Deconinck AE et al. (2000), FOG acts as a repressor of red blood cell devel...

XB-ART-11187

Development 2000 May 01;12710:2031-40. doi: 10.1242/dev.127.10.2031.

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FOG acts as a repressor of red blood cell development in Xenopus.

Deconinck AE , Mead PE , Tevosian SG , Crispino JD , Katz SG , Zon LI , Orkin SH .

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Members of the GATA family of zinc-finger transcription factors have critical roles in a variety of cell types. GATA-1, GATA-2 and GATA-3 are required for proliferation and differentiation of several hematopoietic lineages, whereas GATA-4, GATA-5 and GATA-6 activate cardiac and endoderm gene expression programs. Two GATA cofactors have recently been identified. Friend of GATA-1 (FOG-1) interacts with GATA-1 and is expressed principally in hematopoietic lineages, whereas FOG-2 is expressed predominantly in heart and brain. Although gene targeting experiments are consistent with an essential role for FOG-1 as an activator of GATA-1 function, reporter assays in transfected cells indicate that FOG-1 and FOG-2 can act as repressors. We have cloned a Xenopus laevis homologue of FOG that is structurally most similar to FOG-1, but is expressed predominantly in heart and brain, as well as the ventral blood island and adult spleen. Ectopic expression and explant assays demonstrate that FOG proteins can act as repressors in vivo, in part through interaction with the transcriptional co-repressor, C-terminal Binding Protein (CtBP). FOG may regulate the differentiation of red blood cells by modulating expression and activity of GATA-1 and GATA-2. We propose that the FOG proteins participate in the switch from progenitor proliferation to red blood cell maturation and differentiation.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 ctbp2 fgf2 gata2 gata3 gata4 gata6 odc1 smad1 zfpm1 zfpm2

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	Fig. 3. Analysis of xFog expression pattern by RNA in situ hybridization on developmentally staged (albino) embryos. The pattern of xFog expression (A-E) in developing Xenopus embryos is a combination of the xGata gene expression patterns (F-I). (A) At stage 15, expression starts in the presumptive heart and the presumptive ventral blood island, possibly extending up into the presumptive pronephric region, but excluded from the presumptive liver. P, posterior. (B) At stage 23, expression is pronounced in heart primordia, similar to xGata-6 (F). (C) At stage 27, expression is high in heart tube, and in the ventral blood island (indicated by stars, very similar to xGata-2, shown in G). Expression is also seen in midbrain and hindbrain, and in the otic vesicle (indicated by white arrow, similar to xGata-3 shown in H).(D) At stage 33, xFog continues to be highly expressed in the heart and ventral blood island. The faint staining in the region around the midline is either the pronephros, similar to xGata-3 (H) or a blood vessel. Expression is also seen in the eyes and brain (similar to xGata-3 (E,J) At stage 38, expression of xFog is pronounced in heart and surrounding tissue (similar to xGata-4 in I), midbrain and the posterior region of the ventral blood island. (F) xGata-6, stage 23; (G) xGata-2, stage 28; (H) xGata-3, stage 26; (I) xGata-4, stages 38 (bottom) and 40 (top); (J) xFog, stage 38, cleared embryo. (K,L) Transverse section of a stage 29 embryo. xFog is expressed in the mesodermal layer of the ventral blood island (original magnification: (K) ´100, (L) ´400; v, ventral; d, dorsal; se, sensorial ectoderm; en, endodermal yolk mass; m, mesodermal layer.
	zfpm1 (zinc finger protein, multitype 1) gene expression in Xenopus laevis embryos, NF stage 33, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	gata3 (GATA binding protein 3 ) gene expression in Xenopus laevis embryos, NF stage 33, assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	Fig. 5. Ectopic expression of FOG-2 in whole embryos can block hemoglobin production. Fertilized eggs were injected with 500 pg Fog-2 RNA per blastomere at the 2- or 4-cell stage. Injected embryos and age-matched control siblings were stained in o-dianisidine to examine the level of hemoglobin in the ventral blood island.
	Fig. 4. Induction of xFog expression by growth factors. (A) Semiquantitative RT-PCR analysis at stage 24, of xFog expression after growth factor induction in animal cap assays. Key: −, no reverse transcriptase; Ac, Activin; B, BMP-4; Sm, Xmad; Vg, AVg; F+, bFGF; F−, without growth factor; W, whole embryo. BMP- 4, Smad and Avg each induce xFog, whereas xGata-1 is induced by BMP-4 and Smad, only. ODC is the RNA loading control. (B) In whole embryos, BMP-4 signaling increases the area of xFog expression (top panel; ventralized embryos) compared to uninjected sibling controls (bottom panel).
	Fig. 7. Overexpression of FOG-2 and xFOG inhibits erythropoiesis. The interaction with CtBP appears to be, at least in part, responsible for this inhibition. (A,C,E) Anesthetized tadpoles; (B,D,F,G-J) Tadpoles stained with o-dianisidine. Uninjected (A) tadpoles have beating hearts that pump red blood, whereas Fog-2-injected (C) tadpoles have a beating heart (indicated by black arrow) that pumps very pale blood cells. Fog∆CtBP-injected (E) tadpoles have a beating heart that pumps very red blood. Globin-expressing red cells, stained with o-dianisidine, are noticeable along the tail vein and in the heart in uninjected tadpoles (B,G, indicated by arrows), in tadpoles injected with a negative control (3′-5′) xFog RNA (H, white arrows point to staining in major veins), but not in Fog-2 or xFog- injected tadpoles (D,I,J). Tadpoles injected with Fog∆CtBP (F) display normal o- dianisidine staining.
	Fig. 8. In the absence of CtBP binding, FOG-2 stimulates stem cell proliferation, but maturation and differentiation is not occurring as normal. (A,B,D,E,G,H) Tadpole blood, stained with o-dianisidine and cytospun (original magnification A,D,G, 00; others, 00). (C,F,I) May Grwald Giemsa (MGG) staining of tadpole blood smears. Tadpoles injected with Fog-2 have fewer red blood cells (D), which are paler when stained with o-dianisidine, and rounder (E), compared to uninjected (A,B). Tadpoles injected with Fog∆CtBP have a striking increase in number of red blood cells (G), but not all these blood cells are normal in appearance (H, arrowhead) when compared to stage-matched wild-type sibling controls. Red blood cells collected from wild-type tadpoles have compacted nuclei (C), whereas blood cells from tadpoles injected with Fog-2 look developmentally delayed (F). A mixture of normal and developmentally delayed blood cells (arrow and arrowhead, respectively) are found in tadpoles injected with Fog∆CtBP (I).