Opposing Effects of Activin A and Follistatin on Developing Skeletal Muscle Cells

Activin and the activin-binding protein follistatin modulate a variety of biological processes and are abundant at sites of muscle development. Activin and follistatin were expressed in developing chick pectoral musclein vivoand in primary cell culture. Addition of recombinant activin inhibited muscle development in a dose-dependent manner as measured by the number of nuclei in myosin heavy chain positive cells and creatine phosphokinase activity. Conversely, follistatin potentiated muscle development. The effects of activin were found to be distinct from those of the related protein transforming growth factor (TGF) β1. Muscle development was repressed by activin at all time points investigated and did not recover with the removal of activin following a limited exposure. In contrast, while myogenic differentiation in TGFβ1 was initially repressed, muscle marker expression recovered to control levels—even in the continued presence of TGFβ1. Fibroblast growth factor (FGF) had little effect on inhibiton of muscle development caused by activin A. However, inhibition of development produced by TGFβ increased with increasing concentrations of FGF. Finally, early expression of myoD and myf5 mRNA by muscle cultures in the presence of activin and follistatin was analyzed. Activin-treated cultures expressed reduced myoD and myf5 levels at 1.5 days after plating. Myf5 levels in follistatin-treated cultures were elevated, but, surprisingly, these cultures showed a reduction in myoD levels. These data suggest that endogenously expressed activin and follistatin are important modulators of muscle development.     

Independent specialisation of myosin II paralogues in muscle vs. non-muscle functions during early animal evolution: a ctenophore perspective

ABSTRACT: BACKGROUND: Myosin II (or Myosin Heavy Chain II, MHCII) is a family of molecular motors involved in the contractile activity of animal muscle cells but also in various other cellular processes in non-muscle cells. Previous phylogenetic analyses of bilaterian MHCII genes identified two main clades associated respectively with smooth/non-muscle cells (MHCIIa) and striated muscle cells (MHCIIb). Muscle cells are generally thought to have originated only once in ancient animal history, and decisive insights about their early evolution are expected to come from expression studies of Myosin II genes in the two non-bilaterian phyla that possess muscles, the Cnidaria and Ctenophora. RESULTS: We have uncovered three MHCII paralogues in the ctenophore species Pleurobrachia pileus. Phylogenetic analyses indicate that the MHCIIa / MHCIIb duplication is more ancient than the divergence between extant metazoan lineages. The ctenophore MHCIIa gene (PpiMHCIIa) has an expression pattern akin to that of "stem cell markers" (Piwi, Vasa...) and is expressed in proliferating cells. We identified two MHCIIb genes that originated from a ctenophore-specific duplication. PpiMHCIIb1 represents the exclusively muscular form of myosin II in ctenophore, while PpiMHCIIb2 is expressed in non-muscle cells of various types. In parallel, our phalloidin staining and TEM observations highlight the structural complexity of ctenophore musculature and emphasize the experimental interest of the ctenophore tentacle root, in which myogenesis is spatially ordered and strikingly similar to striated muscle formation in vertebrates. CONCLUSION: MHCIIa expression in putative stem cells/proliferating cells probably represents an ancestral trait, while specific involvement of some MHCIIa genes in smooth muscle fibres is a uniquely derived feature of the vertebrates. That one ctenophore MHCIIb paralogue (PpiMHCIIb2) has retained MHCIIa-like expression features furthermore suggests that muscular expression of the other paralogue, PpiMHCIIb1, was the result of neofunctionalisation within the ctenophore lineage, making independent origin of ctenophore muscle cells a likely option.     

Identification of a regulatory function for an orphan receptor in muscle: COUP-TF II affects the expression of the myoD gene family during myogenesis.

