XB-IMG-78949
Xenbase Image ID: 78949
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Fig. 2. XNr1/Cripto and caAlk4 induce endogenous Cer mRNA. Embryos were injected with synthetic mRNA for XNr1 and Cripto or caAlk4 into one ventral blastomere of a 4–8 cell stage embryo. Non-cardiogenic VMZ explants were dissected at onset of gastrulation (stage 10.25–10.5) then either frozen immediately for subsequent RNA isolation or grown in culture and frozen when age matched siblings had reached stages 10.5, 11, 13 and 19 and analyzed for mRNA levels by quantitative RT-PCR (data presented represent the average of two technical replicates each from two of the 6 biological replicates performed). (A) Plot depicts the fold induction of Cer in injected cells relative to uninjected controls, after normalization to levels of the ubiquitously expressed Ef1α transcript. (B–I) Examples of Cer induction detected by in situ hybridization relative to the AD546 (B, C, G, H, I) or β-galactosidase (D–F) lineage labels that mark the progeny of injected cells. XNr1 and Cripto induced expression of Cerberus in mesendodermal cells in a manner that was largely cell autonomous (note nearly complete overlap of Cerberus with AD546 in merged image (C) and large number of purple stained nuclei in Cerberus-expressing tissue (D, E, green arrows)). Neighboring cells that did not express the injected mRNAs often expressed Cer, as can be seen in panel E by the presence of nuclei visualized by DAPI but without β-galactosidase lineage label stain (blue arrows). In contrast, cells in the deep endoderm (D, E, red arrows) and cells in the pigmented epithelium (F) never expressed Cer despite expressing the injected β-galactosidase lineage label. Injection of caAlk4 also induced expression of Cerberus (G–I); however, induction was almost entirely cell non-autonomous [note lack of overlap between injected cells (red) and Cer-expressing cells (blue)], suggestive that chronic misactivation of the Nodal pathway is incompatible with Cer expression. Scale bars in panels B–F represents 20 μM and in panels G–I 100 μM. Image published in: Foley AC et al. (2007) Copyright © 2007. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |