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Fig. 1
The islet2a TALEN mutant.
(A) The sequence of homozygous islet2a105 genomic DNA revealed a 13-nuc deletion (at position *) that removed a restriction enzyme site and introduced a frameshift. (B) Schematic of wildtype and mutant Islet2a proteins. The islet2a105 sequence predicts a truncated Islet2a protein product lacking the homeobox (HD) and LIM2 (L2) domains and containing only a portion (~70%) of the LIM1 (L1) domain. The Isl11/2 monoclonal antibody used in this study was raised against a carboxy-terminus sequence not present in the predicted mutant protein. (C-F) Expression of islet2a mRNA (C, D) and Isl1/2 immunoreactivity (E, F) in wildtype (wt) and homozygous mutant (mut) islet2a 28 hpf embryos. Images are lateral views of embryos, with dorsal up and anterior to the left. Bright field and fluorescent images have been merged. (C, D) A tissue that expresses islet2a, but not islet1 or islet 2b, was used to assess the efficacy of the TALEN mutation. The proctodeum (P), just caudal to the posterior end of the yolk sac extension (yse) emerges during embryonic stages and develops later into the anal passage. In both wildtype (n = 6) and mutant (n = 8) embryos, the proctodeum expressed islet2a mRNA. (E, F) The Isl1/2 antibody immunolabeled the proctodeum in wildtype (E; n = 10) but not mutant (F; n = 7) 28 hpf embryos. Scale bar in F, for C-F: 25 μm.