Fig. 2

Isolation and transcriptomic profiling of pioneer cells from NTN1^{NTN1 − T2A−GFP/+} reveals novel pioneer cell markers with regulated expression during OFC. (a) Schema for optic fissure transcriptomic profiling strategy: manually dissected fissures (red hatched lines) from HH28 NTN1^{NTN1 − T2A−GFP/+} embryos were dissociated and FACS sorted prior to bulk RNA sequencing. (b) The GFP + ve and GFP-ve sorted cell populations from NTN1^{NTN1 − T2A−GFP/+} eyes were defined by gating based on using non-fluorescent WT control samples (i.e. no GFP). (c) GFP + ve and GFP-ve sorted NTN1^eGFP cells were further separated using spectral flow cytometry to subtract auto fluorescent cells from the GFP + ve population (red arrows). (d) Volcano plot of all DEGs defined by -log10 p-value and Log2 fold change. There were 433 GFP + ve enriched DEGs (LFC₂ > 1.0) and 1268 DEGs enriched in GFP-ve (LFC₂ < -0.5). GFP + ve DEGs included known pioneer cell markers NTN1 and PAX2, whereas GFP-ve DEGs included periocular mesenchyme markers ALX1 and EMX2. (e) Fluorescence in situ hybridisation confirmed NTN1 and PAX2 mRNA expression was localised to pioneer cells (arrowheads) at HH28. In contrast ALX1 and EMX2 mRNA expression were identified in periocular mesenchyme (arrows). (f) Box plots for novel pioneer cell markers identified in this study, with log2 fold change and adjusted-P values indicated. (g) Colorimetric in situ hybridisation for novel OFM genes at HH28 showed pioneer cell specific expression (arrowheads) in the open fissure at HH28 before fusion. Non pioneer cell is expression indicated by arrows.