COUP-TF II is an 'orphan steroid receptor' that binds a wide variety of AGGTCA repeats and represses thyroid hormone (T3) and retinoid dependent trans-activation; however, very little is known of its functional and/or developmental role during mammalian cell differentiation. T3 and retinoids have been demonstrated to promote terminal muscle differentiation via activation of the muscle specific myoD gene family (myoD, myogenin, myf-5 and MRF-4). The myoD gene family can direct the fate of mesodermal cell lineages, repress proliferation, activate differentiation and the contractile phenotype. Hence, we investigated the expression and functional role of COUP-TF II during muscle differentiation. Proliferating C2C12 myoblasts expressed COUP-TF II mRNA which was repressed when cells were induced to differentiate into post-mitotic multinucleated myotubes by serum withdrawal. Concomitant with the decrease of COUP-TF II mRNA was the appearance of muscle specific mRNAs (e.g. myogenin, alpha-actin). We show that Escherichia coli expressed full length and truncated COUP-TF II bound in a sequence specific manner to the T3 response elements (TREs) in the myoD and myogenin regulatory HLH genes [Olson (1992) Dev. Biol. 154, 261-272]; and the TRE in the skeletal alpha-actin contractile protein gene. COUP-TF II diminished the homodimeric binding of the thyroid hormone receptor and the heterodimeric binding of thyroid hormone and retinoid X receptor complexes to these TREs. Constitutive over-expression of COUP-TF II cDNA in mouse C2C12 myogenic cells suppressed the levels of myoD mRNA and blocked the induction of myogenin mRNA, whereas constitutive expression of anti-sense COUP-TF II cDNA significantly increased the steady state levels of myoD mRNA and hyper-induced myogenin mRNA. These studies demonstrate for the first time (i) that COUP-TF II, functions as a physiologically relevant antagonistic regulator of myogenesis via direct effects on the myoD gene family and (ii) direct evidence for the developmental role of COUP-TF II during mammalian cell differentiation.     

Comparative genomic analysis of genes encoding translation elongation factor 1B(alpha) in human and mouse shows EEF1B1 to be a recent retrotransposition event

We have characterized genomic loci encoding translation elongation factor 1B(alpha) (eEF1B(alpha)) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Consequences of Hox gene duplication in the vertebrates: an investigation of the zebrafish Hox paralogue group 1 genes

As a result of a whole genome duplication event in the lineage leading to teleosts, the zebrafish has seven clusters of Hox patterning genes, rather than four, as described for tetrapod vertebrates. To investigate the consequences of this genome duplication, we have carried out a detailed comparison of genes from a single Hox paralogue group, paralogue group (PG) 1. We have analyzed the sequences, expression patterns and potential functions of all four of the zebrafish PG1 Hox genes, and compared our data with that available for the three mouse genes. As the basic functions of Hox genes appear to be tightly constrained, comparison with mouse data has allowed us to identify specific changes in the developmental roles of Hox genes that have occurred during vertebrate evolution. We have found variation in expression patterns, amino acid sequences within functional domains, and potential gene functions both within the PG1 genes of zebrafish, and in comparison to mouse PG1 genes. We observed novel expression patterns in the midbrain, such that zebrafish hoxa1a and hoxc1a are expressed anterior to the domain traditionally thought to be under Hox patterning control. The hoxc1a gene shows significant coding sequence changes in known functional domains, which correlate with a reduced capacity to cause posteriorizing transformations. Moreover, the hoxb1 duplicate genes have differing functional capacities, suggesting divergence after duplication. We also find that an intriguing function 'shuffling' between paralogues has occurred, such that one of the zebrafish hoxb1 duplicates, hoxb1b, performs the role in hindbrain patterning played in mouse by the non-orthologous Hoxa1 gene.     

