sox10 mutants show severe and early defects in enteric nervous system development. Lateral views (A, C, E, G, I, K, M, O, Q, S, U, W, Y, AA, AC, AE, AG, AI) or transverse sections (B, D, F, H, J, L, N, P, R, T, V, X, Z, AB, AD, AF, AH, AJ) of whole-mount in situ hybridizations of anterior trunk at 35 (A–L), 45 (M–X) and 60 hpf (Y–AJ) for enteric nervous system markers sox10 (A, B, G, H, M, N, S, T, Y, Z, AE, AF), phox2b (C, D, I, J, O, P, U, V, AA, AB, AG, AH) or ret (E, F, K, L, Q, R, W, X, AC, AD, AI, AJ) in wild-type (A–F, M–R, Y–AD) and sox10 (cls) mutant (G–L, S–X, AE–AJ) embryos. At all stages, wild-type ENS progenitors express all three genes (arrows), whereas sox10 mutants show occasional sox10-positive cells in this location, but do not express phox2b nor ret. As an internal positive control, note that expression of phox2b in the hindbrain (*, C, D, I, J) and ret in the ventral neural tube (#, E, K) is not affected. Note too that in wild-type embryos ret is expressed in both a chain of ENS progenitor cells (arrow and inset in E, Q and AC) and more medially in the tightly packed cells of the forming pronephros (partially out of focus in this focal plane; arrowhead, E, Q); the former is absent in sox10 mutants, whereas the latter is unaffected (K, W, AI). ENS progenitor marker expression extends more posteriorly along the gut with increasing age (e.g. compare C, O, AA). sox10 expression consistently extends more posteriorly than the other two markers at equivalent stages (e.g. compare A, C, E). Transverse sections reveal changes in progenitor distribution around gut primordium with age. ENS progenitors at 35 hpf initially lie in contact with gut primordium (arrow, B), forming two longitudinal columns migrating along the gut, but have spread around the developing gut by 60 hpf (Z). Note the identical positioning of sox10, phox2b and ret positive progenitors with respect to the developing gut (e.g. N, P, R). ENS progenitors are not usually seen in sections of cls mutant embryos (H, J, L, T, V, X, AF, AH, AJ), although expression in CNS remains. Interestingly, sox10 and phox2b expression is prominent and overlapping in the pectoral fin mesenchyme (p) of wild-types (N, P), whereas phox2b appears to be strongly reduced in cls mutants (T, V). NB All whole-mount embryos treated with PTU to inhibit melanin synthesis. g, gut primordium.
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