Embryonic temperature and the relative timing of muscle-specific genes during development in herring (Clupea harengus L.)
Gatty Marine Laboratory, Division of Environmental and Evolutionary Biology, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, Scotland
Present address: Department of Anatomy and Physiology, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland
*Present address: Centre for Biomolecular Sciences, School of Chemistry, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland (e-mail: gkt{at}st-and.ac.uk)
Accepted August 13, 2001
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Summary |
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Key words: herring, Clupea harengus, temperature, muscle, MyoD, myogenin, myosin heavy chain.
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Introduction |
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There are four members of the MyoD gene family of myogenic regulatory factors (MRFs) in vertebrates. Gene knock-out experiments in the mouse have established that MyoD proteins are components of a highly complex regulatory network that play a pivotal role in myogenesis (Rudnicki et al., 1993; Hasty et al., 1993). The primary MRFs, MyoD and myf-5, are involved in muscle lineage determination whereas the secondary MRFs, myogenin and MRF4, play a role in initiating and stabilising the differentiation programme (Perry and Rudnicki, 2000). MRFs share a highly conserved basic region, which mediates DNA binding, and a helixloophelix domain that regulates dimerisation with the universal proteins E12 and E47 coded by the E2A gene (Ma et al., 1994). The resulting heterodimer has a high affinity for the E-box motif present in the promoter region of the majority of muscle-specific genes (Watabe, 2001). Accessory proteins involved in muscle differentiation include a family of cysteine-rich proteins (CRPs) with a LIM domain that form a complex with the MyoD/E protein complex (Kong et al., 1997). The cloning and expression of MRFs, E-proteins and CRP isoforms have been reported in a very restricted number of fish species [for a review, see Watabe (Watabe, 2001)].
Fertilisation of the eggs is external in the majority of teleosts, and the rate of development is therefore strongly influenced by the prevailing temperature. There is also evidence that temperature affects the relative timing of various aspects of the muscle developmental programme (Johnston et al., 1996). We have studied myogenesis in a spring-spawning stock of Atlantic herring (Clupea harengus L.) from the Firth of Clyde, Scotland (Johnston et al., 1995; Johnston et al., 1997; Johnston et al., 1998). Historical records indicate that sea temperatures during deposition of the eggs range from 5 to 10°C and increase by several degrees during the period of embryonic development (Jones and Jeffs, 1991). At 515°C, the cranial-to-caudal progression of multinucleated myotube formation occurred at similar somite stages (Johnston et al., 1995). In contrast, subsequent aspects of differentiation, as demonstrated by the appearance of myofibrils and the development of acetylcholinesterase staining at the neuromuscular junctions, occurred at later somite stages as the incubation temperature was reduced (Johnston et al., 1995; Johnston et al., 1997).
Recent studies indicate that the degree of developmental plasticity of myogenesis associated with temperature variation differs between herring populations spawning at different times of the year (Johnston et al., 2001). Although transitory in nature, changes in the relative timing of differentiation with temperature may be of considerable ecological importance. Clyde herring larvae were found to exhibit more advanced developmental characters at shorter body lengths, including expression of adult myofibrillar protein isoforms (Johnston et al., 1997) and the formation of fin rays and associated muscles (Johnston et al., 1998; Johnston et al., 2001), when hatching from eggs incubated at 12°C compared with 5°C. Over the length range 12.518.0 mm, larvae hatching from eggs incubated at 12°C had a more advanced carangiform style of swimming and superior fast-start performance than fish hatching from eggs incubated at 5°C, in spite of the fish having the same thermal experience following hatching (Johnston et al., 2001).
The objective of the present study was to test the hypothesis that temperature influences the relative timing of transcription of the MRFs and myosin heavy chain gene, thereby providing a potential mechanism for the delay in the appearance of myofibrils at low temperatures.
