Figure 4.

tab2 Knockdown in Zebrafish

(A) Schematic organization of the tab2 gene in the zebrafish genome. Two alternative splice isoforms as detected by PCR on the reverse-transcribed mRNA are shown. Introns are dotted lines, exons full boxes (coding sequences in black, noncoding sequences in orange). The target sites of the sbMO) and tbMO are indicated by double arrowheads. The lower schema indicates the effect of sbMO injection on the mature tab2 mRNA (isoforms 1 and 2).

(B) Gel electrophoresis of the product of PCR on cDNA extracted from nonmorphant (WT) or 2 ng sbMO-injected zebrafish embryos 24 hpf and a DNA marker for size comparison, showing one band around 2000 bp in the wild-type situation, and two additional fragments in the morphant situation.

(C) Effect of TAB2 sbMO injection on the amount of correctly spliced mRNA.

(D) Wild-type AB embryo at 48 hpf.

(D′) Noninjected embryo at 48 hpf expressing GFP in vascular endothelial cells under the influence of an flk promoter (flk-GFP).

(E) Morphant embryo at 48 hpf. Note the enlarged pericardial sac (arrow).

(E′) Morphant flk-GFP embryo at 48 hpf. Note the thin and elongated heart.

(F) Phenotypical classification of sbMO-injected zebrafish embryos at 12 hpf. Defects in epiboly progression are evident, and progression of the yolk-syncytial layer is schematically displayed below. Phenotypes are ordered from severe to normal. Class I: anterior-posterior gradient is not evident. Class II: the yolk-syncytial layer has not progresses until the vegetal pole; two different embryos are shown to illustrate the continuum in phenotypes in class II. Class III: wild-type. Left, posterior; up, dorsal.

(G) Distribution of phenotypes at 12 hpf dependent upon the sbMO dose injected. Y axis: percentage of embryos in each class as indicated by the color code. X axis: amount of TAB2 sbMO injected at the one-cell stage. At least 42 embryos were successfully injected for each dose. In total, over 230 embryos were successfully injected.

(H) Phenotypical classification of sbMO-injected zebrafish embryos at 24 hpf. Phenotypes are ordered from severe to normal. Class I: death. Other classes are as described in the main text. Lateral images: up, anterior.

(I) Distribution of phenotypes at 24 hpf dependent upon the sbMO dose injected. Y axis: percentage of embryos in this class. X axis: amount of TAB2 sbMO injected at the one-cell stage. At least 42 embryos were successfully injected for each dose. In total, over 230 embryos were successfully injected. Injection of 4 ng of cMO did not increase the frequency of abnormal phenotypes in comparison to noninjected or 0 ng-injected control embryos (not shown).