Fig. 3

Suppression of red blood cell formation in zebrafish embryos by antisense MOs targeted to the bty mRNA. (A–B) DIC microscopy of zebrafish embryos in vivo 28 hpf (Nikon Eclipse 800). (A) Non-injected, wild-type embryo. The arrow shows circulating erythrocytes in the heart. (B) Wild-type embryo injected at the two cell stage with 10 ng of Zebb302 antisense MO targeted to the bty start codon and 21 residues downstream. Although the heart beat normally, circulating blood cells were absent (arrow). (C–E) Hemoglobin detection by o-dianisidine, whole embryo lateral view (32 hpf). Fixed and stained embryos were micrographed in 70% glycerol/PBS using the Nikon dissecting microscope. (C) Danieau′s injected wild-type embryo. The circulation stained reddish brown when reacted with o-dianisidine, indicating the presence of hemoglobin-expressing red cells. (D) Antisense MO Zebb 302a, 5 ng. Note the nearly complete absence of red blood cells. (E) Control 5-bp mismatch MO Zebb302e, 5 ng. Red blood cells were present at wild-type levels. (F–H) Hemoglobin detection by o-dianisidine, view of venous circulation over yolk to heart. Embryos were micrographed as in panels (C–E). (F) Uninjected wild-type embryo. (G) Antisense MO Zebb302a, 10 ng. (H) Control 4-bp mismatch MO Zebb302c, 10 ng. (I–K) Whole-mount in situ hybridization of MO-treated zebrafish to α-globin antisense RNA (32 hpf). Micrographs were recorded using a Nikon Eclipse 800 in DIC mode. (I) Uninjected embryo. (J) Embryo injected with 10 ng of Zebb302 antisense MO. (K) Embryo injected with 15 ng of control 4-bp mismatch MO Zebb302b. The arrows indicate the position of the heart. Globin-positive red cells were greatly reduced in the experimental MO-injected embryo.