In Vivo Morpholino Screen in Zebrafish Identifies Eight Regulators of Hematopoiesis
(A) MOs were injected into one-cell stage embryos and number of erythrocytes assessed with o-dianisidine staining. For eight genes, injection of MOs resulted in a reduced number of blood cells in circulation at 48 hr postfertilization (hpf). For clarity representative images illustrating three different phenotypes (unaffected [left], mild [middle], and severe [right]) are shown with the number of embryos in each group indicated in the lower, left corner. The number in the upper left corner of the very left image shows the total number of embryos used in each experiment. The predominant phenotype is framed in red. Black arrow indicates hemoglobin staining in the control.
(B) Wild-type zebrafish embryo at 72 hpf. Confocal images presented in (C) were taken of the caudal hematopoietic tissue, indicated by the boxed region.
(C) MOs were injected into the one-cell stage transgenic Tg(cd41:EGFP) zebrafish embryos and assayed for their effect on the number of presumed hematopoietic stem cells (cd41low) and thrombocytes (cd41high; white arrows) at 72 hpf. For march2 (n = 44), max (n = 63), smox (n = 60), pttg1lp (n = 50), emilin1 (n = 65), and sufu (n = 65), a severe decrease of cd41 positive cells was observed. Ncor2 (n = 53) depletion resulted in a mild phenotype, and pdzk1ip1l (n = 49) and nfatc1 (n = 60) MO injected embryos showed no phenotype. See also Tables S4–S7 and Figure S4.
Fig. S4 Representative pictures at 72 hpf of 5 control transgenic Tg(cd41:EGFP) zebrafish embryos or injected with MOs. The GFP positive cells represent the number of presumed hematopoietic stem cells (cd41low) and thrombocytes (cd41high). For march2 (n=44), max (n=63), smox (n=60), pttg1lp (n=50), emilin1 (n=65) and sufu (n=65), a severe decrease of cd41 positive cells was observed. Ncor2 (n=53) depletion resulted in a mild phenotype, and pdzk1ip1l (n=49) and nfatc1 (n=60) MO injected embryos showed no phenotype.
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Reprinted from Developmental Cell, 20(5), Tijssen, M.R., Cvejic, A., Joshi, A., Hannah, R.L., Ferreira, R., Forrai, A., Bellissimo, D.C., Oram, S.H., Smethurst, P.A., Wilson, N.K., Wang, X., Ottersbach, K., Stemple, D.L., Green, A.R., Ouwehand, W.H., and Göttgens, B., Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators, 597-609, Copyright (2011) with permission from Elsevier. Full text @ Dev. Cell