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Materials and methods |
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Embryos were sampled approximately every five somites by removing the chorion with fine forceps and fixing overnight at 4°C in 4 % (m/v) paraformaldehyde in phosphate-buffered saline (PBS) in 0.1 % (v/v) dimethyl pyrocarbonate (DMPC). The embryos were then washed twice in DMPC PBS, once in 50 % DMPC PBS/50 % methanol (v/v) and stored at 75°C in 100 % methanol.
cDNA cloning, probe synthesis and in situ hybridisation
Total RNA was isolated from juvenile herring fast (Myod and MyHC probe synthesis) and slow (myogenin probe synthesis) skeletal muscle using an RNeasy Midi kit (Qiagen). First-strand cDNA synthesis was carried out using a 3' rapid amplification of cDNA ends (Race) system (Gibco BRL Life Technologies) in which synthesis was initiated at the poly(A) tail using Adapter Primer. PCR amplification was carried out using Abridged Universal Amplification Primer (AUAP) and gene-specific primers designed from various vertebrate species (see Table 1). A 100 µl polymerase chain reaction (PCR) contained 0.5 µg of first-strand cDNA, 10 µl of 10x Taq polymerase buffer (500 mmol l1 KCl, 100 mmol l1 Tris-HCl, pH 9.0, and 1.0 % Triton X-100) (Promega), 1.5 mmol l1 MgCl2, 0.2 mmol l1 dNTP mix, 0.2 µmol l1 each of forward and reverse primer and 1 unit of Taq polymerase (Promega). Using a DNA thermal cycler (Techne, Cambridge, UK), reaction conditions were 94°C for 2 min followed by 30 cycles, for Myod and MyHC, of 1 min of denaturation at 94°C, 1 min of annealing at 59°C and 1 min of extension at 72°C. Reaction conditions for myogenin involved a 30-cycle touchdown PCR protocol of 94°C for 30 s, 63°C for 30 s reducing by 0.3°C with every cycle and 72°C for 30 s. After the last cycle, extension of incomplete products was carried out by holding the samples at 72°C for 7 min.
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Plasmids were linearised using the restriction enzymes Not1 and Spe1 (Promega). Probes were synthesised using digoxigenin-UTP (DIG) and bacteriophage T7/T3 RNA polymerases (Boehringer Mannheim). Antisense probes were produced with Not1 linearisation and T3 RNA polymerase.
Embryos were processed for in situ hybridisation following standard procedures (Schulte-Merker et al., 1992; Joly et al., 1993). MyoD and MyHC antisense probes were used on 20, 22 and 15 embryos from the 5°C, 8°C and 12°C groups, respectively. Myogenin antisense probes were used on 18, 14 and 16 embryos from the 5°C, 8°C and 12°C groups, respectively. Sense probes were tested on three embryos of varying ages from each group. Treatment in 50 µg ml1 proteinase K was carried out for 23 min. Hybridised transcripts were detected using a DIG nucleic acid detection kit (Boehringer Mannheim). Embryos were photographed on a Leica MZ8 microscope with a RS Photometrics Coolsnap digital camera using Openlab (Improvision).
Two 15-somite stage embryos reared at 8°C and stained for MyoD and two 46-somite stage embryos reared at 8°C and stained for myogenin were embedded in wax and cut to produce 10 µm transverse and sagittal sections.
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Results |
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Discussion |
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We cloned and sequenced partial-length cDNAs of MyoD, myogenin and fast muscle MyHC from herring. When comparing herring and the few fish species studied to date, the bHLH regions of MyoD and myogenin were highly conserved, with percentage identities comparable to those reported between carp and other vertebrates (Kobiyama et al., 1998). Using cRNA probes and in situ hybridisation, we found that expression of the MyoD transcript in herring embryos occurred initially within the adaxial cells of the presomitic mesoderm before progressing to the adaxial cells and the posterior regions of newly formed somites (Fig. 4). Similar expression patterns have been reported in zebrafish and trout (Weinberg et al., 1996; Delalande and Rescan, 1999). However, in herring, we detected initial MyoD transcript in as few as three somites (Fig. 4A), whereas in zebrafish it was simultaneously observed in the first 57 somites (Weinberg et al., 1996). In trout, Delalande and Rescan (Delalande and Rescan, 1999) found that the expression patterns were fulfilled by two nonallelic genes, TMyod and TMyoD2. Increased gene copy number is a common feature in fish and is thought to have arisen from multiple duplications of the fish genome (Meyer and Schartl, 1999). Further study would be required to determine whether more than one MyoD gene existed in herring.
