide exchange factor like domain. (C) qPCR analysis of bcar3 gene expression in ectodermal explants isolated at stage 10 from embryos that had been injected with Gata2 MO or Fog RNA and cultured to stage 12. The level of expression of bcar3 was normalized to that in ectoderm from uninjected controls. (D) Gata2 MO (40 ng) or control MO (40 ng) were injected into both cells of two-cell embryos and ventral ectoderm and ventral mesendoderm were dissected at stage 12–13. RNA was extracted from tissue from 10 pooled explants in three independent experiments and bcar3 gene expression was analyzed by qPCR. The level of expression of bcar3 was normalized to that in ectoderm from control MO injected embryos. VE: ventral ectoderm, VM: ventral mesendoderm, ND: not detected. (E) Fog RNA (500 pg) was injected into one cell of two-cell embryos and expression of bcar3 was examined at stage 15 using WMISH. Uninjected embryos were used as controls. In each row, dorsal, lateral and ventral views of the same embryo are shown. Brackets indicate the Fog RNA injected side. (F) Gata2 MO (40 ng), control MO (40 ng), or Gata2 MO (40 ng) and Gata2 RNA (1 ng) together was injected into both cells of two-cell embryos and ectoderm was explanted when embryos reached stage 10. When sibling embryos reached stage 12, RNA was isolated from ectoderm for qPCR analysis. The level of expression of bcar3 was normalized to that in ectoderm from control MO injected embryos. Image published in: Green YS et al. (2016) Copyright © 2016. Image reproduced with permission of the Publisher, Elsevier B. V.
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