Figure 1.
The morpholino can block the translation of both nb and nbl transcripts.
Live zebrafish embryos at 24 hpf. Mosaic EGFP expression in embryos injected at the 1–2 cell stage with 10 pg/nl nblpEGFP reporter construct (A) or 12.5 pg/nl nbpEGFP (C). No EGFP signal is detectable in embryos co-injected with 40 pg/embryo nblpEGFP and 0.8 pmol/embryo nb/nbl MO (B) or 50 pg/embryo nbpEGFP and 0.8 pmol/embryo nb/nbl MO (D).
Figure 2.
The nb/nbl knockdown affects erythrocytes development.
A–B. The general morphology of 48 hpf nb/nbl morphants is substantially unaffected (B) when compared to control embryos (A). C. Hematopoietic defects in nb/nbl morphants at 48 hpf: ∼60% of the nb/nbl morphants displayed a severe hematopoietic defect (n = 191). 100% of control embryos was unaffected (n = 93). *** p<0.001 vs std MO. D–I. Detailed images of the blood flow in the trunk-tail region of 48 hpf Tg(gata1:dsRed) embryos injected with std MO (0.8 pmol/embryo; D, E) and nb/nbl MO (0.8 pmol/embryo; G, H). In nb/nbl morphants no circulating red cells are detectable within the trunk vasculature. Whole embryo o-dianisidine staining on 48 hpf std MO embryos (F) and nb/nbl morphants (I). L–M′. Longitudinal semithin plastic sections of std MO (L, L′) and nb/nbl MO-injected embryos (M, M′) at 2 dpf. Fewer elements are detectable within the Dorsal Aorta (DA) and the Caudal Vein (CV) in nb/nbl morphants when compared to control embryos. L′, M′: higher magnification of L, M.
Figure 3.
Expression of hematopoietic genes in nb/nbl morphants.
A–D. Flat mounted 10-ss embryos. Triple WISH were performed on std MO (A, C) and nb/nbl MO-injected embryos (B, D). The expression of myoD in somitic and pre-somitic mesoderm appeared unaltered in nb/nbl MO embryos (B, D). The expression of krox20 in the rombomeres 3 and 5 is unaffected in nb/nbl morphants (B, D). A–B. The expression of scl appears reduced in the posterior PLM (black arrowheads). C–D. gata1 expression is reduced in nb/nbl morphants, particularly in the posterior PLM (black arrowheads). E–N. Lateral view of 22–24 hpf std MO embryos (E, G, I, M) and nb/nbl morphants (F, H, L, N). In nb/nbl morphants at 22 hpf gata1 expression is reduced in the ICM (F); ikaros and βe1 globin are also downregulated at 24 hpf (H, L). The myeloid marker pu.1 is expressed at normal levels in nb/nbl MO-injected embryos (N).
Figure 4.
dsRed+ erythroid cells are dramatically reduced at 28–30 hpf.
A–H. Tg(gata1:dsRed) embryos injected with std MO (A, B, E, F) and nb/nbl MO (C, D, G, H) were examined by confocal microscopy between 24–30 hpf. Fluorescent images (B, D, F, H) were merged with bright field images (A, C, E, G). In nb/nbl morphants at 24–26 hpf red fluorescent erythroid cells are present within the ICM (C, D) but at 28–30 hpf the overall fluorescence of nb/nbl morphants appears strongly reduced (G, H). I–N. Whole-mount double immunofluorescence on Tg(gata1:dsRed) nb/nbl morphants at 28–30 hpf to detect caspase-3 activation (green) and DsRed (Red). Single optical section of nb/nbl morphants obtained by confocal microscopy (20× magnification, I). I. White spots, indicating double positive cells, have been pseudocoloured according to the region of interest (ROI1 in O). The sub-image area, shown in detail in panels L–N, is highlighted by the white box. L–N. Insets of single channel fluorescent images (L, M) and merge (N) are shown. Activated caspase-3 overlaps with some dsRed+ cells (white arrowheads, L). O. Fluorogram shows the degree of colocalization between red signals (dsRed) and green signals (activated caspase-3); colocalization is indicated by the region of interest (ROI1).
Figure 5.
Erythroblasts fail to differentiate into erythrocytes in apparently unaffected nb/nbl morphants.
A–B. Bright-field microscopy of blood cells in the caudal arteries of living 48 hpf std MO embryos (A) and nb/nbl morphants (B). nb/nbl MO-injected embryos with no apparent hematopoietic defects show the presence of abnormal-shaped blood cells (white arrowheads) in the blood flow (B). C–D, G–H. Wright-Giemsa staining of circulating embryonic red blood cells from controls and nb/nbl morphants at 52 hpf (C, D) and 3 dpf (G, H). Erythroid cells of apparently unaffected nb/nbl MO injected embryos were larger, showed a large nucleus and had more basophilic cytoplasm indicating the presence of maturation defects (D, H). Asterisks indicate representative erythroid cells with maturation defects. E–F. WISH using gata1 on 48 hpf std MO (E) and nb/nbl MO-injected embryos (F). gata1 expression persists in red blood cells of nb/nbl morphants revealing the presence of immature red cells (black arrowhead).
Figure 6.
nb and nbl mRNAs rescue the hematopoietic phenotypes of nb/nbl morphants.
A–B. Percentages of Tg(gata1:dsRed) with hematopoietic defects obtained in rescue experiments (A) and corresponding percentages of embryos which rescue the hematopoietic phenotype (B). Injection of nb/nbl MO (0.8 pmol/embryo) produced ∼68% of Tg(gata1:dsRed) embryos with hematopoietic defects at 48 hpf (n = 142). Co-injection of 1 ng/embryo of numb-EGFP mRNA and 0.8 pmol/embryo nb/nbl MO (n = 114) produced ∼35% of embryos with hematopoietic defects (A) that correspond to ∼49% of rescue (B). ∼49% of embryos co-injected with 170 pg/embryo of numblike mRNA and 0.8 pmol/e nb/nbl MO (n = 105) showed hematopoietic defects (A) corresponding to ∼28% of rescue (B). * p<0.05 vs nb/nbl MO no defects, **p<0.01 vs nb/nbl MO no defects, #p<0.05 vs nb/nbl MO defects, ##p<0.01 vs nb/nbl MO defects.