Logan M and Mohun T (1993), Induction of cardiac muscle differentiation in ...

XB-ART-22459

Development 1993 Jul 01;1183:865-75.

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Induction of cardiac muscle differentiation in isolated animal pole explants of Xenopus laevis embryos.

Logan M , Mohun T .

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We have isolated a cDNA fragment encoding a portion of the myosin heavy chain alpha-isoform (XMHC alpha) in the amphibian, Xenopus laevis. The XMHC alpha transcript is highly enriched in adult heart RNA and is expressed exclusively in embryonic heart tissue. It therefore provides a tissue-specific marker for cardiac muscle differentiation during early embryogenesis. Using an RNAase protection assay, we 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. Whole-mount in situ RNA hybridisation indicates that expression of the XMHC alpha gene is restricted to the developing heart primordium. XMHC alpha gene expression can also 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 XMHC alpha transcripts are detected in explants incubated with basic FGF, despite the induction of myotomal muscle differentiation. Activin-induced explants show a similar temporal pattern of XMHC alpha 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.

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Species referenced: Xenopus laevis
Genes referenced: actl6a eef1a2 fgf2 fubp1 inhba mt-tr myh6 myod1 tbx2 trna

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	Fig. 1. Sequence of a Xenopus MHCa cDNA fragment. (A) Nucleotide sequence of a Xenopus MHC cDNA fragment isolated from adult heart cDNA. The sequence is derived from two overlapping clones obtained by RT-PCR (see Methods) and has been submitted to the EMBL database. The encoded polypeptide fragment is shown along with the termination codon (*) and putative polyadenylation signal (underlined). (B) Alignment of the Xenopus MHC polypeptide sequence with the carboxy terminal region of other vertebrate MHC sequences. The XMHCa sequence most closely resembles the human (Yamauchi-Takihara et al., 1989), mouse (EMBL accession number M74751), rat (McNally et al., 1989), rabbit (Sinha et al., 1984) and hamster (Liew and Jandreski, 1986) MHCa proteins in the final seven residues. The human (Yamauchi-Takihara et al., 1989), mouse (EMBL accession number M74752), rat (Kraft et al., 1989) and hamster (Jandreski et al., 1988) MHCb isoforms are also shown, along with the b-like newt MHCc/s (Casimir et al., 1988) and the cardiac-specific chick VMHC1 (Bisaha and Bader, 1991) sequences.
	Fig. 2. Expression of XMHCa in adult frog tissues. The distribution of XMHCa mRNA in adult frog tissues was analysed by RNAase protection assay (A) and compared with that of the cardiac actin gene transcript (B). M, size markers (HinfI-digested pBR322); P, undigested probe; lane 1, tRNA control; lanes 2-7, stomach, oviduct, lung, liver, skeletal muscle, heart RNA, respectively; lanes 8-9, tailbud (stage 24) and tadpole (stage 37/8) embryo RNA, respectively. 10 mg of total RNA was used in each assay, except in the case of the heart sample which comprised only 1 mg to avoid overexposure of the autoradiogram. Fulllength, protected fragments for each probe are indicated. As an internal control, a probe for the highly abundant EF1a was included in the XMHCa assay.
	Fig. 3.Expression of XMHCa Xenopus embryos. The prevalence of cardiac actin (A), XMyoD (B) and XMHCa transcripts (C) in early embryos was analysed by RNAase protection assay. Myotomeenriched dorsal fragments were dissected from tailbud or early tadpole embryos and compared with ventral pieces that included the heart anlage (D). P, undigested probe; t, 10 mg tRNA control; lanes 2, 4, 6, 8 and 10, dorsal fragments (stages 26, 28, 30, 32 and 34, respectively); lanes 3, 5, 7, 9 and 11, ventral fragment from the same embryos. Total RNA from four fragments was analysed in each lane. These comprised approximately similar amounts, as determined using the EF1a probe (data not shown). Full-length, protected fragments for each probe are indicated. The cardiac actin probe gives several partial protection products resulting from cross hybridisation with cytoskeletal actin transcripts. A prominent, dorsal-specific band (open triangle) is obtained with the XMHCa probe.
	Fig. 4. Distribution of XMHCa mRNA in Xenopus embryos. Whole-mount in situ hybridisation was used to examine the spatial distribution of XMHCa transcripts in tailbud and tadpole embryos. Specific staining (purple) is readily distinguished from the natural pigmentation (brown/black) of the embryos. (A) Developmental series comprising (from top to bottom) embryo stages 29, 35 and 38. Staining for XMHCa can be seen in the ventral region immediately anterior to the gut. This corresponds to the developing heart anlage. (B) Anterior region of a stage 35 tadpole. XMHCa expression is clearly restricted to the conus arteriosus, heart chambers and sinus venosus that have formed from the endocardial tube.
	Fig. 5. Induction of XMHCa expression in animal pole explants. Activin-induced animal pole explants were assayed for the presence of cardiac actin (A), XMyoD (B) and XMHCa transcripts (C) using an RNAase protection assay. P, undigested probe; lane 1, 10 mg tRNA; lanes 2-8, total RNA from induced explants cultured until sibling embryos reached stages 18, 22, 26, 30, 34, 38 and 42, respectively; lane 9, 3 mg of total tadpole RNA (stage 42). Full-length, protected fragments for each probe are indicated, as is the myotome-specific band (open triangle) obtained with the XMHCa probe. The equivalent of four explants were analysed in each assay and contained similar amounts of total RNA, as judged by the level of EF1a transcripts (C).
	Fig. 6. XMHCa expression in explants is induced by activin A but not by bFGF. Blastula animal pole explants were induced with bFGF or activin A and cultured until control embryos reached stage 42. Total RNA was assayed for cardiac actin (A) and XMHCa mRNA (B) by RNAase protection. P, Undigested probe; lane 1, 10 mg tRNA; lane 2, uninduced explants (control for bFGF); lanes 3-7, explants induced with 10, 20, 40, 80 and 120 units/ml of Xenopus recombinant bFGF; lane 8, uninduced explants (control for activin); lanes 9-11, explants induced with 8, 32 and 80 units/ml of human recombinant activin A. The equivalent of four explants were analysed in each assay. The level of cardiac actin mRNA detected should be normalised by reference to the signal obtained for the cross-hybridising cytoskeletal actin transcripts (A). In the XMHCa assays (B), the EF1a probe was included to monitor the relative amounts of explant RNA.
	Fig. 7. XMHCa expression is induced in discrete foci within cultured explants. Whole-mount in situ hybridisation was used to examine the distribution of XMHCa transcripts in activin-induced animal pole explants. Discrete purple foci of signal (arrows) can be seen in fused aggregates of explants (A) which were dissected from pigmented embryos. No signal was detected in explants that failed to fuse, nor in explants that were cultured in isolation (B).