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Calmodulin-dependent protein kinase IV mediated antagonism of BMP signaling regulates lineage and survival of hematopoietic progenitors

Melinda J. Walters^1,, Gary A. Wayman^2,, John C. Notis², Richard H. Goodman², Thomas R. Soderling² and Jan L. Christian^1,*

¹ Department of Cell and Developmental Biology, and
² Vollum Institute, L215, Oregon Health Sciences University, School of Medicine, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USA
These authors contributed equally

View larger version (46K): [in a new window]   Fig. 1. Misregulation of BMP signaling in Xenopus embryos inhibits erythropoiesis. RNAs encoding ß-gal, tBR, caALK3 or BMP4 were injected near the ventral (VMZ) or dorsal marginal zone (DMZ) of 4-cell embryos. (A) Tailbud stage 32 embryos were stained for globin RNA (purple) by in situ hybridization. BMP-injected embryos were subdivided into those with normal dorsal-ventral pattern (DAI 5) and those with a ventralized phenotype (DAI 0). Ventral views of embryos are shown except for the BMP-injected, DAI 0 embryo for which ventral and lateral views are provided. (B) Northern blot analysis of globin expression. Levels of globin transcripts, normalized relative to levels of EF-1 transcripts, are expressed as percentage of control, below each lane.

View larger version (53K): [in a new window]   Fig. 2. BMP signaling in ectodermal cells regulates erythropoiesis. (Top) Two dorsal animal pole (A1) blastomeres of 32-cell embryos were injected with ß-gal RNA alone or together with dominant negative (tBR) or constitutively active (caALK3) BMP receptor RNAs as illustrated. (A) Tailbud stage 32 embryos were stained for ß-gal activity (red stain, white arrows) and then for globin RNA (purple stain, black arrows) by in situ hybridization. (B) Control embryos (ß-gal), as well as embryos in which BMP signaling was misregulated in A4 or A1 blastomeres, were analyzed for expression of globin by northern blot hybridization. Levels of globin transcripts, normalized relative to levels of EF-1 transcripts, are expressed as a percentage of control, below each lane.

View larger version (47K): [in a new window]   Fig. 3. Misregulation of BMP signaling does not perturb specification of hematopoietic fate. Embryos were injected with RNA encoding caALK3 (near the VMZ) or tBR or Smad6 (near the DMZ) at the 4-cell stage. Expression of SCL was analyzed at neurula (St. 18) and tailbud (St. 32) stages by northern blot hybridization. The blot was stripped and reprobed for globin, and then EF-1 transcripts. The difference in SCL levels in tailbud stage embryos injected with Smad6 as opposed to tBR was not reproducible.

View larger version (50K): [in a new window]   Fig. 4. Activation and inhibition of BMP cause distinct hematopoietic defects. (A) Wright-giemsa-stained blood collected from control tadpoles or from tadpoles in which BMP signaling had been misregulated. Black arrows indicate RBCs; white arrows indicate WBCs. (B) The mean number (± s.e.m.) of WBCs (white bars), RBCs (black bars) and total blood cells (shaded bars) present in three random fields of blood from at least 30 control or experimental embryos is shown. (C) Ratio of WBCs to RBCs, calculated from data shown in B. (D) TUNEL analysis of apoptotic cells (arrows). Nuclei were stained with propidium iodide. (E) Percentage apoptotic cells (mean ± s.e.m.) determined by counting total and TUNEL-positive cells in three random fields of blood from at least 24 embryos.

View larger version (35K): [in a new window]   Fig. 5. CaM KIV antagonizes hematopoietic functions of BMPs downstream of receptor activation. (A,C) The mean number (± s.e.m.) of WBCs (white bars), RBCs (black bars) and total blood cells (shaded bars) present in three random fields of cytospin blood preparations from at least 30 control embryos or embryos injected with RNA encoding mutant forms of BMP and/or CaM KIV as indicated: caKIV, constitutively active form of CaM KIV; caKK, its upstream kinase; DnKIV, dominant negative form of CaM KIV. (B,D). The average ratio of WBCs to RBCs (± s.e.m.), calculated from the data in A and C.

View larger version (39K): [in a new window]   Fig. 6. Constitutively active CREB phenocopies hematopoietic defects caused by gain of CaM KIV or loss of BMP function. Embryos were injected with RNAs encoding ß-gal (control), or mutant forms of CREB as indicated. (A) Expression of globin in stage 28 embryos analyzed by whole-mount in situ hybridization. (B) The mean number (± s.e.m.) of WBCs (white bars), RBCs (black bars) and total blood cells (shaded bars) present in three random fields of cytospin blood preparations from at least 30 control or experimental tadpole stage embryos.

View larger version (29K): [in a new window]   Fig. 7. CaM KIV and BMP compete for limiting amounts of CBP. (A,C,E) The mean number (± s.e.m.) of WBCs (white bars), RBCs (black bars) and total blood cells (shaded bars) present in three random fields of cytospin blood preparations from tadpole stage embryos that had been injected with RNA encoding ß-galactosidase (control), mutant forms of CaM KIV, BMP and CREB, or wild type CBP, either alone or in combination, as indicated. (B,D,F) Average ratio of WBCs to RBCs (± s.e.m.), calculated from the data in A, C and E.

View larger version (43K): [in a new window]   Fig. 8. Model for CaM KIV-mediated inhibition of BMP signaling (details in text). Nuclear component of an active BMP signal in which the Smad complex is linked to a hematopoietic-specific transcription factor (purple box) by the coactivator CBP. CaM KIV inhibits interactions between the Smad/CBP complex and other transcriptional components by activating a downstream substrate such as CREB. Endogenous levels of CaM KIV activity partially repress BMP-mediated transcription in ectodermal cells, leading to expression of a secreted protein that promotes erythroid fate and survival. Abnormally high levels of this gene product lead to excessive myeloid fate, abnormally low levels lead to apoptosis of RBCs.