nautilus

EVOLUTIONARY HOMOLOGS

Table of contents

Nautilus homologs: DNA binding specificity

Basic helix-loop-helix (bHLH) proteins perform a wide variety of biological functions. Most bHLH proteins recognize the consensus DNA sequence CAN NTG (the E-box consensus sequence is in bold) via the DNA-binding basic region (BR) but acquire further functional specificity by preferring distinct internal and flanking bases. In addition, induction of myogenesis by MyoD-related bHLH proteins depends on myogenic basic region and BR-HLH junction residues, both of which are unessential for binding to a muscle-specific site, implying that their BRs may be involved in other critical interactions. An investigation has been carried out to see whether the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins (Daughterless in Drosophila) prefer distinct CAN NTG sites. In MyoD, the myogenic BR residues establish specificity for particular CAN NTG sites indirectly, by influencing the conformation through which the BR helix binds DNA. An analysis of DNA binding by BR and junction mutants suggests that an appropriate BR-DNA conformation is necessary but not sufficient for myogenesis, supporting the model that additional interactions with this region are important. The sequence specificities of E2A and Twist proteins require the corresponding BR residues. In addition, mechanisms that position the BR allow E2A to prefer distinct half-sites as a heterodimer with MyoD or Twist, indicating that the E2A BR can be directed toward different targets by dimerization with different partners. These findings indicate that E2A and its partner bHLH proteins bind to CAN NTG sites by adopting particular preferred BR-DNA conformations, from which they derive differences in sequence recognition that can be important for functional specificity (Kophengnavong, 2000).

In part, the specificity with which bHLH proteins function derives from preferential recognition of different classes of CAN NTG sites by different bHLH protein subgroups. The HLH segment consists of a parallel, left-handed, four-helix bundle. The BR is unstructured in solution but when bound to DNA, it extends N terminally from the HLH segment as a helix that crosses the major groove. Crystallographic analyses have revealed some differences in how these proteins bind DNA. For example, in Myc family and related bHLH proteins, an arginine residue at BR position 13 specifies recognition of CACGTG sites by contacting bases in the center. However, it still is not understood how bHLH proteins that have a different amino acid at BR position 13 bind preferentially to distinct CAN NTG sites or how bHLH proteins establish differences in flanking sequence selectivity that can be of biological importance (Kophengnavong, 2000 and references therein).

Many bHLH proteins that lack R13, including MyoD and other E2A partners, can bind to similar DNA sequences in vitro but they act on different tissue-specific genes. Cooperative or inhibitory relationships with other transcriptional regulators might contribute to this specificity, but it is not likely to derive entirely from other lineage-specific factors, because MyoD can induce myogenesis in many different cell types. Initiation of myogenesis by MyoD and other myogenic bHLH proteins depends on three residues that are located within the BR and the BR-HLH junction (A5, T6, and K15). These residues, which are referred to in this study as myogenic are not essential for binding a muscle-specific site in vitro or in vivo, suggesting that they are involved in other critical interactions. These interactions have been proposed to involve distinct cofactors and the unmasking of an activation domain in MyoD or the myogenic cofactor MEF2. In the MyoD-DNA structure, K15 is oriented away from the DNA, but A5 and T6 face the major groove and could not contact other proteins directly. However, the latter two residues could influence protein-protein interactions indirectly, by affecting how the BR helix is positioned on the DNA. Although substitutions at these positions might not substantially impair binding to particular CAN NTG sites, it is important to determine whether they might have more subtle influences on sequence specificity that could reflect conformational effects (Kophengnavong, 2000 and references therein).

The myogenic residues A5 and T6 establish the characteristic MyoD sequence preference, which includes a CAGCTG core. Individual substitutions at these BR positions simultaneously alter preferences for multiple bases that MyoD does not contact directly, indicating that these preferences are determined indirectly, by how the BR helix is positioned on the DNA. This mechanism is distinct from the standard model for sequence specificity, in which preferred bases are contacted directly. The corresponding BR residues are also required for the sequence preferences of E2A proteins, which can recognize either of two distinct half-sites depending on their dimerization partner. E2A homodimers and E2A-MyoD heterodimers bind to asymmetric sites that include a CACCTG core. In contrast, as a heterodimer with the bHLH protein Twist, E2A binds preferentially to half of the symmetric sequence CATATG. The preference of E2A for the former asymmetric sites depends not only on the BR sequence but also on BR positioning that involves the junction region. An analysis of DNA binding by MyoD and E2A junction and BR mutants indicates that a MyoD-like sequence specificity is associated with, but not sufficient for, myogenesis. This supports the model that the BR-junction region is also involved in other critical interactions. The results suggest that E2A and its partner bHLH proteins bind DNA by adopting a limited number of preferred BR conformations, each of which is associated with a characteristic DNA sequence preference. They also indicate that binding of cofactors to the MyoD BR might be influenced by how it is positioned on the DNA and are consistent with the idea that relatively subtle differences in binding sequence recognition can modulate bHLH protein activity in vivo (Kophengnavong, 2000).

