Fig. 6

Intestinal smooth muscle development is coincident with enteric neuron differentiation: (A) Circular smooth muscle cells are first identified at 74 hpf by desmin immunohistochemistry (lateral view of a the mid intestine; whole-mount projection with anterior–posterior axis aligned horizontally). (B) Cross-sections at this stage reveal a discontinuous layer of desmin+ smooth muscle cells. (C) Enlarged inset from (B) (arrowhead) shows absence of longitudinal fibers. (D) Lateral view of the 120 hpf mid intestine, desmin immunohistochemistry; same orientation as A. Well-defined longitudinal smooth muscle cells are now evident. (E,F) Cross-sections reveal a nearly continuous inner layer of circular smooth muscle fibers and individual longitudinal smooth muscle cells peripheral to the circular muscle layer. Arrowhead points to area from E that is enlarged in (F). (G) Whole-mount view of a 74 hpf intestine processed for Hu immunohistochemistry shows enteric neuron cell bodies along the lateral borders of anterior, mid and posterior intestine. (H) Acetylated tubulin immunohistochemistry at this stage shows early axonal projections of these cells. (I–L) Hu and tubulin stainings at 96 (I,J) and 120 hpf (K,L) show proliferation and medial migration of enteric neurons and elaboration of their axonal projections.