The MLC1v gene provides a transgenic marker of myocardium formation within developing chambers of the Xenopus heart.
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.
PubMed ID: 15736168
Article link: Dev Dyn.
Genes referenced: irx4 mlc1 myh4 myh6 myl2 myl3 myl4 tbx1
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|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.|