Abstract
Purpose :
During eye formation, cells and tissues undergo dramatic movements to generate the precise 3-dimensional structure that is crucial for visual function. We lack a comprehensive understanding of the dynamic cell and tissue interactions governing eye morphogenesis, which have implications for structural malformations underlying vision impairment disorders. To uncover new genetic loci and processes shaping the eye, we carried out a haploid forward genetic screen in zebrafish for eye morphogenesis mutants.
Methods :
A zebrafish haploid forward genetic screen was carried out in which the shutdown corner (sco) mutant was isolated. 4D imaging datasets were acquired via confocal microscopy, and cell tracking performed using our LongTracker software. Antibody staining was used to visualize extracellular matrix (ECM). CRISPR mutagenesis was used to screen genes in the deletion interval for their ability to phenocopy the sco phenotype.
Results :
In a haploid screen, we isolated shutdown corner (sco), which exhibits an intriguing novel eye morphogenesis defect: the retina and lens make initial contact normally, but then fail to detach, abolishing the space between the retina and lens. Contact between the prospective retina and lens is necessary for reciprocal signaling and coordinated invagination, but how these tissues detach is unknown. The retina-lens space is where the hyaloid vasculature forms: in sco, the hyaloid vasculature exhibits delayed abnormal development. 4D cell tracking of sco mutants reveals that neighboring cells in the retina and lens move normally. ECM molecule deposition (e.g. laminin and hyaluronic acid binding protein) is altered, however, and preliminary transmission election microscopy suggests aberrant features at the retina-lens interface. Subsequent retinal development is impaired. RNAseq analysis revealed a 10 Mb deletion (~100 genes) in sco. Using CRISPR screening in the deletion interval, we identified the causative gene: the chondroitin sulfate proteoglycan versican a (vcana).
Conclusions :
The shutdown corner (sco) mutant uncovers a previously unappreciated, genetically regulated step of eye morphogenesis: retina-lens detachment, which is necessary for proper retina and hyaloid vasculature development. The chondroitin sulfate proteoglycan versican is the causative gene, and we are now working to determine the molecular mechanism by which versican facilitates retina-lens detachment.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.