Shah RR , Koniski A , Shinde M , Blythe SA , Fass DM , Haggarty SJ , Palis J , Klein PS .

1R01DA028301 NIDA NIH HHS , 1R01GM076621 NIGMS NIH HHS , 1R01HL110806-01 NHLBI NIH HHS , P30 CA016520 NCI NIH HHS , R01 DA028301 NIDA NIH HHS , R01 GM076621 NIGMS NIH HHS , R01 HL110806 NHLBI NIH HHS , R01DK09361 NIDDK NIH HHS , R25 CA101871 NCI NIH HHS , R25 CA101871-07 NCI NIH HHS

	Fig. 1. HDAC inhibition blocks primitive erythropoiesis in Xenopus laevis. A: Circulating RBCs visualized in tadpoles (stage 43) by benzidine stain- ing (blue) for hemoglobin. RBCs are also present in pronephros (arrow) and in heart (A ventral view). B: Treatment of gastrulating embryos (stage 102) with the class I HDAC inhibitor VPA blocks blood formation, indicated by benzidine staining (B ventral view). C: Numbers indicate stages during gastrulation at which VPA was administered; in all conditions, VPA was removed at the end of gastrulation (stage 12). Benzidine-positive RBCs are readily visible within the heart and great vessels in controls and when VPA was added at stage 10.5 or later. D,E: Expression of the erythrocyte marker alpha-T1 hemoglobin in the VBI detected by WISH in tailbud embryos (stage 31). In VPA-treated embryos (E), globin expression is dramatically reduced, with residual staining restricted to the aVBI. F: Embryos were treated with control buffer or VPA during the gastrula stage and globin expression in stage-28 embryos was assessed by quantitative RT-PCR.
	Fig. 2. VPA inhibits the development of erythroid progenitors. A,B: The primitive erythroid marker runx1 is expressed in the VBI in early tailbud (stage 26) embryos (A) and is reduced after VPA treatment (B). Runx1 is also expressed in the olfactory placodes (arrows in A and B). C: In control late neurulae (stage 19, anterior view, with dorsal oriented to the top), runx1 is expressed in precursors of the aVBI, which lie in a ventral-anterior domain at this stage. D: VPA markedly reduces runx1 expression within presumptive hematopoietic cells in neurula-stage embryos (same orienta- tion as C). E,F: Dorsal view (anterior to the left). Expression of runx1 in Rohan Beard cells in the same embryos as C and D is not affected by VPA. G: Ventral views, anterior to the right. VPA treatment (H) and (J) reduces expression of erythroid markers scl (G, H0 and gata1 (I, J) in the VBI of tailbud embryos (stage 31). K: runx1 expression in whole embryos is reduced with VPA treatment at late neurula stage (stage 18), as deter- mined by qRT-PCR (data represent the mean of 4 independent experiments, normalized to ODC).
	Fig. 3. Marker analysis with VPA treatment. Embryos were treated with buffer control (A,C,E,G, I, K, M,O,Q,S,U,W, Y, AA) or VPA during stage 102 (B,D,F,H,J,L,N,P,R,T,V,X, Z, BB). Whole mount in situ hybridization performed for the indicated markers shows no change in expression with VPA treatment. myod (A,B), vent2 (C,D), nkx2.5 (G,H), xrx1 (I,J), xbra (K,L), slug (Q,R), tbx20 (S,T), otx2 (U,V), ventral view. xpo (E,F), lim (W,X), krox20 (O,P) lateral view, anterior to the left, hoxb9 (M,N); dorsal view, anterior to the left. Also unaffected by VPA treatment were VBI-derived mye- loid marker mpo (Y, Z) and vascular marker aplnr (AA, BB), lateral view, anterior to the right. myod, vent2, xpo, xbra: late gastrula. nkx2.5, xrx1, slug, hoxb9: neurula stages. lim1, krox20, mpo, aplnr, tbx20, otx2: tailbud stages.
	Fig. 4. HDAC inhibition prevents hematopoietic induction in ectodermal explants downstream of bmp4 and gata1. A: Expression of bmp-4 (A, B), chordin (C,D), and noggin (E,F) in late gastrula stage embryos is unaffected by VPA (B, D, F); orientation: posterior view, dorsal to the top. G: Quantitative RT-PCR of ectodermal explants harvested at tailbud stages (stage 281). G: Injection of bmp4 mRNA (2 ng) induced globin expres- sion and VPA inhibited this effect. H: Injection of gata1 mRNA (200 pg) induced globin expression and VPA treatment inhibited this effect. I: Injec- tion of bmp4 induced expression of runx1 and VPA blocked this effect. J: VPA upregulated endogenous expression of ncam in uninjected explants.
	Fig. 5. VPA inhibits primitive erythroid progenitor emergence in mouse yolk sac explants. A: EryP-CFC (mean 6 SEM) per 1,000 yolk sac explant cells cultured in vitro for 48 hr with 0, 0.4, and 0.8 mM VPA. B: Number of live cells (mean 6 SEM) per yolk sac explant with 0, 0.4, and 0.8 mM VPA.
	Fig. 6. VPA-mediated developmental defects are due to inhibition of class I HDACs. A: runx1 expression in the anterior ventral region of late neu- rula stage control embryos (stage 18). B, C: TSA (B) and VPA (C) suppress runx1 expression. D: The inactive VPA analog VPM does not inhibit runx1 expression. E: bmp4 mRNA (2 ng) induction of globin expression in ectodermal explants is blocked by TSA (bmpSA). F: VPA (class I HDAC inhibitor), CI-994 (selective inhibitor of HDACs 1, 2, and 3), and C60 (IC50 for HDACs 1 and 2>>HDAC3) increase global acetylation of his- to>>H3 at lysine 9/14 (H3K9/14Ac) at 24 hr post-treatment, as demonstrated by Western blotting. Bottom: Total H3 (loading control). G: Lateral view of stage-45 tadpoles treated with vehicle (DMSO) control (G), C60 (H), VPA (I), and CI-994 (J) from late neurula (stage 19) to tailbud (stage 31) stages.
	Fig. 7. Runx1 expression rescues VPA-mediated hematopoietic defects. A: Benzidine staining of hemoglobin (blue) in untreated stage-43 tad- pole. B: Treatment with VPA inhibits RBC development. C: Injection of runx1 mRNA partially recovered erythrocyte development in VPA-treated embryos. D: Percentage of benzidine-positive tadpoles for uninjected (n1⁄420), VPA-treated (n1⁄425), VPA-treated runx1 (n1⁄421), and runx1 expres- sion alone (n1⁄421). This experiment was repeated with 4 separate clutches of embryos and the table shows data from all 4 experiments. Intensity of benzidine stain was reduced in VPA and VPAunx1 groups (as shown in C). E: Expression of globin was detected by WISH in all (23/23) control tadpoles, 0/20 VPA-treated tadpoles, and 7/18 VPA-treated, runx1-expressing tadpoles.

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