Proliferation-deficient retwmr1/+ ENCCs highlight cell density-associated migration mechanism during ENS formation. (A,B) Average speed of vanguard cell migration reveals a reduction in retwmr1/+ ENCC speed during the key cell migration window of 48-60 hpf (n=6 per condition; Wilcoxon signed-rank test, P=0.00027). (C,D) Average distance to three nearest neighbors was calculated for each ENCC within an individual population and then averaged across conditional replicates and plotted versus hpf for both ret+/+ and retwmr1/+ fish (n=3 per condition). Significantly higher distances between neighbors were observed among retwmr1/+ ENCCs at 48-60 hpf (Wilcoxon signed-rank test, P=0.00940) and 61-72 hpf (Wilcoxon signed-rank test, P=0.00870), indicative of lower cell density. (E) Percent proliferating ENCCs was calculated using whole-mount immunofluorescence performed using Tg(−8.3phox2bb:Kaede) fish in combination with anti-pHH3 and anti-Kaede antibodies to calculate the percent pHH3+, Kaede+ ENCCs at 60 hpf, 72 hpf and 80 hpf. retwmr1/+ fish displayed a lower percentage of proliferating ENCCs at 60 hpf (Wilcoxon signed-rank test, *P=0.02). NS, not significant. Box plots show median values (middle bars), and first to third interquartile ranges (boxes). Whiskers indicate 1.5× the interquartile ranges; dots indicate mean values per hpf within the given time window. (F) Graphic depicts density-dependent migration model in which more densely spaced ret+/+ ENCCs display higher speeds of migration than less densely spaced retwmr1/+ ENCCs.
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