Humans are largely dependent upon cone-mediated vision. However, death or dysfunction of rods, the predominant photoreceptor subtype, results in secondary loss of cones, remodeling of retinal circuitry and blindness. The changes in circuitry may contribute to the vision deficit and undermine attempts at restoring sight. We exploit zebrafish larvae as a genetic model to specifically characterize changes associated with photoreceptor degenerations in a cone-dominated retina. Photoreceptors form synapses with two types of second order neurons, bipolar cells and horizontal cells. Using cell-specific reporter gene expression and immunolabeling for postsynaptic glutamate receptors, significant remodeling is observed following cone degeneration in the pde6cw59 larval retina but not rod degeneration in the Xops:mCFPq13 line. In adults, rods and cones are present in approximately equal numbers, and in pde6cw59 mutants glutamate receptor expression and synaptic structures in the outer plexiform layer are preserved, and visual responses are gained in these once-blind fish. We propose that the abundance of rods in the adult protects the retina from cone degeneration-induced remodeling. We test this hypothesis by genetically manipulating the number of rods in larvae. We show that an increased number and uniform distribution of rods in lor/tbx2bp22bbtl or six7 morpholino-injected larvae protect from pde6cw59-induced secondary changes. The observations that remodeling is a common consequence of photoreceptor death across species, and that in zebrafish a small number of surviving photoreceptors afford protection from degeneration-induced changes provides a model for systematic analysis of factors that slow or even prevent the secondary deteriorations associated with neural degenerative disease.
The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species.
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