Smith SJ et al. (2005), The MLC1v gene provides a transgenic marker of ...

XB-ART-2263

Dev Dyn 2005 Apr 01;2324:1003-12. doi: 10.1002/dvdy.20274.

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The MLC1v gene provides a transgenic marker of myocardium formation within developing chambers of the Xenopus heart.

Smith SJ , Ataliotis P , Kotecha S , Towers N , Sparrow DB , Mohun TJ .

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Many details of cardiac chamber morphogenesis could be revealed if muscle fiber development could be visualized directly within the hearts of living vertebrate embryos. To achieve this end, we have used the active promoter of the MLC1v gene to drive expression of green fluorescent protein (GFP) in the developing tadpole heart. By using a line of Xenopus laevis frogs transgenic for the MLC1v-EGFP reporter, we have observed regionalized patterns of muscle formation within the ventricular chamber and maturation of the atrial chambers, from the onset of chamber formation through to the adult frog. In f1 generation MLC1v-EGFP animals, promoter activity is first detected within the looping heart tube and delineates the forming ventricular chamber and proximal outflow tract throughout their development. The 8-kb MLC1v promoter faithfully reproduces the embryonic expression of the endogenous MLC1v mRNA. At later larval stages, weak patches of EGFP fluorescence are found on the atrial side of the atrioventricular boundary. Subsequently, an extensive lattice of MLC1v-expressing fibers extend across the mature atrial chambers of adult frog hearts and the transgene reveals the differing arrangement of muscle fibers in chamber versus outflow myocardium. The complete activity of the promoter resides within the proximal 4.5 kb of the MLC1v DNA fragment, whereas key elements regulating chamber-specific expression are present in the proximal-most 1.5 kb. Finally, we demonstrate how cardiac and craniofacial muscle expression of the MLC1v promoter can be used to diagnose mutant phenotypes in living embryos, using the injection of RNA encoding a Tbx1-engrailed repressor-fusion protein as an example.

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Species referenced: Xenopus laevis
Genes referenced: irx4 mlc1 myh4 myh6 myl3 myl4 myl7 nppb tbx1

