Premature glial differentiation is the source of the ENS defect in Spot animals. Premature glial differentiation is the source of the ENS defect in Spot.

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Premature glial differentiation is the source of the ENS defect in Spot animals. Premature glial differentiation is the source of the ENS defect in Spot animals. (A) Dissected embryonic intestines of an allelic series of Spot;G4-GFP animals at E10.5, E11.5, E12.5 and E15.5 (for each stage, n=4 per genotype). Where relevant, arrowheads point to the migration front. Asterisks indicate a lack of ENS progenitors in the cecum of SpotTg/+ and SpotTg/Tg embryos relative to wild type (WT, G4-GFP control). G, vagal ganglion; S, stomach; C, cecum; A, anus. (B) Live imaging and quantification of ENCC movement in colons of SpotTg/Tg;G4-RFP (SpotTg/Tg) and G4-RFP (WT) embryos at E12.5 and E13.5. Mean speed was calculated from individual cells at the tip of migrating arms over a 6-h period. N, number of colons analyzed; n, number of cells tracked; P, Student's t-test P-value; ns, not significant. (C) Directionality of ENCC movement in colons of SpotTg/Tg;G4-RFP (SpotTg/Tg) and G4-RFP (WT) embryos (quantified in B) relative to the mesentery axis. Likelihood ratio test P-value for circular data is indicated. (D) Proliferation of Sox10-positive ENCCs in the proximal intestine of SpotTg/Tg;G4-RFP (SpotTg/Tg) and G4-RFP (WT) E12.5 embryos (n=3 per genotype; Student's t-test). (E) Quantification of undifferentiated progenitors (Sox10-positive S100β-negative), as well as glial-fated (Sox10-positive S100β-positive) and neuronal-fated (HuC/D-positive) cells, within the ENCC population in the proximal colon of SpotTg/Tg;G4-RFP (SpotTg/Tg) and G4-RFP (WT) E15.5 embryos (n=5 per genotype). ***P<0.0001 (Student's t-test). (F) Confocal projections of the developing ENS (combining Sox10-positive and HuC/D-positive cells) within the proximal small intestine of SpotTg/Tg;G4-RFP (SpotTg/Tg) and G4-RFP (WT) E13.5 embryos, showing the precocious appearance of the glial marker S100β in Sox10-positive cells of SpotTg/Tg embryos (n=3 per genotype). Scale bars: 200 µm (A, E10.5–E12.5); 500 µm (A, E15.5); 50 µm (F). Karl-F. Bergeron et al. Dis. Model. Mech. 2016;9:1283-1293 © 2016. Published by The Company of Biologists Ltd