Expression of XMyoD protein in early Xenopus laevis embryos.

A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

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.     

Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133

The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.     

Cloning of hibernation-related genes of bullfrog (Rana catesbeiana) by cDNA subtraction

Seven genes specifically expressed during hibernation in the bullfrog (Rana catesbeiana) were cloned from a subtracted cDNA library constructed from livers of winter bullfrogs. Those genes were fibrinogen alpha-subunit, fibrinogen gamma-subunit, complement component C3, alpha-1-microglobulin/bikunin precursor (AMBP), transferrin, apoferritin middle subunit and one novel gene. Northern hybridization has indicated that these seven genes were specifically induced or enhanced in winter. Above all, expression of the novel gene was specifically induced in winter in liver, though the expression of that was neither induced in bullfrog nor Xenopus laevis by cold treatment. The novel gene, which was designated as rc-hirp (Rana catesbeiana hibernation-related protein), encoded 420 base pairs length and a putative protein of 139 amino acid residues. Annual analyses of the expression of these genes have suggested that the seven winter-specific genes are playing an important role in hibernation processes.     

Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.