Cox WG and Neff AW (1995), Cardiac myosin heavy chain expression during he...

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Differentiation 1995 Apr 01;584:269-80.

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Cardiac myosin heavy chain expression during heart development in Xenopus laevis.

Cox WG , Neff AW .

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Muscle-specific gene expression in the heart during Xenopus development was investigated using reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization to detect transcripts of the gene for the cardiac myosin heavy chain (CMHC). RT-PCR analysis determined that CMHC transcripts are present in the cardiac mesoderm at state 13, demonstrating that muscle-specific gene expression in the primitive myocardium has begun by the early neurula stage, approximately 30 h before the heart beat begins. Xenopus, therefore, is similar to amniotes and mammals in that cardiac precursor cells begin to express muscle-specific gene transcripts soon after commitment to the cardiac myocyte lineage. The earliest CMHC gene transcripts can be detected in the heart using whole-mount in situ hybridization is early tailbud stage 28, which coincides with the onset of heart tube morphogenesis. CMHC gene expression was also detected in skeletal muscle: RT-PCR analysis determined that CMHC transcripts are transiently expressed in the somite during the initial phases of skeletal muscle differentiation. Furthermore, CMHC mRNAs are expressed in a subset of head muscles of the feeding tadpole. CMHC gene expression is induced in ectodermal cells of the animal cap in blastula-stage embryos injected with synthetic MyoD or Myf5 RNA, suggesting that the CMHC gene contains regulatory elements that are responsive to the activity of those skeletal-muscle-specific transcription factors.

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Species referenced: Xenopus laevis
Genes referenced: actc1 myf5 myh4 myh6 myod1 odc1

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	Fig. 1. A Nucleotide sequence of the Xenopus cardiac myosin heavy chain (XCMHC) cDNA. Differences in nucleotide sequence from XMHCa [40] are blocked. The stop codon is marked (***) and the poly adenylation site is in bold. XCMHC-specific oligonucleotide primer sequences are underlined. B Comparison of the decduced amino acid sequence of Xenopus cardiac MHC with other Xenopus MHC genes. The Xenopus MHC genes cloned to date, with the exception of XMHCa, are skeletal isoforms; their similarity to XCMHC in a @-amino acid overlap is 79.7% (E19) [55], 84.3% (E3) [55], 72.7% (A7) [55]. The LM [ 101 isoform is 72% similar to XCMHC in a 44-amino acid overlap. There are three amino acid differences between XCMHC and XMHCa. C Comparison of the deduced amino acid sequence of XCMHC to cardiac MHCs of other vertebrates. XCMHC was found to be similar to the aand P-cardiac MHC genes of mouse (87.5%. 85.9%) [21,54], rat (85.9%. 85.9%) [43], and human (85.9%. 85.9%) [35,74] in a 64-amino acid overlap. XCMHC is 73.4% similar to the avian ventricular MHC in this region [61
	Fig. 2. Detection of CMHC transcripts by Northern blot analysis. Equal amounts of total RNA from stage-40 (sr.40) heart, stage 40 somite, and adult heart (whole) were hybridized to a 32P-labeled cDNA probe for a-cardiac actin and CMHC. Hybridization with an a-cardiac actin probe served as a positive control for tissues not expressing CMHC. Equal amounts of 28s RNA in each lane is shown. CMHC is a 7-Kb transcript present in stage-40 and adult heart
	Fig. 3A, B. Reverse transcriptase-polymerase chain reaction (RT-PCR) detection of CMHC, a-cardiac actin, El9 MHC and ornithine decarboxylase (ODC) mRNAs during embryogenesis. Analysis of stage-9 to -14 embryos (A) and stage-I5 to -40 heart and somite (B). Animal cap (C),m arginal zone (M), vegetal core (Vc), dorsal (D), ventral (V), heart (H), somite (8. Poly A+ RNA was isolated from dissected tissues, reverse transcribed, and subjected to PCR with gene specific primers. Expression of El9 MHC served as a marker for dorsal mesoderm and was assayed only in the stage-9 to stage-14 series. At stage 15, the heart primordia are close to fusing at the ventral midline, making dissection of the cardiac mesoderm from the dorsal mesoderm easy. Thus, stage- 15 to -40 tissues were not assayed for El9 expression. Detection of acardiac actin served as a positive control for mesoderdmuscle not expressing CMHC. ODC amplification served as an internal control (see Methods) as well as a positive control for tissues not expressing muscle-specific markers
	Fig. 4. Whole-mount in situ hybridization detection of localized CMHC expression in stage-28 (upper) and stage-34 (lower) animals. Stage 28 is the earliest stage when CMHC expression can be detected in the heart (arrow) using this method of detection. At heart-beat stage 34, the entire heart tube is labeled (arrow). No labeling of the myotome was observed
	Fig. 5A-C. Whole-mount immunohistochemical detection of MHC expression. The MF20 antibody used detects sarcomeric MHCs. A A stage-3 I embryo showing detection of MHC in the heart (arrow). B In the stage-28 embryo, labeling of the heart (area befween the bars) was not obvious. However, when viewed with confocal microscopy (C), labeling of the heart was observed
	Fig. 6A-D. Localized expression of CMHC and a-cardiac actin in the stage-42 embryo detected by wholemount in situ hybridization. A The upper embryo is labeled with CMHC probe whereas the lower embryo shows a-cardiac actin labeling. The arrow points to the anterior lymph heart. B Dorsal view showing CMHC expression in the anterior pair of lymph hearts (arrowheads).C Ventral view showing CMHC expression in a subset of head muscles. D Ventral view showing a-cardiac actin expression in the head. In C and D, labeling of the heart (large arrowhead), laryngis (thin arrow), quadrato- hyo-angularis (thick arrow), orbitohyoideus (arrowhead), and interhyoideus (double arrowhead) is shown. Closer examination of the CMHC-labeled heart showed that both the ventricle and the atrium were labeled
	Fig. 7. RT-PCR detection of a-cardiac actin, CMHC, MyoD, and ODC transcripts in the head muscles (hm, interhyoideus, orbitohyoideus), heart (H), and somite (5') of the stage-37/38 embryo. Poly A+ RNA was isolated from these muscles, reverse transcribed, and subjected to PCR with gene-specific primers. Detection of a-cardiac actin mRNA served as a positive control. Amplification of OM3 message served as iln internal control (see Methods)
	Fig. 8. RT-PCR detection of CMHC expression in animal cap ectoderm. Blastula-stage animal cap ectoderm was collected from embryos injected with synthetic RNA for EFla, Myf5, or MyoD and subjected to RT-PCR analysis. Gene-specific oligonucleotide primer pairs for Myf5, MyoD, CMHC, El9 MHC, and ODC were used. Expression of CMHC and El9 MHC was detected only in caps taken from Myf5- or M yoD-injected embryos. Amplification of ODC message served as an internal control (see Methods)