Wayman GA , Walters MJ , Kolibaba K , Soderling TR , Christian JL .

CA78593 NCI NIH HHS , DK44239 NIDDK NIH HHS , HD01167 NICHD NIH HHS , P01 DK044239 NIDDK NIH HHS

	Figure 1. Sequence alignment of Xenopus and murine CaM KIV. The predicted amino acid sequence of Xenopus CaM KIV (xCaM KIV) is aligned with murine CaM KIV (mCaM KIV). The conserved threonine (T) within the activation loop is circled, the catalytic domain is boxed and shaded, and the autoinhibitory/CaM-binding domain is boxed. These sequence data are available from GenBank/EMBL/DBBJ under accession no. AF316822.
	Figure 2. Temporal and spatial expression of Xenopus CaM KIV during embryogenesis. Equivalent amounts of RNA, which were isolated from (A) ovary or developmentally staged embryos or from (B) various regions dissected from staged embryos, as illustrated or described below, were analyzed for expression of xCaM KIV and ODC (a loading control) by RT-PCR. Dor, dorsal; Ven, ventral; Ecto, ectoderm; Mesend, mesendoderm; Ant, anterior one third; Mid, middle one third; Post, posterior one third.
	Figure 3. Whole-mount in situ analysis of xCaM KIV expression in Xenopus embryos. (A) Animal pole view of a gastrula stage 13 embryo. (B) Dorsal view of a neurula stage 24 embryo. (C) Lateral view of a tailbud stage 34 embryo. (D) Lateral view or transverse sections at the level of the (E) midbrain, (F) hindbrain, and (G) spinal cord of stage 39 tadpoles. Strong staining is noted in portions of the hindbrain (HB), midbrain (MB), Rohon-Beard sensory neurons (R-B), olfactory placode (OP), and cranial nerves (CN) V, VII, and IX.
	Figure 4. Enzymatically active CaM KK is present in Xenopus embryos. Whole Xenopus embryos, at the indicated developmental stages, and dissected dorsal and ventral halves of early gastrulae were homogenized and four embryo equivalents were assayed for induction of Ca2+–CaM-independent activation of recombinant CaM KIV (graph) or were subjected to Western blot analysis (insets). The control lane on the Western blot contains 50 ng of purified rat CaM KKα. Relative CaM KK activity is presented as the mean fold ± SD stimulation of CaM KIV activity as determined in triplicate assays.
	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.
	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.
	Figure 7 Misregulation of CaM KIV activity after the onset of gastrulation does not disrupt erythropoiesis. (A) RNA encoding β-galactosidase (β-gal) was injected either alone or together with mutant CaM kinase RNAs near the animal pole of two-cell embryos. At the early gastrula stage (stage 10), ectoderm was isolated from injected embryos and cocultured with VMZ explants from uninjected sibling embryos until control tailbud stage 32. (B) Left three images show photographs of stage 32 ectoderm/mesoderm recombinants that were stained for β-galactosidase activity (red punctate stain) followed by in situ hybridization to detect globin transcripts (purple stain, black arrows). The rightmost image shows a Northern blot of globin expression in whole-control embryos or in recombinants of ventral mesoderm with ectoderm made to express mutant CaM kinases, as indicated above each lane. The blot was stripped and rehybridized with an EF-1α probe as a loading control. (C, left) Western blots of protein extracted from uninjected (control) embryos and embryos injected with plasmid expression vectors encoding mutant CaM kinases, as indicated. Endogenous CaM KK protein is readily detected throughout development, whereas ectopic CaM KKc protein (lower band) is first detected at stage 10. Endogenous CaM KIV protein is not detectable on the exposures shown. (C, right) RT-PCR analysis of globin expression in uninjected (control) embryos and embryos injected with plasmid expression vectors encoding mutant CaM kinases, as indicated above each lane.
	Figure 7 Misregulation of CaM KIV activity after the onset of gastrulation does not disrupt erythropoiesis. (A) RNA encoding β-galactosidase (β-gal) was injected either alone or together with mutant CaM kinase RNAs near the animal pole of two-cell embryos. At the early gastrula stage (stage 10), ectoderm was isolated from injected embryos and cocultured with VMZ explants from uninjected sibling embryos until control tailbud stage 32. (B) Left three images show photographs of stage 32 ectoderm/mesoderm recombinants that were stained for β-galactosidase activity (red punctate stain) followed by in situ hybridization to detect globin transcripts (purple stain, black arrows). The rightmost image shows a Northern blot of globin expression in whole-control embryos or in recombinants of ventral mesoderm with ectoderm made to express mutant CaM kinases, as indicated above each lane. The blot was stripped and rehybridized with an EF-1α probe as a loading control. (C, left) Western blots of protein extracted from uninjected (control) embryos and embryos injected with plasmid expression vectors encoding mutant CaM kinases, as indicated. Endogenous CaM KK protein is readily detected throughout development, whereas ectopic CaM KKc protein (lower band) is first detected at stage 10. Endogenous CaM KIV protein is not detectable on the exposures shown. (C, right) RT-PCR analysis of globin expression in uninjected (control) embryos and embryos injected with plasmid expression vectors encoding mutant CaM kinases, as indicated above each lane.
