mNCCs migrate into aganglionic guts to contribute to the ENS. (A) Whole-mount preparation of an E13.5 Wnt1Cre;R26tdTom gut. Arrowhead and arrow indicate pioneer extrinsic axons and the wavefront of eNCCs, respectively. (B) Whole-mount preparation of an E13.5 Wnt1Cre;R26tdTom;Ednrb-/- gut showing the loss of typical rostral–caudal eNCC chain integrity along the gut. The arrowhead indicates pioneer extrinsic axons. (B’) Magnification of the boxed area in panel B, showing 1 mNCC (arrowhead) migrating in or out of the midgut. (B’’) Magnified view of another boxed area in panel B, showing a solitary NCC clump within the midgut. NCC-derived neurons (TOM+/PHOX2Bstrong/TUJ1+), NCC-derived glia (TOM+/PHOX2Bweak/TUJ1-), and NCCs (TOM+/PHOX2Bstrong/TUJ1-) are indicated by green arrowheads, red arrowheads, and white arrowheads, respectively. (C and D) Segmental ENS within the aganglionic guts of E14.5 and E15.5 Wnt1Cre;R26tdTom;Ednrb-/- embryos. Red arrows indicate the migratory wavefront of eNCCs. White arrows indicate pioneer pelvic ganglion-arising axons within the hindgut. (C’) Magnified view of the boxed area in panel C, showing mNCC-derived neurons (TOM+/TUJ1+) and glia (TOM+/B-FABP+). (C’’) Magnified view of another boxed area in panel C showing mesenteric (arrowheads) and enteric (arrow) neuronal clumps. (D’) Magnified view of the boxed area in panel D, showing extrinsic axon-associated SCPs (TOM+/B-BFAP+). CC, cecum; ME, mesentery.