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	Figure 4. The onset of ventricular cardiac expression for the MLC1v promoter detected in MLC1v::EGFP transgenic embryos. A-E: Ventral views of the heart-forming region of a single MLC1v::EGFP animal at stage 32 (A), stage 34 (B), stage 37 (C), stage 40 (D), and stage 43 (E). F, G: Left lateral views of the same tadpole at stage 37, depicting the whole animal (F) and detail of the head (G). H, I: The heart, dissected from a stage 48 transgenic tadpole (H, bright field image) reveals its chamber myocardium-restricted EGFP fluorescence (I, dark field image). H, heart; IH, interhyoid facial muscle; S, somites; A, atria; V, ventricle; OT, outflow tract.
	MLC1v promoter activity observed in MLC1v::EGFP transgenic frog hearts. A-E: The heart of an 80 day old, recently-metamorphosed froglet. Terminal anaesthesia was used to stop the animal's heart beat and its pericardium removed by dissection to reveal the cardiac chambers. The atria are filled with blood, ventricle part-filled. A, B: Bright-, and dark field views of the entire heart. C-E: High magnification views of the single ventricle (C), left atrium (D), and right atrium and proximal outflow tract (E). F-H: The heart, dissected from a 39 week old juvenile frog, showing bright EGFP fluorescence in the ventricle and some myocardial fibres of the atrial chambers (F). High magnification views of the left atrium (G) and proximal outflow tract (H). A coronary vessel in the outflow tract can be seen as a dark silhouette in (H). Distinct MLC1v-expressing myocardial fibre morphologies are observed in the different compartments of the heart. V, ventricle; LA, left atrium; RA, right atrium; OT, outflow tract; CV, coronary vessel.
	Fig. 1. A: Comparison of whole-mount in situ hybridization analysis of Xenopus MLC1v (A,J), MLC1av (D,N), and MHC alpha (G,R) RNA expression. Left-lateral views of whole embryos are depicted (A) and also higher magnification ventral views of the heart-forming region (J). Anterior is to the left of all images. MLC1v: stage 25 (A), stage 31 (B), stage 38 (C), stage 31 (J), stage 35 (K), stage 38 (L), stage 41 (M). MLC1av: stage 24 (D), stage 33 (E), stage 39 (F), stage 31 (N), stage 32 (O), stage 35 (P), stage 39 (Q). MHC alpha: stage 30 (G), stage 32 (H), stage 39 (I), stage 30 (R), stage 32 (S), stage 35 (T), stage 39 (U). M: The apparent weaker MLC1v staining observed at stage 41 more likely reflects the poor tissue penetration of antisense RNA probes that occurs at later tadpole stages. S, somites; H, heart; IH, interhyoid facial muscle; LH, lymph heart; MLC, myosin light chain. These embryos were obtained using fertilized eggs from an albino female frog; hence, little ectodermal pigment is evident. In all figures presented, the direction of the anteriorosterior (A-P) axis is indicated at the bottom right of key panels (A,J) by a double-headed arrow.
	Fig. 2. The ventricular chamber-restricted embryonic expression of Xenopus MLC1v compared with other cardiac markers. High-magnification, left lateral views of the hearts of tadpoles (A, anterior to the left) that had been rendered transparent using benzyl alcohol/benzyl benzoate treatment, after whole-mount in situ hybridization. A: Cardiac MLC1v mRNA expression at stage 38. B: MLC1av expression at stage 39. C: MHC expression at stage 39. D: MLC2 expression at stage 38. E: Irx4 expression at stage 38. F: B-Type natriuretic peptide precursor (BNF) expression at stage 38. Cardiac domains of gene expression are indicated by red arrows and other domains by black arrows. The apparent differences in outflow tract gene expression of the four sarcomeric muscle proteins shown are due to the changing rostral to caudal position of the proximal outflow tract between stages 38 to 39. Only Irx4 mRNA is absent from the proximal outflow tract. IH, interhyoid facial muscle; V, ventricle; A, atria. G: Transverse sections through Xenopus stage 35 tadpole hearts after whole-mount in situ hybridization for MLC1v and MLC1av mRNA. Four representative sections (10 m) marking progressively posterior cardiac slices are shown for MLC1v (G) and MLC1av (K), with the section number indicated at the bottom-right of each panel. Ec, endocardium; OT, outflow tract; V.Mc, ventricular myocardium; A.Mc, atrial myocardium; LV, liver; MLC, myosin light chain.
	Figure 3. The activity of the Xenopus MLC1v gene promoter in founder generation transgenic tadpoles. A–E: Enhanced green fluorescent protein (EGFP) expression directed by the entire 8-kb MLC1v promoter fragment (A–E) and that resulting from a 1.5-kb proximal MLC1v promoter fragment produced by PacI digestion (F–K). A: Right-lateral view of a stage 45 tadpole showing strong EGFP fluorescence in the heart, mouth, and somites. The somitic fluorescence observed in founder generation, transient-transgenics differs from that of the f1 generation transgenic line and also the endogenous MLC1v RNA expression (see Results section). This ectopic somitic expression is likely the result of nonintegrated, linear-plasmid DNA. B,C: Higher magnification right-lateral view (B) of the heart and ventral view (C) of the same tadpole photographed in A. This animal has an inverse laterality of its visceral organs, as indicated by the direction of heart looping and left-sided gall bladder location. Such situs inversions are frequently observed in laboratory stocks of Xenopus laevis. D,E: The heart, having been dissected from a stage 43 tadpole (D, brightfield image; E, darkfield image) revealing chamber myocardium-restricted EGFP fluorescence. F–K: The 1.5-kb proximal MLC1v promoter fragment drives weak EGFP expression in the same tissues as the full-length promoter, but with a patchy, mosaic distribution, as shown by the stage 46 tadpole depicted. F: Left-lateral view. G,F: High-magnification detail image of a somite (G), whose relative position in the trunk is also indicated by the white box in F. H: Ventral view of the heart. I: Ventral view of the tadpole head and body. J,K: Dissected heart (J, brightfield image) showing patchy ventricular EGFP fluorescence (K, darkfield image). The darkfield images presented in this and all subsequent figures had their input/output levels for the different color channels adjusted individually in Adobe Photoshop. This approach enables the silhouette of the animal to be observed, while giving the EGFP fluorescence a false, blue–green color. IH, interhyoid facial muscle; H, heart; S, somites; GB, gall bladder; A, atria; V, ventricle; OT, outflow tract; MLC, myosin light chain.
	Figure 6. Visceral arch malformations caused by a dominant-negative form of Tbx1, revealed in MLC1v-EGFP embryos. A,B: Heterozygote f2 generation MLC1v-EGFP embryos were injected into both blastomeres at the two-cell stage with 125 pg of RNA encoding a Xenopus Tbx1-EnR fusion protein (plus 125 pg of β-gal RNA, A), or 250 pg of β-galactosidase RNA control (B). Embryos were allowed to develop until stage 47, and their phenotype was assessed by the configuration of the EGFP-fluorescing muscle groups. IH, interhyoid facial muscle; LvH, levator hyoid muscles; VT, ventral transverse muscle; MLC, myosin light chain; EGFP, enhanced green fluorescent protein.