A novel role for lbx1 in Xenopus hypaxial myogenesis

We have examined lbx1 expression in early X. laevis tadpoles. In contrast to amniotes, lbx1 is expressed in all of the myoblasts that contribute to the body wall musculature, as well as in a group of cells that migrate into the head. Despite this different expression, the function of lbx1 appears to be conserved. Morpholino (MO) knockdown of lbx1 causes a specific reduction of body wall muscles and hypoglossal muscles originating from the somites. Although myoblast migratory defects are observed in antisense MO injected tadpoles targeting lbx1, this results at least in part from a lack of myoblast proliferation in the hypaxial muscle domain. Conversely, overexpression of lbx1 mRNA results in enlarged somites, an increase in cell proliferation, but a lack of differentiated muscle. The control of cell proliferation is linked to a strong downregulation of myoD expression in gain-of-function experiments. Co-injection of myoD mRNA with lbx1 mRNA eliminates the overproliferation phenotype observed when lbx1 is injected alone. The results indicate that a primary function of lbx1 in hypaxial muscle development is to repress myoD, allowing myoblasts to proliferate before the eventual onset of terminal differentiation.     

Characterization of SCaMC-3-like/slc25a41, a novel calcium-independent mitochondrial ATP-Mg/Pi carrier

The SCaMCs (small calcium-binding mitochondrial carriers) constitute a subfamily of mitochondrial carriers responsible for the ATP-Mg/P(i) exchange with at least three paralogues in vertebrates. SCaMC members are proteins with two functional domains, the C-terminal transporter domain and the N-terminal domain which harbours calcium-binding EF-hands and faces the intermembrane space. In the present study, we have characterized a shortened fourth paralogue, SCaMC-3L (SCaMC-3-like; also named slc25a41), which lacks the calcium-binding N-terminal extension. SCaMC-3L orthologues are found exclusively in mammals, showing approx. 60% identity to the C-terminal half of SCaMC-3, its closest paralogue. In mammalian genomes, SCaMC-3 and SCaMC-3L genes are adjacent on the same chromosome, forming a head-to-tail tandem array, and show identical exon-intron boundaries, indicating that SCaMC-3L could have arisen from an SCaMC-3 ancestor by a partial duplication event which occurred prior to mammalian radiation. Expression and functional data suggest that, following the duplication event, SCaMC-3L has acquired more restrictive functions. Unlike the broadly expressed longer SCaMCs, mouse SCaMC-3L shows a limited expression pattern; it is preferentially expressed in testis and, at lower levels, in brain. SCaMC-3L transport activity was studied in yeast deficient in Sal1p, the calcium-dependent mitochondrial ATP-Mg/P(i) carrier, co-expressing SCaMC-3L and mitochondrial-targeted luciferase, and it was found to perform ATP-Mg/P(i) exchange, in a similar manner to Sal1p or other ATP-Mg/P(i) carriers. However, metabolite transport through SCaMC-3L is calcium-independent, representing a novel mechanism involved in adenine nucleotide transport across the inner mitochondrial membrane, different to ADP/ATP translocases or long SCaMC paralogues.     

Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Evolution of follistatin in teleosts revealed through phylogenetic, genomic and expression analyses

Follistatin (Fst) inhibits transforming growth factor-β (TGF-B) proteins and is a known regulator of amniote myogenesis. Here, we used phylogenetic, genomic and experimental approaches to study its evolution in teleosts. Phylogenetic analyses suggested that one fst gene (fst1) is common to euteleosts, but a second gene (fst2) is conserved specifically within the Ostariophysi. Zebrafish fst1/2 respectively appear on chromosomes 5 and 10 in two genomic regions, each with conserved synteny to a single region in tetrapods. Interestingly, other teleosts have two corresponding chromosomal regions with a similar repertoire of paralogues. Phylogenetic reconstruction clustered these gene duplicates into two sister clades branching from tetrapod sequences. We suggest that an ancestral fst-containing chromosome was duplicated during the teleost whole genome duplication, but that fst2 was lost in lineages external to the Ostariophysi. We show that Fst1 of teleosts/mammals has evolved under strong purifying selection, but the N-terminal of Fst2 may have evolved under positive selection. Furthermore, the tissue-specific expression of zebrafish fst2 was restricted to fewer tissues compared to its paralogue and the single fst1 orthologue of Atlantic salmon (Salmo salar). Zebrafish fst1/2 may have subfunctionalized relative to non-duplicated vertebrate lineages, as several regions in the fst promoter of tetrapods were conserved with one paralogue, but not both. Finally, we examined the embryonic expression of fst1 in a teleost outside the Ostariophysi (Atlantic salmon). During segmentation, fst1 was expressed in the anterior somite compartment but was excluded from muscle progenitors that strongly expressed myogenic regulatory factors (MRFs). Later, fst1 was expressed in myogenic progenitors of the pectoral fin buds and also within the pax7 ⁺ cell layer external to the myotome. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Muscle-Specific RING Finger (MuRF) cDNAs in Atlantic Salmon (Salmo salar) and Their Role as Regulators of Muscle Protein Degradation