Myogenin expression in herring embryos followed that of MyoD, as has also been found in zebrafish, carp and trout (Weinberg et al., 1996; Kobiyama et al., 1998; Delalande and Rescan, 1999). As in the present study, myogenin transcript expression in trout and zebrafish embryos was never detected in the adaxial cells of the presomitic mesoderm but extended from the adaxial cells of newly formed somites into the posterior compartment of the somite before extending anteriorly into the somite (Weinberg et al., 1996; Delalande and Rescan, 1999). In herring embryos with completed segmentation, the myogenin transcript was also detected in non-myotomal muscle in the head region, as found in trout (Delalande and Rescan, 1999). Weinberg et al. (Weinberg et al., 1996) reported that myogenin transcript levels remained high after those of MyoD had decreased in the more rostral somites of the zebrafish. In contrast, herring myogenin transcript levels showed a very transient expression pattern, decreasing before the transcript levels of MyoD (Fig. 5). Variations in the transcriptional regulation of the MRFs have been noted between fish and other vertebrates (Watabe, 1999). The reasons for differences in MRF expression between fish species are not clear, but may depend on muscle fibre types and/or evolutionary considerations including duplicated genes which may not yet have been recognised.
MyHC transcript expression in the somites of herring embryos lagged behind myogenin expression by approximately nine somites (Fig. 5). Staining produced a characteristic chevron pattern thought to occur from the accumulation of message at the myoseptal ends of the fibres (Ennion et al., 1999). Recent work by Rescan and colleagues (Rescan et al., 2001) has challenged the general conception of muscle differentiation in fish based on that of the zebrafish. Using cRNA probes for slow and fast MyHC in the trout, they found that adaxial cells gave rise not only to slow muscle but also to fast muscle, which differentiated prior to the lateral migration of the slow muscle progenitors. In carp embryos, two MyHC genes, EGGS22 and EGGS24, were found each to exhibit an identical expression pattern within the fast muscle of the somites until 2 weeks post-hatching, when they were replaced by gene transcripts for more developmentally mature isoforms (Ennion et al., 1999). The carboxyl-terminal region of the herring juvenile, fast muscle MyHC showed higher identity (92 %) to the equivalent region of EGGS24 than to that of EGGS22 (86 %).
The patterns of MRF and MyHC transcript expression in herring embryos provided no support for our hypothesis that temperature would affect the timing of transcription with respect to somite stage. In contrast, using reverse transcription and competitive polymerase chain reaction techniques, Yamane et al. (Yamane et al., 2000) found a delayed expression of MRFs in the masseter muscle of the mouse when compared with the tongue, which correlated with the retarded differentiation of the masseter. Although the concentration of MRFs within the somites may impact upon rates of myofibril synthesis, any semi-quantitative analyses of embryonic muscle samples in the present study would be difficult to interpret because of the transient nature and complex patterns of expression observed. No differences in the staining intensity of the MyHC transcript in in situ hybridisations were observed between embryos reared at different temperatures. We suggest, therefore, that the effects of temperature on delayed myofibril synthesis in herring embryos must impact downstream from MyHC transcription either at the level of translation or at the assembly stage.
