Atypical cadherin FAT1 is essential for vertebrate optic fissure closure

Abstract

Purpose : Coloboma consequent to optic fissure closure (OFC) defects is a potentially blinding congenital malformation, affecting 1-3 individuals per 10,000 births. Expression profiling in the developing mouse eye detected Fat among the genes dynamically expressed at the edges of the closing OF. The present study was carried out to investigate the role of Fat1 in OFC.

Methods : In situ hybridization (ISH) was performed on mouse sections using a Fat1 probe. Histology was performed on Fat1^-/- mouse and fat1 knockdown zebrafish embryos. CRISPR/Cas9 technique was used to disrupt the MENA and PDZ binding domains of zebrafish fat1a. Immunofluorescence staining, and super-resolution confocal microscopy were used to study the localization of FAT1 in human primary retinal pigment epithelial (hfRPE) cells. Knock-down of FAT1 in RPE cells was performed using shRNA. Whole exome sequencing was performed on patients with coloboma to explore causative variants.

Results : In mouse, Fat1 and Fat4 mRNA were localized at the edges of the optic fissure margins. Upon histological analysis, Fat1^-/- mice consistently exhibited OFC defects whereas, no OF abnormality was observed in Fat4^-/- animals. Morpholino-mediated knock-down in zebrafish of fat1a but not of fat4 resulted in coloboma. Furthermore, recessive FAT1 mutations were identified in eight individuals affected with coloboma from two families, suggesting an evolutionary conserved role of FAT1 in vertebrate optic fissure closure. To investigate the molecular mechanisms underlying FAT1 role in OFC, we used hfRPE as an in vitro model. Using super-resolution microscopy, we observed FAT1 protein at earliest cell-cell junctions. In vivo, cell processes emanating from the opposing edges of the OF margins, observed by electron microscopy, formed “simple appositional-type” contacts during the early stages of OFC. MENA and ZO1 colocalized with FAT1 at the earliest cell-cell junctions in hfRPE and shRNA mediated knockdown of FAT1 resulted in disruption of their expression pattern. Furthermore, CRISPR/Cas9 mediated disruption of MENA and PDZ binding domains of fat1a was sufficient to cause coloboma in zebrafish.

Conclusions : The spatio-temporal expression of Fat1 in mouse during OF closure, its expression in human RPE cells, and the coloboma phenotypes observed upon targeted disruption in mouse and zebrafish and in patients carrying mutant alleles strongly support an essential role for Fat1 in OF closure.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.