Characterization of zebrafish intestinal smooth muscle development using a novel sm22α-b promoter

Abstract

Smooth muscle cells provide structural support for many tissues and control essential physiological processes, such as blood pressure and gastrointestinal motility. Relatively little is known about the early stages of intestinal smooth muscle development and its relationship to the development of the enteric nervous system, which regulates intestinal motility. Here, we report an evolutionarily conserved 523 base pair regulatory element within the promoter of the zebrafish sm22α-b (transgelin1) gene that directs transgene expression in smooth muscle cells of the intestine and other tissues. Comparative genomic analysis identified a conserved motif within this element consisting of two Serum Response Factor binding sites that is also present in the promoters of many mammalian smooth muscle genes. We established a stable line expressing GFP in smooth muscle cell and used this line to describe lineage relationships among cells within different intestinal smooth muscle layers and their co-development with the enteric nervous system (ENS).

Figure 1

sm22α-b promoter constructs used in this study. A: Promoter constructs (red) and evolutionary conserved regions (ECR, orange) tested for enhancer activity. B: ClustalW alignment of ECR5 from Tetraodon nigroviridis (Tn), Gasterosteus aculeatus (Ga), Oryzias latipes (Ol), and Danio rerio (Dr). 6 (1–6) segments with high conservation are present in all 4 (1, 2, 3, 6) or 3 of the 4 species (4, 5). Segment 3 contains a SRF binding CArG box; segment 6 contains a CArG box and an ETS-protein binding site.

Figure 2

ECR5 of the sm22α-b promoter can drive GFP expression in smooth muscle cells. At 72 hpf (A) expression is detectable in the intestine (arrowheads) heart (*), the rostral notochord (“N”) and trunk muscle cells. Starting at 4 dpf (B–E) expression is present in the intestine (arrowheads B) in the notochord (“N”, C) the dorsal aorta (“DA” D) and the cardinal vein (“CV” D). In the heart (E) GFP is expressed in the bulbus arteriosus (“BA”), the atrium (“A”) and the ventricle (“V”) and the ventral aorta (“VA”). (A, B, D lateral view, C dorsal, E ventral; scalebar in E corresponds to 250 εm in A–C 100 εm in D and 50 εm in E)

Figure 3

Smooth muscle differentiation. GFP expression in smooth muscle of the middle (A, C, E) and posterior (B, D, F) intestine. A–F: large panel: maximum intensity projections of confocal scans through half of the intestine (lateral view); lower panels: top 15 µm to visualize longitudinal layer; right panels: z-section at the position indicated in the lower panel. The longitudinal cell layer is located on top of the circular but is less bright and thus appears below in the large panels. At 72 hpf (A, B) circular layer cells are present but appear undifferentiated, with a round morphology and projections to adjacent cells. Few undifferentiated longitudinal layer cells are present (A, B lower panel). At 4dpf (C, D) and 6dpf (E, F) cells become increasingly directional and differentiate to long strands. Both outer longitudinal and inner circular layer are distinguishable. (scalebar in F: 20 εm, all panels same scale)

Figure 4

Clonal analysis of smooth muscle development. Mosaic expression of the ECR5 -GFP transgene (Tg(sm22α-b: GFP)) in developing smooth muscle cells in a domain in the middle of the intestine (A, C, E, G) and the posterior intestine (B, D, F, H). Frequently patches of 2–3 cells can be found at 72 hpf (A, B). At 80 hpf the cells elongate in circular directions and first longitudinal layer cells become visible in the middle domain (arrowheads C). More longitudinal layer cells develop between 80 hpf and 96 hpf, they are always located close to circular layer cells. (scalebar in H: 25 εm, all panels same scale)

Figure 5

Development of enteric nervous system (ENS) and smooth muscle. 3D reconstruction of smooth muscle cells expressing GFP (green) and ENS cells expressing DsRed (red) (lateral view; insets: z-sections at position indicated by broken line). A, C, E middle, B, D, F posterior intestine at 72 hpf (A, B) and 5 dpf (C–F). Enteric neurons with well-defined axonal projections are detected at 72 hpf while the smooth muscle is still differentiating (A, B) and the cells have a round shape in the posterior (B). At 72 hpf ENS cells are positioned above the smooth muscle cells (A, B). At 5dpf most ENS cells are located above the circular within the longitudinal smooth muscle layer (C, D) axons can often been seen in parallel to longitudinal layer cells. Smooth muscle differentiation appears normal in colourless (cls) mutant larvae without ENS (E, F) (scalebar in F: 20 εm, all panels same scale)