Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY/ABSTRACT The lens of the vertebrate eye becomes transparent by removing the nuclei and organelles of its inner fiber cells. Remarkably, these fiber cells must stay alive and functional without the ability to produce new proteins. Defects in fiber cell differentiation can contribute to lens cataract, the leading cause of blindness worldwide. While much has been learned about the molecular mechanisms that coordinate lens fiber cell development, there are likely many key players yet to be discovered. Our laboratory uses the zebrafish as an efficient screening system to identify novel genes playing a role in this process. We recently used CRISPR/Cas9 gene editing to show that damaging the transcription factor gene, cebpg, and RNA-binding protein genes, csde1 and c1qbp, leads to defects in lens development. Interestingly, in non-lens tissues these three genes play roles that would be relevant to lens development, such as regulating autophagy, protein stability and the stress response. We hypothesize that the protein products of these genes perform similar roles in the lens. In Aim 1 of this proposal, we will characterize the spatial expression of each gene across the developing lens of wild-type fish using RNA-scope, which detects specific mRNA molecules. We will also detail changes in nuclear and mitochondrial loss and the elongation of fiber cells. These experiments will allow us to identify the aspects of differentiation altered by the loss of each gene. Aim 2 of this proposal will use RNA-Seq and RT-qPCR to detail how the lens transcriptome is altered in each mutant. This approach will allow us to determine if the expression of known regulators of lens fiber cell differentiation mechanisms such as autophagy, denucleation or stress response, are changing, which would implicate them as part of the same signaling network as our mutated genes. The transcriptomic analysis will also identify new possible regulators of lens development, which we can then screen with our CRISPR/Cas9 system. Our laboratory is well placed to successfully conduct these proposed experiments, which will provide undergraduate students with excellent training in modern molecular and developmental biology techniques and bioinformatics analysis. Our findings will take advantage of the zebrafish lens system to address broad questions about how cells remodel during tissue differentiation.
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