Volume 8, Issue 1, Pages (January 2005)

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Volume 8, Issue 1, Pages 97-108 (January 2005) Interplay of Pu.1 and Gata1 Determines Myelo-Erythroid Progenitor Cell Fate in Zebrafish Jennifer Rhodes, Andreas Hagen, Karl Hsu, Min Deng, Ting Xi Liu, A.Thomas Look, John P. Kanki Developmental Cell Volume 8, Issue 1, Pages 97-108 (January 2005) DOI: 10.1016/j.devcel.2004.11.014

Figure 1 Movements of pu.1-Expressing Cells (A–D) ISH assays for pu.1 (p, arrow), krox20 (k, rhombomeres 3,5), and myoD (m, somites) expression. Dorsal views of flattened embryos, anterior to left, at (A) 7 somites, (B) 14 somites, (C) 15 somites, and (D) 18 somites. (E–G) Upper panels, dorsal views, anterior to left: live pu.1-GFP transgenic embryo is shown at (E) 13 hpf, (F) 15 hpf, and (G) 20 hpf. Lower panels: corresponding anterior views. (H) Confocal image of GFP+ cells (green) and pu.1 mRNA (red) in a 20 hpf transgenic embryo. Arrow indicates a yellow coexpressing cell. Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)

Figure 2 Pu.1 Is Required for Myelopoiesis (A, B, E, F, I, J) Control or (C, D, G, H, K, L) Pu.1 morphants at 28 hpf. Double ISH assays for (A–D) pu.1 and nkx2.5 (arrowheads), (E–H) mpo and α-globin, (I–L) l-plastin and α-globin (blue and red, as indicated). Left panels: lateral views; right panels: dorsal views; anterior left. Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)

Figure 3 Pu.1 Suppresses Erythroid Development (A, B, E, F, I, and K) Control morphants or (C, D, G, H, J, and L) Pu.1 morphants at (A–D) 21 hpf or (E–H, K, L) 24 hpf. Expression of (A–D) gata1 (blue), (E–H) l-plastin (blue), and (I–L) α-globin (red). Left panels, lateral views; right panels, dorsal views; anterior left. Confocal images of 28 hpf pu.1-GFP transgenic embryos injected with (I) control or (J) Pu.1 morpholinos showing GFP (green) and α-globin (red) expression. Arrowheads indicate coexpressing cells. spadetail mutants injected with (K) control or (L) Pu.1 morpholinos showing gata1 (blue) and l-plastin (red). Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)

Figure 4 Gata1 Suppresses Myelopoiesis (A–C, G, H, K, and L) Control morphants and (D–F, I, J, M, and N) Gata1 morphants were analyzed by double ISH assays for (A–F) pu.1 and gata1, (G–J) mpo and α-globin, and (K–N) l-plastin and α-globin (blue and red, as indicated). Embryos at (A–F) 20 hpf or (G–N) 32 hpf. Left panels, lateral views; middle panels, dorsal views; right panels, magnified lateral views of the posterior ICM; anterior left. Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)

Figure 5 Early Hematopoietic Gene Regulation by Pu.1 and Gata1 Embryos at (A–F) 22 hpf or (G–L) 20 hpf are oriented anterior left in (A–I) dorsal and (J–L) lateral views. Control (left), Pu.1 (middle), and Gata1 (right) morphants were analyzed for the expression of (A–C) scl, (D–F) lmo2, and (G–L) runx1. Arrows indicate ectopic gene expression and arrowheads indicate neural expression. Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)

Figure 6 Cytological Analysis and Transplantation Assays (A–D) Cytospins of sorted GFP+ cells from pu.1-GFP transgenic embryos at (A) 12 hpf and (B) 22 hpf and from gata1-GFP transgenic embryos at (C) 14 hpf and (D) 22 hpf. A promyelocyte (arrowhead) and characteristic myeloid nuclear indentation (arrow) are indicated. (E) Cells from pu.1-GFP embryos injected with the Pu.1 morpholino. (F–H) Magnified confocal images of transplanted anterior-derived pu.1-GFP cells expressing (F) GFP (green), (G) gata1 (red), and (H) GFP with gata1. Arrowhead indicates a coexpressing cell. Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)

Figure 7 Pu.1 Drives Myelopoiesis in cloche Mutants (A–G) Double ISH assays for (A, C, E, and G) l-plastin or (B, D, and F) mpo with gata1 and α-globin (blue and red as indicated), in (A and B) wild-type siblings or (C–G) cloche mutants at 24 hpf. Lateral views, anterior left. (E and F) pu.1 mRNA-injected cloche embryos. (G) Scl mRNA-injected cloche embryo. (H) Genotyping of representative individual embryos (from [E]) by PCR. Wild-type siblings (wt), cloche mutants (clo), or rescued cloche mutants expressing l-plastin (clo-r). (I) Model of MPC regulation. Region-specific, extracellular signals regulate the expression of pu.1 or gata1 and their effects in MPCs. The Pu.1 and Gata1 proteins reciprocally antagonize expression of each other and positively autoregulate their own. Expression of pu.1 or gata1 activates gene expression, indicative of lineage-specific differentiation. Octagons represent proteins, boxes represent genes. Putative positive (arrows) and negative (oval-heads) regulatory effects are indicated. Developmental Cell 2005 8, 97-108DOI: (10.1016/j.devcel.2004.11.014)