Abstract
Abstract: :
Purpose:The CRB1 gene is mutated in 13% of patients with Leber congenital amaurosis and in most cases with autosomal recessive retinitis pigmentosa (arRP) type 12 or arRP with Coats' like exudative vasculopathy. The CRB1 protein consists of 19 EGF-like and 3 laminin A G-like domains, a transmembrane domain and a cytoplasmic domain. Drosophila CRB shows the same overall structure. In this study, we investigated the mRNA expression pattern of mouse Crb1 and explored the functional conservation between the cytoplasmic domains of human and Drosophila CRB. Methods:We isolated mouse Crb1 cDNA, and generated mRNA in situ hybridization probes labeled with 35S-UTP or DIG which were hybridized to mouse eye or brain sections. Using RT-PCR, we cloned two alternatively spliced 3' cDNA ends of human CRB1. We performed GAL4/UAS mediated overexpression of the cytoplasmic domain of human CRB1 in wildtype and crumbs Drosophila embryos. Results:Crb1 is expressed in the retinal progenitor cells, and later on becomes restricted to the differentiated photoreceptor cells where it remains active up to the adult stage. In the developing neural tube, expression of Crb1 is restricted to its most ventral structures, coinciding with the expression domain of Nkx2.2. In the adult brain, Crb1 expression is confined to areas where the production and migration of neurons occurs. Human and Drosophila CRB cytoplasmic domains show 72% amino acid similarity. Overexpression of the cytoplasmic domain of human CRB1 in Drosophila crumbs embryos rescues the mutant phenotype. Large parts of the cuticle are restored. Overexpression of the same construct in wildtype embryos transforms the single-layered epidermis into a multilayered tissue. Conclusion:Expression of the Crb1 gene is restricted to specific parts of the (developing) mouse eye and brain. In the eye, expression is the highest in the photoreceptor cell layer; in the developing brain expression overlaps with Shh and Nkx2.2, suggesting concerted transcriptional regulation of these molecules with Crb1. Overexpression of the cytoplasmic domains of human and Drosophila CRB has similar effects, suggesting that they are functionally conserved despite their evolutionary distance of at least 500 million years.
Keywords: 417 gene/expression • 443 in situ hybridization • 527 protein structure/function