Fig. 3

Generation of lnx2a^Δ70 mutant using TALENs, and the functional redundancy of lnx2 genes. (A) Schematic representation of the lnx2a locus and the TALEN target site in the first protein coding exon (exon 2). TALEN targets (left, orange and right, green), and the lnx2a^Δ70 mutation are shown below the drawing. Next, the protein sequences of WT and the lnx2a^Δ70 frameshift allele are shown. Genotype of lnx2a^Δ70 mutant was analyzed by the genomic PCR (B), and immunoblotting of endogenous Lnx2a protein (C). For validation of the anti-Lnx2a antibody, see SI Appendix, Fig. S2F. (D) Marker gene (try and glu) expression shows little effect in lnx2a^Δ70 null mutants, and in lnx2b-MO–injected embryos at 60 hpf. However, lnx2b-MO injection into lnx2a^Δ70 mutant embryos shows suppression of exocrine markers. (E) Quantification of pancreatic defects by analysis of trypsin expression, classified as in SI Appendix, Fig. S3 D and E for effectiveness of lnx2b-MO. (F–H) lnx2a-MO leads to production of N-truncated Lnx2a protein. (F) RT-PCR using primers F1 and R1 (defined in Fig. 2A) and 48 hpf embryo RNA, followed by sequencing, shows that lnx2a-MO injection results in exon 2 skipping and stabilization of the resulting transcript. lnx2b expression was not changed by lnx2a-MO injection. (G) Endogenous Lnx2a showed the expected size in controls, but a smaller protein was seen in lnx2a-MO–injected embryos, which was identified by mass spectrometry (SI Appendix, Fig. S4) as an N-truncated protein (587 aa) arising from an alternate start site in exon 3. (H) Schematic drawing of exon 2 skipping, alternate translation start site, and N-truncated protein in lnx2a-MO–injected embryos. (I) N-truncated Lnx2a (exon 2-skipped, as shown in F–H) has interfering effect. Tr-mRNA was injected into the WT and lnx2a^Δ70 mutant embryos and analyzed at 48 hpf. (J) Quantification of pancreatic defects in I by analysis of ptf1a expression. Injection of Tr-mRNA into WT embryos has little effect, but leads to the exocrine pancreas phenotype in lnx2a^Δ70 mutants. (Scale bars: D, 100 µm; I, 50 µm.)