Fig. 3
Pathway analyses profiling reveals HGF/Scatter factor activity during active fusion. (a) Consolidated plots showing gene ratios and counts for significant ontology enrichment terms (adj P < 0.05) associated with GFP + ve enriched DEGs using KEGG Pathway, Reactome Pathway, and GO TERMs. (b) STRING Network analysis of protein-protein interactions within the GFP + ve DEG set indicated MET at the centre of interactions and directly connected to its ligand HGF. (c) Box and whisker plots of absolute gene expression level (transcripts per million, TPM y-axis) for MET and HGF in GFP + ve (green plots) and GFP-ve cells (blue plots). (d) Fluorescence in situ hybridisation analyses at prefusion stage HH28 for MET (filled arrows) and HGF (yellow open arrows) in the distal and medial optic fissure margins. Note non-overlapping expression domains. (e) Fluorescence in situ hybridisation analyses during active fusion (HH30) indicated specific expression of MET (arrowheads) in the neural retina pioneer cell domain. (f) Comparison of MET and HGF expression in the fusing OFM revealed HGF expression (arrows) in cells at the junction of the RPE and pioneer cells, which was immediately adjacent to the MET expressing pioneer cells. (g) Immunofluorescence analysis for phosphorylated MET receptor protein (Phospho-Tyr1234/Tyr1235) identified activity at the folding point of the OFM connecting the RPE border cells to the apical regions of pioneer cells (arrows). Signal was not markedly reduced in the fused seam at 100 μm from the fusion plate in the same eye. Large arrowheads in (e) indicate the midline of the OFM; asterisks represent auto fluorescent red blood cells. Abbreviations: RPE, Retinal pigmented epithelium; POM, periocular mesenchyme; NR, neural retina.
