Endoderm and mesodermal hand2 function are required for normal ENS and intestinal smooth muscle development but hand2 is not required for the initial migration of vagal neural crest to the anterior end of the intestine and endoderm is not required for mesodermal hand 2 expression. (A, D, G, J, M, O) wild-type embryos, (B, E, H, K, N, P) cas/sox32 morphant embryos and (C, F, I, L) hand2 morphants. (A–C) Ventral view of the vagal region of 36 hpf embryos that have been hybridized with riboprobes for crestin showing a failure of vagal NCC migration to the anterior end of the intestine in sox32 morphants but no affect on these NCC in hand2 morphants. (D–F) Lateral view of 60 hpf embryos that have been hybridized with riboprobes for phox2b. (G–I) Close up lateral view of the intestine of 60 hpf embryos that have been hybridized with riboprobes for phox2b showing a failure of phox2b expressing cells to populate the entire length of the intestine in cas/sox32 and hand2 morphants. (J–L) Close up lateral view of the intestine of 60 hpf embryos that have been hybridized with riboprobes for myh11 showing a reduction/loss of myh11 expressing intestinal smooth muscle cells in the intestine of cas/sox32 and hand2 morphants. (M, N) Lateral view of the intestine of 72 hpf embryos wholemount in situ hybridized embryos that have been hybridized with riboprobes for α SMA. Black boxes in panels D–F are the regions that are shown in close up in panels G–I. Arrowheads (M) indicate the αSMA expressing cells in the intestine. (O, P) Ventral view of 30 hpf wholemount in situ hybridized embryos that have been hybridized with an antisense probe for hand2. Arrows (A, C) indicate the migrating enteric precursors. White arrowheads (G, I) indicate phox2b expressing cells in the intestine. Black arrowheads (J, L) indicate myh11 expressing cells in the intestine. Black arrowheads (M) indicate the αSMA expressing cells in the intestine Anterior is to the left.
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