Logan, Malcolm Patrick Oliver; (1995) Cardiac muscle differentiation in Xenopus laevis embryos. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes work carried out to initiate a study of cardiogenesis in the amphibian, Xenopus laevis. I have isolated and characterised a cDNA fragment encoding a portion of the myosin heavy chain α-isoform (XMHCα) from Xenopus laevis embryos and characterised another Xenopus cDNA encoding a myosin light chain isoform, XMLC2. Both the XMHC and XMLC2 transcripts are expressed exclusively in adult heart tissue. Whole mount RNA in situ hybridisation indicates that expression of the XMHCα and XMLC2 genes are restricted to the developing heart primordium. They therefore provide tissue-specific markers for cardiac muscle differentiation during early embryogenesis. Using these transcripts as molecular markers, I can detect the onset of cardiac muscle differentiation in an anterior-ventral region of tailbud embryos, many hours before the appearance of a beating heart. XMHCα and XMLC2 gene expression can be induced in isolated animal pole explants of blastulae by treatment with the growth factor, activin A. Induction is dose-dependent, requiring high doses of the growth factor compared with that required for myotomal (skeletal) muscle differentiation. In contrast, no XMHCα transcripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differentiation. In addition, fusion potentiates induction of cardiac muscle differentiation and exposure to activin for just several hours was sufficient to induce markers after days of incubation. Activin-induced explants show a similar temporal pattern of XMHCα gene expression to that found in normal embryogenesis. Furthermore, cells expressing this gene appear clustered in one or two foci within fused explant aggregates, which often show regular, spontaneous contractions after several days in culture. These results show that terminal differentiation of cardiac muscle can occur in growth factor- induced explants and may be distinguished from skeletal muscle differentiation by the dose and nature of the inducing factor. Explant aggregates containing foci of beating cells have been analysed further by histological and immunological techniques. These studies have shown that the amount of induced cardiac muscle is small compared with the amount of induced skeletal muscle within explants. In addition, induction of endoderm within the explant may be required for the formation of heart muscle. Lineage label analysis has been used to follow the fate of cells of the two heart anlage and to show that each anlage is not pre-specified to a particular fate prior to fusion of the two primordia. Lineage labels have also been used to examine the relative heart-forming potencies of cells from different regions of the heart field and have shown that cells within the field appear equipotent for heart formation. Using an explant culture system and terminal differentiation markers for heart muscle, the restriction of the heart morphogenetic field during tailbud stages, has been studied. In contrast to the results from studies that used a beating-heart assay for heart formation, the restriction of the heart morphogenetic field is not complete by late tailbud, stage 28. Thus, the ability of cells within the heart field to express terminal differentiation markers can be clearly distinguished from their potency to form a beating heart tube, in culture. In addition, analysis by whole mount RNA in situ hybridisation has indicated that restriction of the heart field is apparent in lateral regions after removal of the anterior ventral cells fated to form heart. This suggests that repressive influences on heart formation in vivo originate from outside the heart field.

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