	Figure 7 Misregulation of CaM KIV activity after the onset of gastrulation does not disrupt erythropoiesis. (A) RNA encoding β-galactosidase (β-gal) was injected either alone or together with mutant CaM kinase RNAs near the animal pole of two-cell embryos. At the early gastrula stage (stage 10), ectoderm was isolated from injected embryos and cocultured with VMZ explants from uninjected sibling embryos until control tailbud stage 32. (B) Left three images show photographs of stage 32 ectoderm/mesoderm recombinants that were stained for β-galactosidase activity (red punctate stain) followed by in situ hybridization to detect globin transcripts (purple stain, black arrows). The rightmost image shows a Northern blot of globin expression in whole-control embryos or in recombinants of ventral mesoderm with ectoderm made to express mutant CaM kinases, as indicated above each lane. The blot was stripped and rehybridized with an EF-1α probe as a loading control. (C, left) Western blots of protein extracted from uninjected (control) embryos and embryos injected with plasmid expression vectors encoding mutant CaM kinases, as indicated. Endogenous CaM KK protein is readily detected throughout development, whereas ectopic CaM KKc protein (lower band) is first detected at stage 10. Endogenous CaM KIV protein is not detectable on the exposures shown. (C, right) RT-PCR analysis of globin expression in uninjected (control) embryos and embryos injected with plasmid expression vectors encoding mutant CaM kinases, as indicated above each lane.
	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.
	Figure 8 Misregulation of CaM KIV does not perturb the specification of hematopoietic fate. (A) Whole-mount in situ hybridization analysis of Xaml expression in neurula stage 18 embryos that had been injected near the VMZ at the four-cell stage with RNAs encoding β-galactosidase (control) or mutant CaM kinases. Ventral views are shown, anterior is to the right. (B) RT-PCR analysis of GATA-1 expression in neurula stage 18 and tailbud stage 28 embryos that had been injected near the VMZ at the four-cell stage with RNAs encoding β-galactosidase (control) or mutant CaM kinases as indicated above each lane. ODC is a loading control.
	Figure 8 Misregulation of CaM KIV does not perturb the specification of hematopoietic fate. (A) Whole-mount in situ hybridization analysis of Xaml expression in neurula stage 18 embryos that had been injected near the VMZ at the four-cell stage with RNAs encoding β-galactosidase (control) or mutant CaM kinases. Ventral views are shown, anterior is to the right. (B) RT-PCR analysis of GATA-1 expression in neurula stage 18 and tailbud stage 28 embryos that had been injected near the VMZ at the four-cell stage with RNAs encoding β-galactosidase (control) or mutant CaM kinases as indicated above each lane. ODC is a loading control.
	Figure 9. Activation and inhibition of CaM KIV causes distinct defects in hematopoiesis. (A) Low (top row) and high (bottom row) magnification views of Wright-Giemsa–stained cytospin preparations of blood collected from control tadpoles or from tadpoles in which CaM KIV activity had been misregulated in ventral cells. Black arrows, WBCs; white arrows, abnormal RBCs. (B) The mean number (± SEM) of RBCs (black bars), WBCs (open bars), and total blood cells (shaded bars) present in three random fields of cytospin blood preparations from control or experimental embryos are shown. At least 100 embryos from three independent experiments were averaged for each point. (C) The ratio of WBCs to RBCs were calculated from the data shown in B. (D) Northern blot analysis of myeloperoxidase expression in tadpole stage 41 embryos in which CaM KIV activity was misregulated. Ethidium bromide staining of the RNA gel before transfer is shown as a loading control. (E) Apoptotic cells in cytospin preparations of blood collected from control and experimental animals were detected using a TUNEL assay. Nuclei of cells were stained with propidium iodide. Arrows indicate TUNEL-positive cells. (F) The percent of blood cells that are apoptotic in control and experimental animals, as determined by counting the total number of cells and the number of TUNEL-positive cells present in three random fields of cytospin blood preparations from at least 24 embryos. The data presented are the mean ± SEM.
	camk4 (calcium/calmodulin-dependent protein kinase IV) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior right, dorsal up.

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