Gdf6a, Encoded by the Zebrafish Out of Sight Locus, is a Key Regulator of Eye Size

Abstract

Purpose: : A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the zebrafish out mutant, and to determine whether mutations in the out gene cause microphthalmia in humans.

Methods: : The out^m233 mutant strain was originally recovered in a large-scale ENU mutagenesis screen. A bulk segregant mapping analysis was performed by the Mutant Mapping Facility at the University of Louisville. Positional candidate genes were selected from the zebrafish genome sequence and analyzed by direct sequencing. Apoptotic cell death was evaluated by acridine orange staining and TUNEL. The presence of several retinal cell types was analyzed with immunohistochemical markers. The human GDF6 gene was screened in 200 patients with coloboma, microphthalmia or anophthalmia by direct sequencing.

Results: : In this study we show that the severe reduction of eye size in the out mutant is caused by a mutation in the zebrafish gdf6a gene. The overall retinal architecture of out mutants appears largely intact, and immunohistochemical studies confirm that all major cell types are present in out retinae. The reduced eye size in out mutants is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. In a recent study a segmental deletion encompassing the GDF6 gene was identified in a coloboma patient. Our study confirms that loss of GDF6 is likely the cause of the ocular phenotype in this patient. We also show, however, that mutations in GDF6 are a rare cause of ocular malformations, since no pathogenic variants were detected in 200 patients with microphthalmia, anophthalmia or coloboma.

Conclusions: : GDF6a is a key regulator of vertebrate eye size but not of the formation of retinal architecture. The patterning of retinal neurons does not require a precise regulation of eye size growth.

Keywords: development • positional cloning