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Figure 5
Misregulation of CaM KIV activity in Xenopus embryos inhibits erythropoiesis. (A) RNAs encoding constitutively active CaM KK and CaM KIV (KKc/KIVc), DnCaM KIV (DnCaM KIV), or β-galactosidase (Control) were injected near the VMZ or DMZ of four-cell embryos, as illustrated in the schematic diagram. Western blots of extracts from injected and uninjected embryos were probed with an antibody directed against CaM KIV.
(B) Ventral views of tadpole stage 46 embryos are shown in the top row; arrows indicate the position of the heart. The bottom two rows show ventral views of tailbud stage 32 embryos that have been stained for globin RNA (purple) by in situ hybridization.
(C) Northern blot analysis of globin expression in tailbud stage 29 and tadpole stage 39 embryos that had been injected at the four-cell stage with RNAs encoding β-galactosidase (Control) or mutant CaM kinases, as indicated above each lane. Bands on the gel were visualized with a phosphorimager and quantitated using the MacIntosh IP lab gel program. Levels of globin transcripts, expressed as percent of control levels, are indicated below each lane. Ethidium bromide staining of the RNA gel before transfer is shown to demonstrate that relatively equivalent amounts of RNA are present in each lane. |
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Figure 6
CaM KIV plays a non-cell–autonomous role in regulating hematopoiesis. (A) Two dorsal midline animal pole blastomeres of 32-cell embryos were injected with β-galactosidase (β-gal) RNA alone or with mutant CaM kinase RNAs, as illustrated. Embryos were cultured to tailbud stage 31 and stained for β-galactosidase activity (red stain, white arrows) and then for β-globin RNA (purple stain, black arrows) by in situ hybridization. Lateral (top row) and ventral (bottom row) views of each embryo are shown. (B) Northern blot analysis of globin expression in whole tailbud stage 31 embryos in which CaM KIV activity was misregulated in the progeny of two dorsal midline (A1) or ventral midline (A4) animal pole blastomeres of 32-cell embryos. Relative levels of globin transcripts were quantitated as described in the legend to Fig. 5 and are expressed as percent of control below each lane. Ethidium bromide staining of the RNA gel before transfer is shown as a loading control. |
