Fig. S1

c-myb mutant hematopoietic cells are intrinsically defective. (A) Fluorescence microscopic representation of wild type and c-myb mutant embryos transgenic for gata1:DsRed and ikaros:eGFP reporters at 1 dpf; the caudal hematopoietic tissue is shown. Note the reduced number of fluorescent cells in the intermediate cell mass (ICM; indicated by white oval) of mutant embryos. (B) PCR assay for the detection of transgenes in wild type (wt) or c-myb mutant (c-myb^-/-) fish; nTg, non-transgenic fish; dTg, double-transgenic for gata1:DsRed and ikaros:eGFP reporters. (C) Fluorescence microscopic appearance of the thymic region (T) of wild type (wt), cmyb mutant (c-myb^-/-) fish and c-myb mutant fish transplanted with wild type zebrafish whole kidney marrow cells 4 weeks after transplantation (zf^wt > c-myb^-/-), all fish additionally double-transgenic for gata1:DsRed and ikaros:eGFP reporters (dTg). The bright red-fluorescent spots are caused by non-cellular background. (D) Reconstitution of hematopoiesis after transplantation of zebrafish wild type cells into c-myb mutants. Histological sections of thymi (T) of wild type and reconstituted animals are shown; G; gills. The inset is a 4x higher magnification indicating the dense repopulation with small lymphocytes. Abbreviations are as in (A) and (B). (E-G) Flow cytometric profiles of zebrafish of the indicated genotypes additionally transgenic for gata1:DsRed and ikaros:eGFP reporters, recorded using an AriaFusion instrument. Light scatter profiles are shown in the left-most columns; fluorescence profiles are indicated for the four major cell populations (red, erythrocytes, second columns; blue, lymphocytes, third columns; green, myelomonocytic cells, fourth columns; magenta, progenitor cells, fifth columns). (E) Flow cytometric profiles of wild type zebrafish (data representative of n=4). (F) Flow cytometric profiles of c-myb mutant zebrafish (data representative of n=3). (G) Flow cytometric profiles of c-myb mutant zebrafish reconstituted with non-transgenic wild type cells (data representative of n=6). Scale bars, 50 µm.