A stable Netrin-1 fluorescent reporter chicken reveals cell-specific molecular signatures during optic fissure closure.

Coloboma is a developmental eye disorder characterised by failure of epithelial fusion during optic fissure closure (OFC). The incomplete identification of genes and molecular pathways that directly regulate fusion restricts current understanding of OFC mechanisms and coloboma causation. The recent identification of pioneer cells at the edges of the fissure margins provides an opportunity to molecularly characterise OFC. Here we describe the generation of a pioneer cell reporter chicken line (NTN1-T2A-eGFP) by targeting green fluorescent protein into the Netrin-1 locus using CRISPR/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (NTN1T2A-eGFP/+) embryos in which GFP localisation faithfully replicated endogenous NTN1 expression. Furthermore, all NTN1T2A-eGFP/+ embryos and hatched birds were phenotypically normal. We then optimised the isolation of GFP expressing cells from embryonic eyes using spectral fluorescence cell-sorting and performed transcriptomic profiling that revealed multiple new pioneer cell markers. Our approach implicated several novel pathways, including HGF/MET signalling, that plausibly mediate fusion in OFC. This work is the first to characterise pioneer cells directly at the molecular level and provides a new chicken reporter line with broad experimental utility. Optic fissure tissue from 10x NTN1-T2A-GFP gene edited Hy-Line chicken embryos at Hamburger-Hamilton stage 28 were pooled for each biological replicate. For each replicate, NTN1-expressing GFP-positive cells and the control GFP-negative cells were sorted. Subsequently, mRNAseq was performed on each replicate. A total of 5 biological replicates of each GFP-positive and GFP-negative cells were included in the study. Differential gene expression analysis was conducted by comparing the gene expression profile of GFP-positive cells to the control GFP-negative cells.