Fig. 2

Characterization of the tjp3/zo-3 morpholino phenotype. Zebrafish embryos were injected with a control morpholino (Cmo), a splice site (MO) or a translation start morpholino (ATGmo) against tjp3/zo-3. (A) Both tjp3/zo-3 splice and translation start morpholino injected embryos present the same phenotypes. At 1 dpf embryos had shorter and curved tails with an expanded blood island (b, arrowhead). Some individuals showed an overall reduction in body length and the beginning of pericardial edema (c, arrow). By 2 dpf, pericardial edema was pronounced (e, f, arrows) and many individuals had no blood circulation. Morphants had curved tails and the size of the tailfin was reduced (e, f, arrowheads). (B) tjp3/zo-3 splice morpholino inhibits tjp3/zo-3 mRNA splicing. RT-PCR analysis was performed on embryos injected either with control morpholino (Cmo), or tjp3/zo-3 splice morpholino (MO) at 1 dpf. In control morpholino injected embryos, only one product of 97 bp corresponding to the properly spliced exon1/2 sequence was amplified (lane 3). In embryos injected with tjp3/zo-3 splice morpholino and selected for normal phenotypes, a 210 bp fragment containing the first intron sequence was amplified in addition to the 97 bp WT fragment (lane 2). Abnormal phenotypes were characterized by the absence of the 97 bp WT fragment and the presence of the 210 bp exon1/intron1/exon2 fragment (lane 1). (C) The tjp3/zo-3 splice and translation blocking morpholino reduce Tjp3/Zo-3 protein levels. Protein from whole 6 hpf (lanes 1-3) or 2 dpf (lanes 4-8) embryos was extracted and blotted with anti-zebrafish Tjp3/Zo-3 antibody. At 6 hpf, low levels of Tjp3/Zo-3 protein were detected in embryos injected with a control morpholino (Cmo, lane 1). No Tjp3/Zo-3 protein could be detected in embryos injected with tjp3/zo-3 splice morpholino (MO, lane 2) or tjp3/zo-3 translation blocking morpholino (ATGmo, lane 3). At 2 dpf both splice morpholino and ATG morpholino injected embryos were selected for morphant phenotypes. These individuals showed a strong reduction of Tjp3/Zo-3 protein levels (lanes 5 and 6) while splice morpholino injected embryos without phenotype had normal levels of Tjp3/Zo-3 protein (lane 7). WT embryos injected with Tjp3/Zo-3 expression vector served as a positive control (lane 8).