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Jonathan M. Skarie, Brian A. Link; Development of Genetic Tools to Study Anterior Segment Dysgenesis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2351.
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Anterior segment dysgenesis (ASD) describes a clinical spectrum of congenital malformations in anterior ocular structures, including those important for intraocular pressure maintenance. ASD can be found in a number of genetic syndromes, such as Axenfeld Rieger (AR) and Nail-Patella (NP) syndromes, and results in increased risk for glaucoma. While genes have been identified for many of these syndromes (ie PITX2 and FOXC1 for AR, LMX1B for NP), the molecular and developmental pathways disrupted are not clearly understood. The purpose of this study was to develop tools to study the molecular basis for ASD.
Genomics and PCR were used to identify and amplify the promoter regions of zebrafish foxC1.2 and lmx1b.1. Transgenic zebrafish were developed using Gateway cloning technology and the tol2 recombination system. Time-lapse fluorescent/confocal microscopy was used to monitor real time ocular development. Morpholinos were used for gene knockdown studies.
Stable transgenic lines expressing fluorescent proteins under the control of the lmx1b.1 and the foxC1.2 promoters were created. Expression patterns in both lines correlated with previously published expression patterns for each gene. Each line labeled distinct populations of peri-ocular mesenchyme cells during anterior segment development. Decreased numbers of GFP positive cells were found after knockdown of foxC1.1/.2. Expression of GFP persisted into adulthood in both lines. Transgenic lines with these promoters driving gal4 have also been developed to allow for tissue specific modulation of target genes.
We have developed and characterized a series of genetic tools relevant to known human disease which will be used to investigate molecular mechanisms leading to ASD. These tools have particular advantages of labeling distinct populations of cells and allow for this process to be monitored in real time. In addition, the identified promoter regions have provided information about the regulation of the foxC1 and lmx1b pathways. Finally, the persistence of specific expression into the adult will make these tools amenable to studying regulation of aqueous humor and intraocular pressure in the adult animal.
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