Fig. 2

Fig. 2 In D. rerio, expression of a putative dominant negative Irf6 causes severe gastrulation defects and late defects in the pectoral fin and skin. (A) RT-PCR analysis of efficacy of splice blocker MO targeting D. rerio irf6. RNA was harvested from 22 hpf embryos that were either uninjected or injected at the single cell stage with 5 ng of irf6 exon 2/intron 2 MO. First strand cDNA was synthesized and PCR carried out with forward primer in exon 1 and reverse primer in exon 3, yielding, from uninjected embryos (un), a single band of expected size, and from MO-injected embryo a second, smaller band (arrowhead). The MO-dependent PCR product was excised and sequenced and found to reflect loss of the first coding exon, which encodes the first 60 amino acids, including half of the predicted DNA binding domain. In e2i2 MO injected embryos, a substantial fraction of irf6 mRNA, possibly corresponding to maternal stores, is spliced correctly (asterisk). - RT, negative control lacking reverse transcriptase (B) Western blot analysis of efficacy of MO targeting translation of Irf6. All lanes show lysates from 16 hpf embryos injected at the two-cell stage with in vitro transcribed irf6 RNA altered to include most of the 5′ UTR and to encode full length Irf6 with a carboxy-terminal Myc tag. -, embryos not injected with MO. AUG, RNA-injected embryos subsequently with 5 ng of irf6 AUG MO; 5′UTR, RNA-injected embryos subsequently with 5 ng of irf6 5′UTRb MO. In a similar Western blot experiment, embryos injected with 20 ng of a 5-base pair mismatch variant of the AUG MO were comparable to embryos not injected with MO (not shown). (C) Schematics of full-length Irf6 and the DNA binding domain variant, which contains the amino terminal 115 amino acids. IAD, Interferon Association Domain (Taniguchi et al., 2001). (D) Frames from a time-lapse video of a water-injected control, left, and an irf6DBD-injected embryo, right, taken at the indicated times. In the latter, thinning of the DEL is delayed (most obvious at the animal pole), as is vegetal migration of the DEL (white line, extent of DEL migration). Finally, when siblings are at about 80% epiboly (9 h), the irf6DBD-injected embryo ruptures near the animal pole. Arrowhead indicates first area of embryo rupture. Staging was analyzed in 10 embryos in three separate experiments with equivalent results. (E) Histogram showing percentage of embryos at 24 hpf of the indicated class that were normal (black), or dead (red), or with head and tail defects (grey). (F, G) Ventral views of 4 dpf embryos stained with alcian-green. (F) Uninjected embryo. (G) irf6DBD-injected embryos showing pharyngeal cartilage elements are present but smaller than normal and pectoral fins (arrowheads) are reduced. (H, I) Dorsal views of live, (H) uninjected embryo and, (I) irf6DBD-injected embryos at 4 dpf. The later shows blistered skin (arrows) and abnormal fins (arrowheads). (J–M) Lateral views of, (J, L) uninjected and, (K, M) irf6DBD-injected embryos at shield stage. (J, K) Embryos processed to reveal expression of zfk8, a marker of the EVL. In the irf6DBD-injected embryo, the vegetal limit of the EVL (arrowhead) is at the position appropriate for dome stage. (L, M) YSL nuclei labeled with sytox green, revealing that in, M) the irf6DBD-injected embryo vegetal migration of the YSL has stalled at dome stage.