Studies with human cultured cells have shown that, although myosin heavy chain is one of the first myofibrillar proteins to be expressed, its characteristic A-band structure appears much later in differentiation and requires a cytoskeletal scaffold (van der Ven et al., 1999). Myosin is thought to polymerise directly into thick filaments 1.6 µm long since shorter precursors are not observed in differentiating myotubes (Fischman, 1970). Thin filaments, the other major component of the myofibril, are arranged in an anti-parallel polarised manner in each sarcomere with the barbed ends at the Z-line and the pointed ends in the middle of the sarcomere (Huxley, 1960). The barbed capping protein (CapZ) is thought to function early in myofibril assembly to nucleate actin filament assembly and to establish filament polarity by aligning the barbed ends of the filaments with the Z-line (Schafer et al., 1995). Tropomodulin is associated with the capped ends of actin filaments and is also expressed early in differentiation prior to the cross-linking of the filaments to the Z-line by -actinin (Almenar-Queralt et al., 1999).
Another key protein involved in filament assembly and alignment is the giant sarcomeric protein titin that extends from the Z-line to the M-line (Gregorio et al., 1999). Titin has a role in directing the assembly of sarcomeres and maintaining sarcomere integrity by providing mechanical linkages with other sarcomeric proteins. Cell culture studies to characterise a functional knock-out of titin resulted in a failure of thick filament formation and the absence of ordered actin/myosin arrays, although the sarcomeric proteins were expressed (van der Ven et al., 2000). There is also evidence that the cytoskeletal scaffolding protein nebulin has an important role in the accessibility/exchangeability of actin into nascent myofibrils (Nwe and Shimada, 2000). Thus, there are numerous potential targets for temperature to impact on the assembly of myofibrils downstream from the transcription of the myosin heavy chain gene, involving either the transcription of other genes and/or movement of the cytoskeletal scaffold/sarcomeric proteins. The complexity of the assembly mechanism may make it relatively more susceptible to disruption by temperature change than earlier stages in muscle differentiation.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Almenar-Queralt, A., Gregorio, C. C. and Fowler, V. M. (1999). Tropomodulin assembles early in myofibrillogenesis in chick skeletal muscle: evidence that thin filaments rearrange to form striated myofibrils. J. Cell Sci. 112, 11111123.
Blagden, C. S., Currie, P. D., Ingham, P. W. and Hughes, S. M. (1997). Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev. 11, 21632175.
Chen, Y. H., Lee, W. C., Cheng, C. H. and Tsai, H. J. (2000). Muscle regulatory factor gene: zebrafish (Danio rerio) myogenin cDNA. Comp. Biochem. Physiol. B 127, 97103.[Medline]
Christ, B. and Ordahl, C. P. (1995). Early stages of chick somite development. Anat. Embryol. 191, 381396.[Medline]
Currie, P. D. and Ingham, P. W. (1998). The generation and interpretation of positional information within the vertebrate myotome. Mech. Dev. 73, 321.[Medline]
Cushing, D. H. (1990). Plankton production and year-class strength in fish populations an update of the match mismatch hypothesis. Adv. Mar. Biol. 26, 249293.
Delalande, J. M. and Rescan, P. Y. (1999). Differential expression of two nonallelic MyoD genes in developing and adult myotomal musculature of the trout (Oncorhynchus mykiss). Dev. Genes Evol. 209, 432437.[Medline]
Devoto, S. H., Melancon, E., Eisen, J. S. and Westerfield, M. (1996). Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development 122, 33713380.
Ennion, S., Wilkes, D., Gauvry, L., Alami-Durante, H. and Goldspink, G. (1999). Identification and expression analysis of two developmentally regulated myosin heavy chain gene transcripts in carp (Cyprinus carpio). J. Exp. Biol. 202, 10811090.
Fischman, D. A. (1970). The synthesis and assembly of myofibrils in embryonic muscle. Curr. Topics Dev. Biol. 5, 235280.[Medline]
Gallego, A., Heath, M. R., McKenzie, E. and Cargill, L. H. (1996). Environmentally induced short-term variability in the growth rates of larval herring. Mar. Ecol. Prog. Ser. 137, 1123.
