Expression of muscle LIM protein during early development in Xenopus laevis

We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.     

Expression of the Ca2+-binding protein, parvalbumin, during embryonic development of the frog, Xenopus laevis

A cDNA segment encoding the Ca2+-binding protein, parvalbumin, was isolated with the use of antibodies, from a lambda gtll expression library of Xenopus laevis tadpole poly(A)+ RNAs. The bacterially expressed beta-galactosidase-parvalbumin fusion protein of one lambda recombinant shows high affinity 45Ca2+ binding. The sequence of the tadpole parvalbumin is highly similar to previously characterized beta-parvalbumins of other organisms. Data from protein and RNA blotting experiments demonstrate that parvalbumin is absent in oocytes, eggs, and early staged embryos, and only becomes expressed during embryogenesis at the time of myogenesis. The protein can be detected in individual developing muscle cells and in muscle fibers of tadpole tail muscles. A simple method is also described for the isolation of neural tube-notochord-somite complexes from Xenopus embryos.     

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk.     

Isoform diversity of giant proteins in relation to passive and active contractile properties of rabbit skeletal muscles

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between approximately 3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type-dependent size differences of < or =70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.     

The slow isoform of Xenopus troponin I is expressed in developing skeletal muscle but not in the heart

In birds and mammals three isoforms of troponin I (TnI) exist; a slow (TnIs), a fast (TnIf) and a cardiac (TnIc). Although each of these isoforms is expressed in the adult forms of these organisms in a muscle fiber-type-specific manner, the gene encoding TnIs is also expressed within the developing heart of these vertebrates. Herein, our results demonstrate that the developing heart of Xenopus laevis, unlike its counterpart in birds and mammals, does not express the gene encoding the TnIs isoform and that the expression of this gene, as well as the one encoding the Xenopus TnIf isoform, is restricted to skeletal muscle.     

Comprehensive analysis of titin protein isoform and alternative splicing in normal and mutant rats

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle.     

Differential expression of two skeletal muscle beta-tropomyosin mRNAs during Xenopus laevis development

A cDNA clone for a Xenopus laevis skeletal muscle beta-tropomyosin (beta-TMad) isoform was isolated from an adult skeletal muscle cDNA library. Sequence analysis revealed that this clone corresponded to a second beta-tropomyosin mRNA distinct from the one that was previously characterized (beta-TMemb). The two skeletal beta-TM mRNAs originate from distinct genes and are differentially expressed during development. Beta-TMemb mRNA is expressed only in the somites of the early embryo while beta-TMad mRNA is expressed in pre-metamorphic tadpoles and adult skeletal muscles. We have isolated the promoter region of the beta-TMemb gene and shown that a DNA construct containing 2.9 kb of promoter region is properly expressed after injection in the embryo.     

Sexually differentiated,androgen-regulated,larynx-specific myosin heavy-chain isoforms in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>; comparison to <Emphasis Type="Italic">Xenopus laevis</Emphasis>

We have shown that the sarcoplasmic myosin heavy-chain (MyHC) isoform xtMyHC-101d is highly and specifically expressed in the larynx of the aquatic anuran, Xenopus tropicalis. In male larynges, the predominant MyHC isoform is xtMyHC-101d, while in females, another isoform, xtMyHC-270c, predominates. The X. tropicalis genome has been sequenced in its entirety, and xtMyHC-101d is part of a specific array of xtMyHC genes expressed otherwise in embryonic muscles (Nasipak and Kelley, Dev Genes Evol, in press, 2008). The administration of the androgen dihydrotestosterone increases the expression of xtMyHC-101d in juvenile larynges of both sexes. Using ATPase histochemistry, we found that in adults, X. tropicalis male laryngeal muscle contains only fast-twitch fibers, while the female laryngeal muscle contains a mix of fast- and slow-twitch fibers. Juvenile larynges are female-like in fiber type composition (44% slow twitch, 56% fast twitch); androgen treatment increases the percentage of fast-twitch fibers to 86%. xtMyHC-101d predominates in larynges of dihydrotestosterone-treated juveniles but not in larynges of untreated juveniles. We compared the larynx-specific expression of xtMyHC genes in X. tropicalis to the MyHC gene expressed in X. laevis larynx (xlMyHC-LM) by sequencing the entire xlMyHC-LM gene. The androgen-regulated xtMyHC that predominates in the male larynx of X. tropicalis is not the gene phylogenetically most similar to xlMyHC-LM at the nucleotide level but is instead a similar isoform found in the same MyHC array and expressed in the embryonic muscle.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.     

SCUBE3 (Signal Peptide-CUB-EGF Domain-containing Protein 3) Modulates Fibroblast Growth Factor Signaling during Fast Muscle Development

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.     

Hypaxial muscle migration during primary myogenesis in Xenopus laevis.

In contrast to many vertebrates, the ventral body wall muscles and limb muscles of Xenopus develop at different times. The ventral body wall forms in the tadpole, while limb (appendicular) muscles form during metamorphosis to the adult frog. In organisms that have been examined thus far, a conserved mechanism has been shown to control migratory muscle precursor specification, migration, and differentiation. Here, we show that the process of ventral body wall formation in Xenopus laevis is similar to hypaxial muscle development in chickens and mice. Cells specified for the migratory lineage display an upregulation of pax3 in the ventro-lateral region of the somite. These pax3-positive cells migrate ventrally, away from the somite, and undergo terminal differentiation with the expression of myf-5, followed by myoD. Several other genes are selectively expressed in the migrating muscle precursor population, including neural cell adhesion molecule (NCAM), Xenopus kit related kinase (Xkrk1), and Xenopus SRY box 5 (sox5). We have also found that muscle precursor migration is highly coordinated with the migration of neural crest-derived melanophores. However, by extirpating neural crest at an early stage and allowing embryos to develop, we determined that muscle precursor migration is not dependent on physical or genetic interaction with melanophores.     

Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis

We have used immunocytological techniques to examine the developmental expression of the Ca2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells.     

Histopathological effects induced by paraquat during Xenopus laevis primary myogenesis

The oxidative agent paraquat induced tail abnormalities during Xenopus laevis development. Specimens exposed from blastula to the tadpole stage revealed pear-shaped myocytes and irregular intersomitic boundaries. The histological feature of the axial musculature was evaluated in embryos sampled at significant stages of the primary myogenesis. During the somitogenesis PQ-treated embryos showed normal appearing myotomes, but reduced PAS activity in the post-rotating myotomal cells, and myoblasts with slight vacuolations. Once etched from the vitelline envelope, embryos showed severely altered myoblasts with irregular cellular apexes, heavy sarcoplasmic vacuolations, pyknotic nuclei and disorganizing intersomitic boundaries. Myotomes with many necrotic myocytes containing disorganized contractile material and heavily malformed intersomitic boundaries characterized the late myogenic stages. Our results evidence the heaviest PQ histopathological effects to affect myogenesis of post-etched embryos, suggesting a possible linkage between the swimming activity and the oxidative damage to muscle tissue.     

Nebulin isoforms of extraocular muscle

The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and α-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL. This work was supported by grants from NIH-NHLB HL073089 to C.L.M. and NEI/NIH EY12998 to F.H.A.