Whitesell et al., 2014 - An alpha-smooth muscle actin (acta2/alphasma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells. PLoS One   9:e90590 Full text @ PLoS One

Fig. 1

Acta2 promoter/enhancer construct design and expression in zebrafish.

(A) A zebrafish (Dr) enhancer/promoter construct was constructed from the proximal promoter and first intron sequence of the zebrafish acta2 gene, and contains three highly conserved CArG binding sites also found in the mouse (Mm) acta2 proximal promoter and first intron. (B) Comparison of zebrafish CaRG boxes A and B in zebrafish, tilapia and medaka. (C,D) By wholemount in situ hybridization, acta2 shows strong expression in the gut (g) at 72 hpf (B), and expressed in the gut, swim bladder (sb), ventral aorta (va), floor plate (fp), aortic arch arteries (aaa), and bulbus arteriosus (ba) at 100 hpf (C). (E,F) Co-localization of wholemount in situ hybridization acta2 and anti-GFP staining of the acta2:GFP transgene shows strong expression in the aortic arch arteries (aaa) at 100 hpf. (G,H,I) 4 dpf acta2:EGFP transgenic fish (H) stained with Tagln rabbit polyclonal antibody (G). Merge (I) shows co-localization between acta2:GFP and Tagln. Arrowheads in G–I depict vascular mural cells. Scale bar in G represents 20 μm.

Fig. 2

Morphology of vascular and visceral mural cells in acta2:EGFP transgenic fish.

(A) Ventral pharyngeal region of a 4 dpf double transgenic Tg(acta2:EGFP)ca7; Tg(kdrl:mCherry)ci5 (mural cells are green and endothelial cells are red) zebrafish shows extensive mural cell coverage of the ventral aorta (VA) and lesser coverage on the smaller aortic arches (AA) or opercular artery (ORA). (B) Wholemount adult ventral aorta and attached afferent branchial arteries shows extensive smooth muscle coverage. (C) Lateral view of the gut (g) and swim bladder (b) of a 14 dpf double transgenic Tg(acta2:EGFP)ca7; Tg(kdrl:mCherry)ci5 zebrafish shows radial and circumferential smooth muscle on both gut and swim bladder, but sparse mural cells on the dorsal aorta (DA) and no visible cells on the posterior cardinal vein (PCV). Scale bar in A represents 25 μm. Scale bar in B and C represents 100 μm.

Fig. 3

Smooth muscle markers are restricted to the developing cardiac outflow tract by 56 hpf.

(A) At 56 hpf, acta2 expression is restricted to the developing BA. (B,C) Double transgenic Tg(acta2:EGFP)ca7; Tg(kdrl:mCherry)ci5 embryo shows expression of EGFP in both the atrium and ventricle of the heart at 56 hpf, but not in the BA. (D) acta2 expression is evident at 78 hpf in the BA in both wholemount and cross section (E) and in transgenic animals (F). (G–I) Expression of acta2 continues to be restricted to the BA and ventral aorta (VA) at 100 hpf by in situ hybridization and in transgenic fish. (J–O): Cross sections of the 22 dpf BA show a multilamellar arterial phenotype as visualized by hematoxylin and eosin staining (J), in situ hybridization of acta2 (K) and transgenic GFP (nuclei stained blue with DAPI, L). The bulbus vascular wall consists of three layers: an inner intima, middle media, and outer adventitia (Ad, separated by black lines in J). The intima is endothelial (arrowheads point to nuclei of endothelial cells). The media consists of 3–4 cell-thick layers of vascular smooth muscle cells (M, arrows point to nuclei of SMCs). In comparison to the BA, the vascular wall of the VA at 22 dpf is thin (M) but expresses acta2 by in situ hybridization (N) and GFP in transgenic animals (O). The endothelium of VA is covered by a thin layer of SMCs (arrowheads point to nuclei of SMCs). (P) In situ hybridization of the wholemount adult heart shows strong staining in the bulbus arteriosus, but not ventricle or atrium, which is localized to the myocardial wall in cross section (Q). (R) Wholemount dissected acta2:EGFP transgenic heart shows stronger expression of GFP in the bulbus arteriosus as compared to ventricle. Staining is also continuous with the ventral aorta. In B,C, F, I, and R, green expression is acta2:EGFP transgene. Scale bar in B, C, F, and I is 100 μm. Scale bar in E, H, and Q is 50 μm. Scale bar in K, L, N, and O is 20 μm.

Fig. 4

Mural cell and endothelial development in the ventral head of larval zebrafish.

Confocal micrographs collected from a ventral point of view show a progressive increase in vessel complexity (red, A, C, E, G) and in density of mural cell coverage of aortic arch vessels (green, B, D, F, H) from 4 dpf (A, B), 7 dpf (C, D), 11 dpf (E, F) through 14 dpf (G, H). Heart expression of acta2:EGFP is maintained. aaa = aortic arch arteries; va = ventral aorta; ba = bulbus arteriosus. Scale bar in A represents 100 μm.

Fig. 5

Development of mural cells and endothelial cells as seen in dorsal view.

Confocal micrographs collected from a dorsal point of view show a progressive increase in vessel complexity (red, A, C, E) and in density of mural cell coverage of head vessels (green, B, D, F) at 4 dpf (A, B), 7 dpf (C, D), and 11 dpf (E, F). nc = notochord. Scale bar in A represents 100 μm.

