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Mailin Sotolongo-Lopez, Karen Alvarez-Delfin, James M Fadool; TALEN-mediated mutagenesis establishes a role for six7 in photoreceptor patterning in zebrafish. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1688.
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© ARVO (1962-2015); The Authors (2016-present)
Rod and cone photoreceptors are spatially patterned in most vertebrate species including humans. Previously in a genetic screen, we isolated a locus, lots-of-rods- junior (ljr p23ahub), that regulates rod patterning in the cone-dominated zebrafish retina. Linkage analysis identified six7, a member of sine oculis gene family, as the likely candidate gene. The purpose of this study was to generate targeted mutations of six7 using transcription activator-like effector nucleases (TALENs) to test its role during photoreceptor development.
TALENs were engineered to target the SIX domain of six7. mRNA encoding each TALEN were injected into one-cell stage wild-type embryos. The first exon of six7 was amplified from genomic DNA isolated from TALEN-injected embryos, tail clips of F1 adults or individual F2 larvae, followed by restriction fragment length polymorphism (RFLP) analysis using HaeIII or DNA sequencing. F2 larvae were screened for a rod phenotype by immunolabeling. Complementation testing was performed between ljr p23ahub adults and six7-TALEN carriers.
Injection of mRNAs encoding the TALEN pair into wild-type embryos resulted in the loss of an HaeIII recognition site in the spacer region providing an RFLP to identify molecular lesions in the targeted region. Backcrossing founder animals to wild-type fish revealed that 52% of the founders transmitted one or more novel alleles of six7 to the F1 progeny. Molecular analysis of TALEN-mediated alterations showed insertions and deletions of 4-20 nucleotides that induced frameshift as well as in-frame mutations. Twenty-five percent of the F2 larvae from inter-crosses between F1 carriers of six7-TALEN alleles phenocopied the increase and uniform distribution of rods observed in ljr p23ahub. A correlation between phenotype and genotype was confirmed by RFLP. Complementation testing between carriers of the six7-TALEN alleles and homozygous ljr p23ahub adults resulted in approximately 50% of the progeny displaying the increased rod phenotype providing genetic evidence that ljr p23ahub is indeed an allele of six7.
Targeted mutation of six7 established an unequivocal role for six7 in regulating photoreceptor cell patterning. Future investigations are designed to identify the molecular pathways interacting with six7 in the regulation of rod numbers and patterning.
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