Loss of csf1rb function impairs the development of definitive macrophages. (A-H) WISH of the indicated genes in wild-type and csf1rb−/− siblings at different time points. All lateral views except for G and H, shown in dorsal. The purple arrowheads indicate runx1+ HSCs along the dorsal aorta. The green and blue arrowheads show c-myb+ hematopoietic progenitors in the CHT and in the pronephros, respectively. Black arrowheads indicate rag1+ or c-myb+ lymphoid progenitors in the thymus. (I-P) Confocal imaging analysis of definitive myelopoiesis in csf1r mutant embryos and larvae. (I) Scheme of the transgenic lines used to discriminate the primitive and definitive myelopoiesis waves in the CHT. (J,K) Quantification of GFP+ DsRed− primitive (J) and GFP+ DsRed+ definitive macrophages (K) in wild-type (n=4), csf1ra−/− (n=4) and csf1rb−/− (n=4) embryos carrying the kdrl:Cre; ßactin:Switch-DsRed; mpeg1:EGFP triple transgene, at the indicated developmental stages. Each point represents the mean±s.e.m. of four individuals. (L-O) Confocal imaging (L-N) and quantification, determined as percentage of the whole GFP+ microglia population (O) of GFP+ DsRed+ definitive macrophages (yellow arrowheads) in the CHT of 6 dpf wild-type and csf1r mutant larvae. Each dot plot represents an independent larva. Significances between groups were calculated using the Kruskal–Wallis test with post hoc Dunn's multiple comparisons [H=8.8 (J); H=7.44 (K); H=7.73 (O)]. *P<0.05. MFs, mononuclear phagocytes. Scale bars: 200 µm (A,B); 150 µm (C,D,G,H); 100 µm (E,F); 50 µm (L-N).
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