Gauvry, L. and Fauconneau, B. (1996). Cloning of a trout fast skeletal myosin heavy chain expressed both in embryo and adult muscles and in myotubes neoformed in vitro. Comp. Biochem. Physiol. B 115, 183190.[Medline]
Gregorio, C. C., Granzier, H., Sorimachi, H. and Labeit, S. (1999). Muscle assembly: a titanic achievement? Curr. Opin. Cell Biol. 11, 1825.[Medline]
Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M., Olson, E. N. and Klein, W. H. (1993). Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364, 501506.[Medline]
Hirayama, Y. and Watabe, S. (1997). Structural differences in the crossbridge head of temperature-associated myosin subfragment-1 isoforms from carp fast skeletal muscle. Eur. J. Biochem. 246, 380387.[Abstract]
Huxley, H. E. (1960). Muscle cells. In The Cell: Biochemistry, Physiology and Morphology, vol. I (ed. J. Branchet and A. E. Mirsky), pp. 365481. New York: Academic Press.
Johnston, I. A., Cole, N. J., Abercromby, M. and Vieira, V. L. A. (1998). Embryonic temperature modulates muscle growth characteristics in larval and juvenile herring. J. Exp. Biol. 201, 623646.[Medline]
Johnston, I. A., Cole, N. J., Vieira, V. L. A. and Davidson, I. (1997). Temperature and developmental plasticity of muscle phenotype in herring larvae. J. Exp. Biol. 200, 849868.
Johnston, I. A., Vieira, V. L. A. and Abercromby, M. (1995). Temperature and myogenesis in embryos of the Atlantic herring Clupea harengus. J. Exp. Biol. 198, 13891403.
Johnston, I. A., Vieira, V. L. A. and Hill, J. (1996). Temperature and ontogeny in ectotherms: muscle phenotype in fish. In Phenotypic and Evolutionary Adaptations of Organisms to Temperature (ed. I. A. Johnston and A. F. Bennett), pp. 153181. Society of Experimental Biology Seminar Series. Cambridge: Cambridge University Press.
Johnston, I. A., Vieira, V. L. A. and Temple, G. K. (2001). Functional consequences and population differences in the developmental plasticity of muscle to temperature in Atlantic herring Clupea harengus. Mar. Ecol. Prog. Ser. 213, 285300.
Joly, J. S., Joly, C., Schulte-Merker, S., Boulekbache, H. and Condamine, H. (1993). The ventral and posterior expression of the even-skipped homeobox gene eve 1 is perturbed in dorsalized and mutant embryos. Development 119, 12611275.
Jones, S. R. and Jeffs, T. M. (1991). Near-surface sea temperatures in coastal waters of the North Sea, English Channel and Irish Sea. Data Report, no. 24. Ministry of Agriculture, Fisheries and Food Research. London: Her Majestys Stationery Office.
Kimmel, C. B., Warga, R. M. and Schilling, T. F. (1990). Origin and organisation of the zebrafish fate map. Development 108, 581594.[Abstract]
Kobiyama, A., Nihei, Y., Hirayama, Y., Kikuchi, K., Suetake, H., Johnston, I. A. and Watabe, S. (1998). Molecular cloning and developmental expression patterns of the MyoD and MEF2 families of muscle transcription factors in the carp. J. Exp. Biol. 201, 28012813.
Kong, Y. F., Flick, M. J., Kudla, A. J. and Konieczny, S. F. (1997). Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol. Cell Biol. 17, 47504760.[Abstract]
Ma, P.-C., Rould, M. A., Weintraub, H. and Pabo, C. O. (1994). Crystal structure of MyoD bHLH domainDNA complex: perspectives on DNA recognition and implications for transcriptional activation. Cell 77, 451459.[Medline]
Meyer, A. and Schartl, M. (1999). Gene and genome duplication in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. Curr. Opin. Cell Biol. 11, 699704.[Medline]
Morin-Kensicki, E. M. and Eisen, J. S. (1997). Sclerotome development and peripheral nervous system segmentation in embryonic zebrafish. Development 124, 159167.
