Fig. S3

Proliferation, cell death and differentiation are all unaffected in ret^hu2846/+ larvae.

(A-C) Proliferation of ENS progenitor cells is equivalent in 48hpf WT (A) and ret^hu2846/+ (B) larvae, shown using GFP from SAGFF234A;UAS:GFP to label pixel area of ENS progenitors and BrdU (following 30 minute BrdU pulse) to quantify proliferating cells per 220um (1000 pixels) of the distal migratory wavefront of progenitors (p = 0.9182) (C). The area (in pixels squared) (D) and roundness (4*area/(π*major axis²)) (E) of the migratory wavefront was quantified, and significant increases in both parameters reflect the fact that the ret^hu2846/+ embryo wavefront is not elongated as is seen in WT (p<0.0001 for both area and roundness). (F-I) TUNEL staining on 42hpf WT (F,G) and ret^hu2846/+ (H,I) larvae does not show any TUNEL⁺ apoptotic cells (red, G,I) within the GFP⁺ migratory stream of ENCCs (F,H). (J-M) Differentiation was assayed in the SAGFF234A;UAS:GFP line by comparing HuC/D expression with GFP expression in 4dpf larvae (J,K) and counting differentiated serotonergic cells (5-HT⁺) within 5dpf larvae (L,M). While HuC/D⁺ cells show expected significantly decreased numbers in ret^hu2846/+ larvae (J, L, p = 0.0096 and p = 0.0045, respectively), the percentage of progenitor cells (GFP⁺Hu^-) cells is not significantly altered (K, p = 0.3785), and the percentage of 5-HT⁺ cells is also not significantly altered (M, p = 0.2188).