Fig. 6

Development of mural cells and endothelial cells as seen in lateral view.

Confocal micrographs collected from a lateral point of view show a progressive increase in vessel complexity (red, A, C, E, G) and in density of mural cell coverage of aortic arch vessels (green, all panels, inset is enlarged in B, D, F, H to show coverage of aortic arches) at 4 dpf (A, B), 7 dpf (C, D), and 11 dpf (E, F). Scale bar in A represents 100 μm.

Fig. 7

Vascular and visceral mural and smooth muscle cells in the trunk.

(A–B) At 4 dpf, acta2:EGFP positive cells (arrows in B) are seen in the ventral portion of the dorsal aorta, but not in other vessels of the trunk. Floor plate (fp) expression of acta2:EGFP is observed in all images. (C–D) At 14 dpf, the distribution of vascular mural cells to the ventral portion of the dorsal aorta only, is still observed. (E) In contrast to the scarce vascular smooth muscle coverage, visceral smooth muscle cells strongly express the acta2:EGFP transgene at 80 hpf. Scale bars represent 100 μm. Green striations are skeletal muscle fibres.

Fig. 8

Lack of co-localization of mural cell and the ectomesenchymal neural crest marker Fli1a.

Confocal images of 4 and 7(acta2:mCherry) and nuclear neural crest marker (fli1a:nEGFPy7) using ventrally staged embryos. (A) Mural cell and neural crest markers are expressed along the ventral aorta (A′) and aortic arch artery region (A′′) of the 4 dpf embryo. (B) Mural cell and neural crest markers are expressed along the ventral aorta (B′) and aortic arch region (B′′) at 7 dpf. There appears to be little to no co-localization of fluorescent markers at both 4 and 7 dpf. Scale bar in A represents 100 μm. Insets (A′, A′′, B′, B′′) are 100 μm in length. VA = Ventral Aorta, AAA = Aortic Arch Arteries. Arrowheads depict cells that no do not co-localize.

Fig. 9

Vascular mural cells of the ventral head are very stable over time.

(A-E) Single images taken from a confocal microscopy timelapse video. Images were collected at 102 hpf (A) and every three hours for 12 hours (B-E). Insets (A′-E′) show a higher magnification of the ventral aorta, where mural cells that are present at the beginning of the timelapse are still present at the end of the timelapse with no cytokinesis. Arrowheads depict mural cells throughout the timelapse that appear to have little movement. Scale bar represents 100 μm.

Fig. S1

The acta2:GFP transgene is expressed surrounding endothelium in the ventral aorta. Single slices of confocal micrograph stacks of double transgenic Tg(kdrl:mCherry; acta2:EGFP) zebrafish embryos at 7 dpf show green acta2:EGFP cells surrounding red kdrl:mCherry expressing endothelial cells in two different regions of the ventral aorta, distal (A) and proximate (B) to the heart outflow tract. The dorsal aorta is depicted in 11 dpf embryos, with green acta2:EGFP cells surrounding red kdrl:mCherry expressing endothelial cells (C) and with individual fluorescent markers (C′ - green acta2:EGFP cells; C′′ red kdrl:mCherry endothelial cells). Scale bar in B represents 20 μm, scale bar in C represents 50 μm.

Fig. S2

Wholemount image of 4 dpf acta2 transgenic zebrafish shows constant smooth muscle and heart expression and variable skeletal muscle expression. Wholemount images of two independent 4 dpf zebrafish embryos using brightfield and fluorescent microscopy. While embryo 1 shows strong visceral smooth muscle expression and heart expression of the transgene, embryo 2 also shows scattered skeletal muscle fiber expression. The expression in skeletal muscle is variable from embryo to embryo and decreases over developmental time.

Fig. S3

In situ hybridization shows expression of acta2 in the Bulbus Arteriosus and Ventral Aorta. Cross sections of 22 dpf zebrafish showing strong acta2 expression in the bulbus arteriosus and ventral aorta. This provides context to Figure 3 K and N. Scale bars are 50 μm.

Fig. S4

Single or double knockdown of FoxD3 or TFAP2a to block neural crest specification results in a reduction in acta2:GFP cells, but also severe ventral head and blood vessel patterning defects. Representative brightfield images of 2 dpf zebrafish embryos show that both double knockdown (dMO) of FoxD3 and TFAP2A (C) or single knockdown (sMO) of FoxD3 (E) or TFAP2A (G), results in hemorrhage which is not present in control (A). Hydrocephalus of the hindbrain ventricle is also observed in dMO and sMO FoxD3. At 4 dpf, confocal microscopy shows that the control has a well-defined heart outflow tract, with mural cell coverage (kdrl:mCherry – red vessels; acta2:EGFP – green mural cells) (B). In dMO there are severe vessel malformations and a reduction in mural cell coverage (D). In the single FoxD3 (F) and TFAP2A (H) morphants, there are also malformations and reduced mural cell coverage, although these are less severe than the double morphant. Scale bar for A, C, E, G represents 200 µm. Scale bar for B, D, F, H represents 100 μm.

Acknowledgments:
ZFIN wishes to thank the journal PLoS One for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS One