Discovery of Genes Critical to Cone Differentiation Using Zebrafish Forward Genetics

Abstract

Purpose: : To discover gene pathways critical to cone photoreceptor differentiation. Zebrafish possess four cone photoreceptor subclasses that are highly conserved in function and development compared to humans. Cone photoreceptor subclasses in zebrafish are arranged in a stereotyped spatial pattern referred to as a ‘cone mosaic’. This feature readily allows detection of aberrant cone differentiation wherein the pattern of the cone mosaic is disrupted. The precision of the zebrafish cone mosaic led us to consider hypotheses that cell adhesion and/or lateral induction of cell fates occur during differentiation of cone subclasses.

Methods: : A forward genetic screen was employed using standard methods. Fish were treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU), which creates random point mutations. We identified homozygous mutants with abnormal cone photoreceptor morphology and a disrupted cone mosaic pattern but no other observable phenotypes in the retina or body morphology. Genetic lesions were identified by linkage analysis followed by a candidate gene approach. Phenotypes were characterized by cell differentiation markers, cell death assays, and multiplex in situ hybridization for cone opsins to define cone ratios. Cellular autonomy of the mutation was assayed in genetically mosaic fish created via blastomere transplantation.

Results: : Linkage analysis and a candidate gene approach have implicated a gene on chromosome 20 as the genetic locus of the mutation in mi215. Another mutant line, mi40, failed to complement mi215 and may be allelic. The phenotype includes disrupted cone differentiation/positioning, an intact inner retina, and only subtle disruptions in ratios of cone subtypes or changes in rate of cell death. The mutation appears to act in a cell-autonomous manner. The candidate gene is known to interact with cell adhesion machinery and the Notch pathway (relevant to lateral induction of cell fates).

Conclusions: : We have isolated mutant lines in which a gene critical to cone photoreceptor development is disrupted. The candidate gene was previously unknown to have any such role. Its known function from other systems is consistent with our hypotheses that cell adhesion and lateral cell fate induction are critical to differentiation and patterning of cone subclasses. Future directions include investigating other members of the cognate cell signaling pathways, characterizing other mutant lines we have isolated, and sequencing of the identified genes in patient panels with cone dystrophies.

Keywords: genetics • photoreceptors • cell adhesions/cell junctions