Cdk5/p35 Activity Is Essential for Eye Growth and Retinal Lamination in Zebrafish

Purpose: Retinal lamination is disrupted in a zebrafish recessive mutant called young (yng) (Link et al., 2000) and a point mutation in a brahma-related gene (brg1) is responsible for this phenotype (Gregg et al., 2003). The inactivation of ERK, a mitogen-activated protein kinase (MAPK), blocks the retinal lamination independent of brg1 mutation. It has been demonstrated ERK controls the neuronal differentiation through the induction of Egr1 and in turn p35, a neuron-specific activator of Cyclin-dependent kinase 5 (Cdk5) (Harada et al., 2001). Cdk5 activity is well known to be responsible for a proper central nervous system (CNS) development, in particular cortical lamination. Although retina is an extension of the CNS, the role of Cdk5 activity in retinal development and differentiation is not clear. The purpose of this study was to investigate the potential role of Cdk5 activity in the brg1-regulated differentiation pathway and retinal lamination process in zebrafish. Methods: Embryonic retinas at 36 and 52 hours post-fertilization (hpf) were dissected from wild type zebrafish and homozygous mutants of yng/brg1. Expression levels of brg1, erk1, erk2, egr1, cdk5 and a putative form of p35 in these samples were determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The p35 gene sequence was inferred from the zebrafish high throughput genome sequence database by a tblastn search using the human p35 protein sequence as the query sequence. The resulting gene sequence with the lowest expectation value (e-140) was used for primer design. Expression of cdk5 was suppressed in zebrafish embryos by microinjection of a morpholino antisense oligonucleotide (MO) which targeted a splice junction of the cdk5 pre-mRNA. The suppression was confirmed by RT-PCR and the corresponding phenotypic changes were observed by microscopy and histology. Results: brg1, erk1, erk2, cdk5 and p35 were down-regulated in the yng/brg1 retinas at 36hpf, while only brg1, erk2, egr1 and p35 were down-regulated in yng/brg1 retinas at 52hpf. There was also a time-dependent increase in the expression of erk1, cdk5 and p35, and a time-dependent decrease in the expression of egr1 in wild type retinas. The suppression of cdk5 expression in the developing embryos by the splice junction MO was found to inhibit eye growth and retinal lamination. <Truncated: Gene Tools database limitation>