XB-ART-21208Genes Dev. June 1, 1994; 8 (11): 1324-34.
The RSRF/MEF2 protein SL1 regulates cardiac muscle-specific transcription of a myosin light-chain gene in Xenopus embryos.
We have examined the role of two RSRF/MEF2 proteins in the onset of skeletal and cardiac muscle differentiation in early Xenopus embryos. In normal development, zygotic expression of SL1 (MEF2D) precedes that of SL2 (MEF2A) by several hours, but neither gene is expressed prior to the accumulation of MyoD and Myf5 transcripts in the somitic mesoderm. Ectopic expression of the myogenic factors in explants of presumptive ectoderm induces expression of both SL1 and SL2, whereas in reciprocal experiments, neither RSRF protein activates the endogenous myoD or Myf5 genes. We conclude that SL1 and SL2 lie downstream of these myogenic factors in the skeletal myogenic pathway. SL1 is distinguished from SL2 in being expressed in the presumptive heart region of the early tailbud embryo, prior to detection of any markers for cardiac muscle differentiation. Furthermore, ectopic SL1 induces the expression of an endogenous cardiac muscle-specific myosin light-chain (XMLC2) gene in cultured blastula animal pole explants, whereas SL2 has no comparable effect. These results demonstrate that in addition to a possible role in skeletal myogenesis, SL1 also acts in vivo as a regulator of cardiac muscle-specific transcription.
PubMed ID: 7926733
Article link: Genes Dev.
Genes referenced: actc1 actl6a mef2a mef2d myf5 myl2 myl7 myod1 tbx2
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|Figure 1. Sequential expression of XmyoD and the RSRF genes during early development. RNAs from successive stages of development from blastula (stage 8) to mid-neurula (stage 15) were analyzed by RNase protection assay for the presence of XmyoD (A), SL1 (B), cardiac actin (C), and SL2 (D) transcripts. Total RNA (101~g) was used for each assay. For each probe, the positions of protected fragments (solid arrows) and undigested probe (open triangles) are inidicated.|
|Figure 2. Ectopic expression of MyoD proteins activates RSRF gene transcription in animal pole explants. Fertilized eggs were injected with RSRF and myogenic factor RNAs, cultured until blastula stage, and dissected to obtain animal pole explants. These were cultured until mid-late neurula stage and analyzed by RNase protection assay for the presence of cardiac actin (A), SL1 (B), and SL2 (C) transcripts. (Lane 1) 10 ~g of tRNA (control); (lanes 2,1 I) uninjected; (lanes 3-5) SLI, SL2, and an equimolar combination of the two, respectively; (lanes 6-8)XE12 in combination with XmyoD (My), Xmyf5 (Mj) and mouse myogenin (Mg), respectively; (lanes 9,10) XE12/XmyoD with SL1 and SL2, respectively; (lane 12) total RNA from neurula (stage 18) embryos. The equivalent of five explants was analyzed in each assay. Approximate size markers were provided by an endlabeled HinfI digest of pBR322 and are shown adjacent to undigested probe. Full-length protected fragments are indicated. The cardiac actin probe cross-hybridizes with ubiquitous cytoskeletal actin transcripts that give rise to a cluster of partial protection products (indicated in A). In B and C, the presence of the injected, synthetic RNA gives rise to a protected band that is larger than that obtained from the endogenous RSRF mRNA. (A background smear was also observed routinely with injected samples). The SL2 probe detects a second endogenous mRNA that is coexpressed with SL2 and produces a smaller protected fragment.|
|Figure 3. SL1 expression in the prospective heart region of tailbud embryos. RNA from anterior-dorsal (D) and anterior-ventral (V) fragments of embryos was analyzed by RNase protection assay for the presence of cardiac actin, XMLC2, SL-1, and SL-2 transcripts (A-D, respectively). The equivalent of five fragments was used in each assay. Samples were also tested for the presence of a myotomal muscle-specific marker (labeled "XMHC") (Logan and Mohun 1993), detected using a probe for XMHCa RNA (E). Explants were dissected from late neurula (stage 18) to late tailbud (stage 28) embryos.|
|Figure 4. (A) Nucleotide sequence of a Xenopus MLC2 cDNA. The nucleotide sequence is derived from two, overlapping cDNA isolates, hXMLC2-5 contained the entire sequence shown except exon 5 (nucleotides 393-441). This was present in a partial cDNA, hXMLC2-12, which lacks the first 260 nucleotides of the sequence. The predicted XMLC2 polypeptide sequence is shown, as is a putative poly(A) addition sequence (bold). (B) Comparison of XMLC2 and other vertebrate MLC2 proteins. The XMLC2 polypeptide sequence is aligned with the human atrial (Hailstones et al. 1992}, human ventricular (EMBL accession $221011, chick (Winter et al. 1985), and rat (Henderson et al. 1989)MLC2 sequences. These are arranged in order of similarity to the frog sequence as determined by use of the UWGCG Pileup program. Only residues that differ from the frog sequence have been shown, and gaps (dashes} have been introduced to permit optimal alignment.|
|Figure 5. XMLC2 expression during development. Total RNA from embryos, cultured explants, and tissues was tested for XMLC2 mRNA by use of an RNase protection assay. (Lane 1) tRNA control; (lanes 2-10) two embryos from neurula to tadpole stages of development (stages 18-35, as indicated); (lanes 11,12) five anterior-dorsal (D) and anterior-ventral (V) regions of swimming tadpoles (stage 37); (lanes 13,14) the equivalent of five blastula animal pole explants treated with 32 or 80 U/ml of activin A and cultured until stage 42; (lanes 15-17) two tadpole (stage 42) heart tubes (EH), 0.5 txg of adult heart (H), and 10 ~g of adult skeletal muscle (Sk). As an internal control, a probe for EF1 a mRNA was included in each assay. An end-labeled HinfI digest of pBR322 was used for approximate size markers.|
|Figure 6. XMLC2 is a marker for terminal differentiation of cardiac muscle in Xenopus embryos. The distribution of XMLC2 RNA was examined by use of albino embryos and digoxigenin-labeled probes. Staining was detected first in stage 28/29 tadpoles in the presumptive heart region and was subsequently confined to the developing heart tube. A stage 32 embryo is shown before (a) and after (b) clearing. In later stages, staining was detected in both atrial and venticular chambers of the beating tadpole heart. No staining was detected with a sense control probe.|
|Figure 7. Ectopic expression of SL1 activates XMLC2 gene transcription in animal pole explants. Cultured animal pole explants from injected embryos were analyzed by RNase protection at the neurula stage for expression of the heart-specific XMLC2 gene. Levels of EFla mRNA were monitored in the same assay. (P) Undigested probe; (lane 1) 10 ~g of tRNA; (lane 2) control explants (uninjected); (lanes 3-5) explants from embryos injected with SL1 and SL2 RNA, individually or in combination (as indicated); (lanes 6, 7) 5 ~tg of late neurula (stage 20) and tadpole (stage 37) RNA, respectively. (Size markers as in earlier figures).|