Nwe, T. M. and Shimada, Y. (2000). Inhibition of nebulin and connectin (titin) for assembly of actin filaments during myofibrillogenesis. Tissue & Cell 32, 223227.[Medline]
Overholtz, W. J., Link, J. S. and Suslowicz, L. E. (2000). Consumption of important pelagic fish and squid by predatory fish in the northeastern USA shelf ecosystem with some fishery comparisons. ICES 57, 11471159.
Perry, R. L. S. and Rudnicki, M. A. (2000). Molecular mechanisms regulating myogenic determination and differentiation. Front. Biosci. 5, D750D767.[Medline]
Rescan, P. and Gauvry, L. (1996). Genome of the rainbow trout (Oncorhynchus mykiss) encodes two distinct muscle regulatory factors. Comp. Biochem. Physiol. B 113, 711715.[Medline]
Rescan, P. Y., Gauvry, L. and Paboeuf, G. (1995). A gene with homology to myogenin is expressed in developing myotomal musculature of the rainbow trout and in vitro during the conversion of myosatellite cells to myotubes. FEBS Lett. 362, 8992.[Medline]
Rescan, P. Y., Gauvry, L., Paboeuf, G. and Fauconneau, B. (1994). Identification of a muscle factor related to MyoD in a fish species. Biochim. Biophys. Acta 1278, 202204.
Rescan, P. Y., Collet, B., Rallier, C., Cauty, C., Delalande, J.-M., Goldspink, G. and Fauconneau, B. (2001). Red and white muscle development in trout (Oncorhynchus mykiss) as shown by in situ hybridisation of fast and slow myosin heavy chain transcripts. J. Exp. Biol. 204, 20972101.
Rudnicki, M. A. and Jaenisch, R. (1995). The MyoD family of transcription factors and skeletal myogenesis. Bioessays 17, 203209.[Medline]
Rudnicki, M. A., Schnegelsberg, P. N., Stead, R. H., Braun, T., Arnold, H. H. and Jaenisch, R. (1993). MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75, 13511359.[Medline]
Schafer, D. A., Hug, C. and Cooper, J. A. (1995). Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments. J. Cell Biol. 128, 6170.[Abstract]
Schulte-Merker, S., Hermann, H. R. K. and Nusslein-Volhard, C. (1992). The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 10211032.
Temple, G. K., Fox, C. J., Stewart, R. and Johnston, I. A. (2000). Variability in muscle growth characteristics during the spawning season in a natural population of Atlantic herring (Clupea harengus). Mar. Ecol. Prog. Ser. 205, 271281.
van der Ven, P. F. M., Bartsch, J. W., Gautel, M., Jockusch, H. and Furst, D. O. (2000). A functional knock-out of titin results in defective myofibril assembly. J. Cell Sci. 113, 14051414.
van der Ven, P. F. M., Ehler, E., Perriard, J. C. and Furst, D. O. (1999). Thick filament assembly occurs after the formation of a cytoskeletal scaffold. J. Muscle Res. Cell Motil. 20, 569579.[Medline]
Watabe, S. (1999). Myogenic regulatory factors and muscle differentiation during ontogeny in fish. J. Fish Biol. 55 (Supplement A), 118.
Watabe, S. (2001). Myogenic regulatory factors. In Fish Physiology, vol. XVIII (ed. I. A. Johnston), pp. 1941. San Diego, CA: Academic Press.
Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A., Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J. and Riggleman, B. (1996). Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 122, 2711280.
Xu, Y., He, J., Wang, X., Lim, T. M. and Gong, Z. (2000). Asynchronous activation of 10 muscle specific protein (MSP) genes during zebrafish somitogenesis Dev. Dyn. 219, 201215.[Medline]
Yamane, A., Ohnuki, Y. and Saeki, Y. (2000). Delayed embryonic development of mouse masseter muscle correlates with delayed MyoD family expression. J. Dent. Res. 79, 19331936.[Abstract]