The selection of proteins destined for degradation by the ubiquitin–proteasome pathway is coordinated by E3 ubiquitin ligases (E3Ub). One group of E3Ubs is described as muscle-specific RING finger (MuRF) molecules. In mammals, these proteins are believed to be central to targetting of muscle proteins for degradation during physiological perturbations such as starvation and inflammatory responses. In fish, the diversity of MuRF sequences is unexplored as is the expression of their mRNAs. In this study, three MuRF1 cDNAs, denoted as MuRF1a, MuRF1b, and MuRF1c, and a single MuRF2 were identified and characterized in Atlantic salmon. The MuRF1 sequences are highly conserved and encode predicted proteins of 349, 350, and 353 amino acids, whereas MuRF2 encodes a longer protein of 462 amino acids. The evolutionary relationship of these sequences with other fish and mammalian molecules shows that MuRF1a and 1b may have arisen from a recent salmonid duplication. The mRNA of MuRFs was expressed in multiple tissues, with highest abundance in white muscle tissue followed by the heart. The expression of MuRFs was modulated after both starvation and immune challenge. Starvation increased expression of all MuRF mRNAs in white muscle, with the greatest increase found in MuRF1a. A proinflammatory stimulation increased expression of MuRF mRNA in muscle and other tissues indicating a role of these proteins in protein degradation during inflammation.     

Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.     

Characterization of two duplicate zebrafish Cb2-like cannabinoid receptors

Several cannabinoid receptors have been detected in many organisms. The best known are CB1, mainly expressed in the central nervous system and CB2 which is almost exclusively expressed in the periphery. Here we report the molecular characterization of two duplicate CB2-like cannabinoid receptors from zebrafish (Danio rerio) (zebrafish Cb2a and zebrafish Cb2b). The amino acid sequences of these receptors present 56% identity with Takifugu rubripes CB2 sequence and 39% with human CB2 sequence and conserve some specific key residues for cannabinoid receptor function. Both duplicate receptors are expressed in peripheral tissues (gills, heart, intestine and muscle), immune tissue (spleen) and also in the central nervous system. Using in situ hybridization techniques zebrafish Cb2 mRNA expression was observed for the first time in the adenohypophysial cells of the rostral pars distalis and proximal pars distalis of the pituitary gland. Given the importance of the existence of duplication of genes in teleosts, the combined analysis of these two new cannabinoid receptors opens a new exciting door to investigate and understand cannabinoid function throughout evolution.     

Sex-specific expression of the X-linked histone demethylase gene Jarid1c in brain

Jarid1c, an X-linked gene coding for a histone demethylase, plays an important role in brain development and function. Notably, JARID1C mutations cause mental retardation and increased aggression in humans. These phenotypes are consistent with the expression patterns we have identified in mouse brain where Jarid1c mRNA was detected in hippocampus, hypothalamus, and cerebellum. Jarid1c expression and associated active histone marks at its 5'end are high in P19 neurons, indicating that JARID1C demethylase plays an important role in differentiated neuronal cells. We found that XX mice expressed Jarid1c more highly than XY mice, independent of their gonadal types (testes versus ovaries). This increased expression in XX mice is consistent with Jarid1c escape from X inactivation and is not compensated by expression from the Y-linked paralogue Jarid1d, which is expressed at a very low level compared to the X paralogue in P19 cells. Our observations suggest that sex-specific expression of Jarid1c may contribute to sex differences in brain function.