XB-ART-25106Development 1991 Feb 01;1112:551-60.
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Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos.
We have cloned a Xenopus cDNA that encodes a homologue of the human myogenic factor, Myf-5. Xenopus Myf-5 (XMyf5) transcripts first accumulate in the prospective somite region of early gastrulae. The pattern of XMyf5 expression is similar to that of the Xenopus MyoD (XMyoD) gene, except that XMyf5 transcripts are largely restricted to posterior somitic mesoderm even before any somites have formed. Transient ectopic expression of XMyf5 activates cardiac actin and XMyoD genes in animal cap cells, but does not cause full myogenesis, even in combination with XMyoD. These results suggest that XMyf5 acts together with XMyoD as one of the set of genes regulating the earliest events of myogenesis, additional factors being required for complete muscle differentiation.
PubMed ID: 1716555
Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a col11a2 myf5 myf6 myod1 myog tbx2
Article Images: [+] show captions
|Fig. 1. Sequence of the XMyf5-2 cDNA. The asterisk indicates the translation stop, and the poly(A)-addition signal is underlined.|
|Fig. 2. Comparison of the predicted XMyf5 protein sequence to those of representatives of the four mammalian myogenic factors, human Myf-5 (Braun et al. 19896), mouse MyoD (Davis et al. 1987), rat MRF4 (Rhodes and Konieczny, 1989) and rat myogenin (Wright et al. 1989). The box surrounds the basic-helix-loop-helix domain; amino acid residues identical to those of XMyf5 are shown in bold. XMyf5 is more closely related to Myf-5 than to the other factors, showing extensive similarity outside the basic-helix-loop-helix region.|
|Fig. 3. XMyf5 expression in early development. (A) Northern blot of RNA from early embryos (two per lane) at timed stages after fertilization, probed successively for XMyf5, XMyoD and cardiac actin (not shown) transcripts (see Materials and methods). (B) Graph plotted from densitometry of appropriate autoradiographic exposures of the blot shown in A, using transcript number measurements for XMyf5 (see Materials and methods) and XMyoD (Hopwood et al. 1989a). The content of XMyf5 and XMyoD transcripts increases before transcripts of the cardiac actin gene first appear. Stages are those of Nieuwkoop and Faber (1967). Embryos were cultured at 23 °C.|
|Fig. 4. XMyf5 transcripts are somite-specific. In situ hybridizations to transverse sections through the posterior parts of late (stlli) gastrulae (A,B) and late (stl8) neurulae (C,D), probed for XMyf5 (A,C) or XMyoD (B,D). Note that the neurula sections are from the 'tail' region used for the northern analysis in Fig. 5 below, and show somite-specific expression in this part of the embryo which was not dissected into its component tissues. The gastrula sections, from wild-type embryos, were bleached with hydrogen peroxide (see Materials and methods); the neurula sections were from albino embryos. Probe concentrations (and exposure times) were: (A,B) 400ctsmin~'^l~1 (one month); (C) TSOctsmin'Vl1 (one week); (D) V"1 (two weeks).|
|Fig. 5. Northern blot of RNA from dissected parts of late (stl8) neurulae, probed successively for transcripts from the XMyf5, cardiac actin (Mohun et al. 1984), and EF-lo- (Krieg et al. 1989) genes. Cardiac actin is a striated muscle marker, and EF-la- shows the relative amounts of total RNA in each lane. Parts were pooled from five dissected embryos; RNA from one whole embryo was analysed for comparison. The dorsal endoderm and notochord lanes were relatively underloaded in this experiment. Two other experiments, in which relatively more endodermal RNA was used, showed that the endoderm does not contain a significant concentration of XMyf5 RNA at stl3 or at stl8 (data not shown). That the notochord does not express XMyf5 was shown by in situ hybridization (Fig. 4A,C). Dl, dorsal; vl, ventral; end, endoderm; mes, mesoderm; ect, ectoderm.|
|Fig. 6. XMyf5 transcripts become restricted to posterior somites before those from the XMyoD gene. Northern blot of RNA from anterior (1), middle (2) and posterior (3) parts of late gastrulae (stl2i), mid-neurulae (stl5) and late neurulae (stl8) probed successively for transcripts from XMyf5, XMyoD, cardiac actin and EF-l<v genes. Parts from six embryos were loaded in each lane.|
|Fig. 7. (A) Clones for expression of XMyf5 in embryos, and point mutant control. Their construction is described in the Materials and methods. BHLH, basic-helix-loophelix region; A23C30, poly(dA)-poly(dC) tracts; dotted line Xenopus /3-globin UTRs. (B) Translation in a rabbit reticulocyte lysate of XMyoD, XMyoD114P, XMyf5 and XMyf5-102P synthetic mRNAs. AutoradiogTaph of 35Slabelled protein. Numbers are MrXl0~3 of marker proteins. The mutant controls are translated as efficiently as the non-mutated mRNAs.|
|Fig. 8. XMyf5 can activate cardiac actin and XMyoD genes in animal cap cells. Animal caps were dissected from RNA-injected embryos and cultured until sibling embryos became late neurulae (stl8), when they were frozen for analysis by northern blotting. RNA from 12 animal caps was pooled and RNA from two animal cap equivalents probed successively for cardiac actin transcripts, injected RNA, and EF-lo-transcripts. The injected RNA was detected using a probe that recognizes the /3-globin 3' UTR that is part of the injected transcripts, but is not present in uninjected embryos at the stage of analysis; injected XMyf5 transcripts are distinguishable from injected XMyoD RNA, because they are smaller. The remaining 10 animal cap equivalents were probed for frww-activated transcripts from the endogenous (endog.) XMyoD gene(s). Note, therefore, that for this panel, five times more total RNA was used in the animal cap samples relative to the whole embryo standards than is indicated by the EF-liy analysis shown. The cardiac actin and XMyoD genes were activated in XMyoD- and XMyf5-injected animal caps, but not in animal caps injected with the point mutant XMyf5-102P RNA. Co-injection of XMyoD and XMyf5 RNAs did not show any synergy in activating the XMyoD and cardiac actin genes.|