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CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Wayman GA, Walters MJ, Kolibaba K, Soderling TR, Christian JL - J. Cell Biol. (2000)

Bottom Line: Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis.These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage.Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

View Article: PubMed Central - PubMed

Affiliation: Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.

ABSTRACT
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

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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.
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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.

Mentions: To look for specific defects in hematopoietic maturation and differentiation, we examined RBC number and morphology in cytospin preparations of peripheral blood samples isolated from stage 45 tadpoles. At this stage of development, all of the circulating blood is derived from the VBI as a result of primitive hematopoiesis (Turpen et al. 1997). In control embryos, ∼80% of circulating cells were RBCs and 20% were white blood cells (WBCs) (Fig. 9A and Fig. B) that are predominantly of the monocyte/macrophage lineage (Ohinata et al. 1990). In embryos where CaM KIV activity was inhibited by injection of RNA encoding DnCaM KIV into either ventral (Fig. 9) or dorsal (data not shown) cells, the total number of circulating blood cells was unchanged, but a greater proportion of these were myeloid, as opposed to erythroid cells (Fig. 9A and Fig. B) such that the ratio of WBCs to RBCs was increased approximately eightfold relative to controls (C). Based on morphology (Fig. 9 A) (Hadji-Azimi et al. 1987) and staining with alpha-naphthyl acetate esterase (data not shown), the majority of WBCs in these embryos, as in controls, were of the monocyte/macrophage lineage. In support of this conclusion, expression of myeloperoxidase, a marker of early embryonic macrophages (Smith, S., and T. Mohun, unpublished data) was increased severalfold in CaM KIV–deficient embryos relative to controls (Fig. 9 D). The increase in the ratio of WBCs to RBCs in embryos injected with DnCaM KIV RNA was specifically caused by a blockade of the CaM KIV branch of the CaM KK cascade, since it was rescued by coinjection of 1 ng of RNA encoding CaM KIVc (Fig. 9 C). These results indicate that embryonic CaM KIV activity is required for hematopoietic stem cell commitment to erythroid, as opposed to myeloid, differentiation.


CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Wayman GA, Walters MJ, Kolibaba K, Soderling TR, Christian JL - J. Cell Biol. (2000)

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.
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Related In: Results  -  Collection

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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.
Mentions: To look for specific defects in hematopoietic maturation and differentiation, we examined RBC number and morphology in cytospin preparations of peripheral blood samples isolated from stage 45 tadpoles. At this stage of development, all of the circulating blood is derived from the VBI as a result of primitive hematopoiesis (Turpen et al. 1997). In control embryos, ∼80% of circulating cells were RBCs and 20% were white blood cells (WBCs) (Fig. 9A and Fig. B) that are predominantly of the monocyte/macrophage lineage (Ohinata et al. 1990). In embryos where CaM KIV activity was inhibited by injection of RNA encoding DnCaM KIV into either ventral (Fig. 9) or dorsal (data not shown) cells, the total number of circulating blood cells was unchanged, but a greater proportion of these were myeloid, as opposed to erythroid cells (Fig. 9A and Fig. B) such that the ratio of WBCs to RBCs was increased approximately eightfold relative to controls (C). Based on morphology (Fig. 9 A) (Hadji-Azimi et al. 1987) and staining with alpha-naphthyl acetate esterase (data not shown), the majority of WBCs in these embryos, as in controls, were of the monocyte/macrophage lineage. In support of this conclusion, expression of myeloperoxidase, a marker of early embryonic macrophages (Smith, S., and T. Mohun, unpublished data) was increased severalfold in CaM KIV–deficient embryos relative to controls (Fig. 9 D). The increase in the ratio of WBCs to RBCs in embryos injected with DnCaM KIV RNA was specifically caused by a blockade of the CaM KIV branch of the CaM KK cascade, since it was rescued by coinjection of 1 ng of RNA encoding CaM KIVc (Fig. 9 C). These results indicate that embryonic CaM KIV activity is required for hematopoietic stem cell commitment to erythroid, as opposed to myeloid, differentiation.

Bottom Line: Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis.These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage.Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

View Article: PubMed Central - PubMed

Affiliation: Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.

ABSTRACT
Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

Show MeSH
Related in: MedlinePlus