α-Smooth muscle actin (αSMA) expression is temporally regulated in the intestine and swim bladder. A,D,G,J,M: αSMA is expressed in the developing gut (arrows) and swim bladder (arrowheads) at 60 (A), 65 (D), and 77 hours postfertilization (hpf) (G), or 4 (J), and 5 days postfertilization (dpf) (M). αSMA expression in the gut is not continuous at 65 hpf (white arrows in inset A), but becomes continuous by 65 hpf (D). G′: αSMA expression is seen in the developing swim bladder at 77 hpf in a dorsal view. B,E,H,K,N: Cross-sections of 60 (B), 65 (E), and 77 hpf (H), or 4 (K) and 5 dpf (N) embryos show αSMA expression in the developing muscularis propria of the gut and swim bladder (arrowhead in E). C,F: Cells expressing αSMA at 60 (C) and 65 hpf (F) are mesenchymal in morphology by hematoxylin and eosin staining. I,L,O: By 77 hpf a layer of circularly arranged cells is seen outside the gut epithelium (arrows in I′ point to nuclei of circularly arranged cells). g, gut; sb, swim bladder.

Temporal regulation of sm22α-b, smtn-b, cpi-17, and nm-mhc-b during the development of visceral smooth muscle. A-D: nm-mhc-b is first seen in the anterior gut at 50 hours postfertilization (hpf; A, arrow). At 3 days postfertilization (dpf), nm-mhc-b is expressed throughout the gut (B, arrow). By 4 dpf, the expression of nm-mhc-b in the gut (arrows) is down-regulated, but continues in the swim bladder (C, arrowhead). In longitudinal section, nm-mhc-b is expressed in a layer of cells surrounding the gut epithelium (D, arrowheads). E-H: SM22α-b expression at 56 hpf (E), 60 hpf (F), and 4 dpf (G) in the developing gut and swim bladder is similar to that of α-smooth muscle actin (αSMA). The inset in F is a dorsal view. Like nm-mhc-b, it is expressed in a layer surrounding the gut epithelium at 3 dpf (H). I,J,L,M: cpi-17 is expressed throughout the gut at 3 dpf (I), while smtn-b is expressed in the anterior gut (L). By 4 dpf, cpi-17 (J) and smtn-b (M) are no longer expressed in the gut, but are expressed in the swim bladder. K,N: In longitudinal section, both genes are expressed in a layer surrounding the gut epithelium.

Smooth muscle marker expression in the juvenile gut and swim bladder. A,B: Tropomyosin is expressed in a circular smooth muscle layer at 4 days postfertilization (dpf; A), and in longitudinal and circular layers at 6 dpf (B). C-F: By15 dpf, tropomyosin staining is strong in a layer surrounding both the gut and swim bladder (C). D,E: A higher power image (D) reveals that the swim bladder has a thin epithelial lining and single layer of smooth muscle cells surrounding it, while the gut (E) at similar magnification has developed a columnar epithelium, and a thicker smooth muscle wall. F: In a whole-mount larva, tropomyosin staining is seen around the swim bladder and gut. G: Whole-mount staining of a 20 dpf larva with α-smooth muscle actin (αSMA) reveals a similar pattern to tropomyosin. H: Longitudinal sections of a 22 dpf larva show extensive smooth muscle layers expressing αSMA. L, lumen; sb, swim bladder; g, gut.

Histology of adult zebrafish gut and swim bladder smooth muscle. A,B: The adult gut has a clear laminar structure of smooth muscle adjacent to the columnar epithelium (ce) consisting of an inner circular smooth muscle layer (csm), and an outer longitudinal smooth muscle layer (lsm). C,D: The adult swim bladder shows a slightly different cellular organization. In addition to an epithelial layer, there is an inner circular smooth muscle layer, and a mixed outer layer of radial smooth muscle (rsm), and longitudinal smooth muscle. For direct comparison between gut and swim bladder, A and C are presented at the same magnification, as are B and D. L, lumen.