Abstract
Purpose: :
The genetic inheritance of glaucoma is often non–Mendelian suggesting that multiple genetic loci interact to affect its progression. Our lab is using zebrafish to model human glaucoma phenotypes and ultimately identify the genes that act as modifiers of an established glaucoma phenotype. LMX1B haploinsufficiency in humans results in Nail Patella Syndrome (NPS) where approximately half of the patients develop glaucoma. To begin the groundwork for studying the mechanisms of LMX1B–dependant glaucoma phenotypes, we sought to characterize the roles of duplicated lmx1b.1 and lmx1b.2 genes in zebrafish eye development.
Methods: :
In situ hybridization was performed to determine the expression pattern of zebrafish lmx1b.1 and lmx1b.2. Morpholinos were used to knock–down lmx1b.1 or lmx1b.2 gene function. The expression pattern of optic stalk and retinal markers in lmx1b.1 morphants was determined by in situ hybridization. Time–lapse confocal microscopy was performed to examine retinogenesis and the contribution of a population of neural crest cells to the lmx1b.1 morphant eye generated in either a HuC:GFP or FoxD3:GFP transgenic background, respectively.
Results: :
Zebrafish lmx1b.1 and lmx1b.2 transcripts were found during early stages of neurogenesis in the ventral forebrain and midbrain–hindbrain boundary (MHB). Zebrafish lmx1b.1 expression was detected at 72 hours post–fertilization (hpf) in ocular mesenchymal cells, a pattern previously observed in higher vertebrates. When injected singly, both lmx1b.1 and lmx1b.2 morpholinos resulted in dramatic ventral retinal defects and MHB disruption. Analysis of pax6 and fgf8 expression in morphants suggests a role for lmx1b.1 in early ocular patterning. Time–lapse confocal microscopy revealed a delay and mislocalization in the appearance of HuC:GFP+ retinal ganglion cells between 29 hpf and 52 hpf in lmx1b.1 morphants. Between 24 hpf and 48 hpf, normal migration of FoxD3:GFP+ neural crest cells into anterior structures of the lmx1b.1 morphant eye was observed with time–lapse confocal microscopy.
Conclusions: :
Examination of morphant embryos reveals novel roles for lmx1b.1 and lmx1b.2 in the zebrafish eye. We propose that early lmx1b.1 expression in the ventral forebrain establishes appropriate regulation of ocular morphogenesis through genes expressed in the optic stalk and ventral retina. Future studies will utilize stable lmx1b transgenic lines to identify interacting genes involved in LMX1B–dependant glaucoma phenotypes. Specific models and the effects of gene dosage on lmx1b function in ocular development for lower and higher vertebrates will be discussed.
Keywords: proteins encoded by disease genes • gene/expression • transcription factors