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Figure 3 Transcriptional profiling in chick optic fissure closure. (a) Transcriptional profiling using microdissected regions of the developing chick eye at E5 (HH.St25-27; pre-fusion), E6 (HH.St27-28; initiation), and E7 (HH.St28-30; during active fusion) revealed multiple DEGs at each stage. (b) NTN1 was the highest expressing gene of 12 fissure-specific DEGs (fissure vs whole eye) throughout all stages of chick OFC (Log2 FC >1.5; FDR < 0.05). These included the known human coloboma associated genes (indicated by #): SMOC1, PAX2, VAX1 and ALDH6, in addition to the coloboma candidates from other animal studies CHRDL1 and CYP1B1 (indicated by •). (c) Clustering for relative expression levels at active fusion stages (HH.St28-30) revealed three independent clusters (2, 3, and 5) where expression levels trended with Fissure >ventral > whole eye. (d) Analysis of normalised mean expression values (TPM, n = 3 technical replicates; error bars = 1 x standard deviation) from clusters 2, 3, five at HH.St28-30 for the Gene Ontology enriched pathways (p<0.0001; Biological fusion [GO:0022610], and Epithelial fusion [GO:0022610]) revealed significant fissure-specific expression for highly expressed (TPM >100) genes - NTN1, FLRT3, CYP1B1 and COL18A1 - in addition to other potential candidate genes for roles in OFC. NTN1 (TPM >200) was the highest expressed fissure-specific DEG identified during active fusion.