A stable NTN1 fluorescent reporter chicken reveals cell specific molecular signatures during optic fissure closure

NTN1 is expressed in a wide range of developmental tissues and is essential for normal development. Here we describe the generation of a Netrin-1 reporter chicken line (NTN1-T2A-eGFP) by targeting green fluorescent protein into the NTN1 locus using CRISPR/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (NTN1T2A-eGFP/+) embryos in which GFP localisation faithfully replicated endogenous NTN1 expression in the optic fissure and neural tube floorplate. Furthermore, all NTN1T2A-eGFP/+ embryos and hatched birds appeared phenotypically normal. We applied this resource to a pertinent developmental context - coloboma is a structural eye malformation characterised by failure of epithelial fusion during optic fissure closure (OFC) and NTN1 is specifically expressed in fusion pioneer cells at the edges of the optic fissure. We therefore optimised the isolation of GFP expressing cells from embryonic NTN1T2A-eGFP/+ eyes using spectral fluorescence cell-sorting and applied transcriptomic profiling of pioneer cells, which revealed multiple new OFC markers and novel pathways for developmental tissue fusion and coloboma. This work provides a novel fluorescent NTN1 chicken reporter line with broad experimental utility and is the first to directly molecularly characterise pioneer cells during OFC