The perplexed mutation is essential for retinal histogenesis and is located on chromosome 20.

Abstract

Abstract: : Purpose:To identify and study genes essential for retinal development in zebrafish. The perplexed mutation affects the transition of proliferating neuroepithelial to postmitotic retinal cells, causing the cells to die as they withdraw from the cell cycle and ultimately preventing proper differentiation and lamination of the retina (Link et al., 2001). Mosaic analysis indicates that perplexed functions in a non cell–autonomous manner, while tissue transplantation experiments suggest that the retinal defect caused by the mutation is intrinsic to the retina. Methods:The recessive perplexed mutation (plx^a52) was identified in screens for retinal lamination defects using F3–generation embryos from ENU mutagenized founders. To produce mapping panels for bulk segregant analysis AB +/plx fish were crossed to TL +/+ fish and resulting plx heterozygous males were backcrossed to AB +/plx females. Wild–type and mutant embryos (plx/plx) were identified and used for DNA isolation. Two pools of 20 wild–type and 20 mutant embryos were used in the bulk segregant analysis. SSLP markers spaced across the genome were amplified by PCR and the products analyzed on agarose gels. Once linkage was detected and confirmed using 96 mutant embryos, two new closely flanking markers were identified. Fluorescent primers were designed for these markers and an additional 530 mutant embryos were genotyped on an automated sequencer. Recombinant embryos were used to further refine the critical region containing the plx mutation. Results:Bulk segregant analysis linked plx to chromosome 20. Fine mapping using the critical recombinants has now restricted the region containing the gene to a 6 centimorgan interval between markers Z4394 and Z8150. Sequence from the current zebrafish genome assembly will be used to design new markers to further refine the interval using linkage mapping before candidate genes are identified. Conclusions:High resolution linkage mapping has localized the perplexed gene to a 6 centimorgan interval on chromosome 20. These mapping results will lead to the cloning of the perplexed gene, which is an important component in normal retinal development. With the increasing efficiency of mapping procedures and the ongoing release of sequence data, forward genetics using zebrafish continues to be a powerful tool for studying the mechanisms involved in vertebrate retinal development.

Keywords: retinal development • mutations • gene mapping