Fig. 6: Pioneer precursors exhibit distinct cellular behavior.

A–C A single confocal Z-slice through the pLL ganglion labeled with FISH probes against ntrk1 (yellow) and nr2f2 (cyan) between 14 and 16.5 hpf. Cell membranes are labeled by cldnB:memgfp, whereas pLL neuroblasts are marked by neurod1:mCherry. Dashed line outlines pLL and otic placodes. Arrows mark leading tip cells upregulating pioneer marker ntrk1. Arrowheads mark neuroblasts co-expressing ntrk1 and nr2f2 (two replicas; n = 10 total animals). D, E Quantification of ntrk1 and nr2f2 expression between 14 and 20 hpf (D) or 30 hpf (E). Dashed red lines indicate cells that coexpress both markers. Note the significant number of co-expressing cells at 14–20 hpf (51/300 cells: 17%) compared to 30 hpf (5/285 cells: 1.8%): p = <0.001, Chi-Square test. F–H Stills from a time series using neurod1:kaede photoconversion at 16 hpf. F–H The first delaminating tip cell was photoconverted and tracked to visualize its peripheral axon. Note that the axon projects into the pLLP. Arrowheads = photoconverted axons, arrows = pioneer axons. I–K A photoconversion of a distal cell. Note the labeled axons lagging behind the pLLP. Arrowheads = photoconverted axons, arrows = pioneer axons. L Schematic summary of photoconversions at 16 hpf. Cell location shows the region where it was photoconverted, and color indicates whether it became pioneer or follower. Delaminating tip cells became a pioneer 4 of 4 times. In labeled distal cells, 7 of 9 became followers, and 2 became pioneers. M–O Stills from a timeseries of two individually labeled neurons between 17 and 25 hpf. M Anterodorsal neurons appear spindle-shaped with early neurite projections. N Their pioneer neurites localize within the pLLP. O Pioneer neurons extend their axons with the primordium during migration. Pioneer neurites marked by arrowheads. P–R Stills from a timeseries of three individually labeled neurons beginning at 17 hpf. Note the absence of neurites in the pLLP through the time series. Follower neurites marked by arrowheads. For experiments shown in (M–R), we imaged 7 embryos that contained 4 pioneers and 7 followers. Membrane = cldnB:memgfp (green). All scale bars = 20 μm. All images scaled the same.