Invertebrate Nautilus homologs

Basic-helix-loop helix factors of the myoD/myf5/myogenin/MRF4 family have been implicated in acquisition and elaboration of muscle cell fates. The embryonic role of C. elegans MyoD resembles that of vertebrate myogenin: in each case, the cells retain muscle identity while executing only part of the differentiation program. There are, nonetheless, significant differences between the two systems: in myogenin-mutant mice, only a fraction of myoblasts undergo muscle differentiation, while in C. elegans hlh-1 null mutants, all embryonic muscle precursors differentiate as muscle. Both myogenic and non-myogenic roles are described for the Caenorhabditis elegans members of this family (CeMyoD), coded for by hlh-1, in postembryonic mesodermal patterning. The postembryonic mesodermal lineage in C. elegans provides a paradigm for many of the issues in mesodermal fate specification: a single mesoblast (‘M’) divides to generate 14 striated muscles, 16 non-striated muscles, and two non-muscle cells. The M-derived body-wall muscles are striated in character; these cells eventually assume positions among the 81 embryonically derived striated muscles used by the animal for locomotion. An analysis of CeMyoD requirements in larval mesoderm formation might be expected to have two advantages in illuminating the function of CeMyoD: (1) the larval myogenic lineage is less complex than the embryonic lineages, and (2) larval mesoderm specification occurs in the context of an already-formed overall body plan; thus it should be possible to follow the specification of cell types in an otherwise normal developmental context. To study CeMyoD function in the M lineage, an embryonic requirement for the protein has to be circumvented. Two approaches were used: (1) isolation of mutants that decrease CeMyoD levels while retaining viability, and (2) analysis of genetic mosaics that have lost CeMyoD in the M lineage. With either manipulation, a series of cell-fate transformations were observed affecting a subset of both striated muscles and non-muscle cells. In place of these normal fates, the affected lineages produce a number of myoblast-like cells that initially fail to differentiate, instead swelling to acquire a resemblance to sex myoblasts (M-lineage-derived precursors to non-striated uterine and vulval muscles). Like normal sex myoblasts, the ectopic myoblast-like cells are capable of migration and proliferation followed by differentiation of progeny cells into vulval and uterine muscle. These results demonstrate a cell-intrinsic contribution of CeMyoD to specification of both non-muscle and muscle fates (Harfe, 1998).

Two aspects of the observed cell-fate transformations were surprising. The first unexpected property of the CeMyoD-deficient M lineage was observed with regard to the transformation of body-wall muscle to sex myoblast. This was less than the 100% expected. Even in mutant animals completely lacking CeMyoD, only a fraction of striated muscles are transformed. The remaining cells adopted their normal striated muscle fate. The process appears to have a stochastic component: individual animals showed different sets of cellular transformations. This suggests a competition between opposing processes, with CeMyoD acting as one of several components contributing to the adoption of a striated muscle fate and the alternative pathway apparently leading to the production of a sex myoblast. The second unexpected property of this lineage regards the transformation of coelomocytes. Coelomocytes are non-muscle macrophage-like cells that inhabit the body cavity of the animal. Although their function is not known, they are known to collect certain xenobiotic molecules and effectively remove them from the coelomic fluid. Coelomocyte precursors fail to differentiate in mutants but instead undergo an extra division to generate cells that can enlarge like sex myoblasts. CeMyoD product is not absolutely required for differentiation of this cell type, since the four embryonically derived coelomocytes form (and collect extracellular GFP) in the absence of CeMyoD. Rather, CeMyoD is required for the specification of this fate only in the postembryonic M lineage (Harfe, 1998).

In vertebrates, striated muscle development depends on both the expression of members of the myogenic regulatory factor family (MRFs) and on extrinsic cellular cues, including Wnt signaling. The 81 embryonically born body wall muscle cells in C. elegans are comparable to the striated muscle of vertebrates. These muscle cells all express the gene hlh-1, encoding HLH-1 (CeMyoD) which is the only MRF-related factor in the nematode. However, genetic studies have shown that body wall muscle development occurs in the absence of HLH-1 activity, making the role of this factor in nematode myogenesis unclear. By ectopically expressing hlh-1 in early blastomeres of the C. elegans embryo, it was shown that CeMyoD is a bona fide MRF that can convert almost all cells to a muscle-like fate, regardless of their lineage of origin. The window during which ectopic HLH-1 can function is surprisingly broad, spanning the first 3 hours of development when cell lineages are normally established and non-muscle cell fate markers begin to be expressed. Maternal factors control zygotic hlh-1 expression. The Caudal-related homeobox factor PAL-1 can activate hlh-1 in blastomeres that either lack POP-1/TCF or that have down-regulated POP-1/TCF in response to Wnt/MAP kinase signaling. The potent myogenic activity of HLH-1 highlights the remarkable developmental plasticity of early C. elegans blastomeres and reveals the evolutionary conservation of MyoD function (Fukushige, 2005).

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in C. elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. Two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naive blastomeres to muscle when overproduced ectopically in the embryo. In addition, genetic null mutants were used to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type (Fukushige, 2006).

Striated muscle development in vertebrates requires the redundant functions of multiple members of the MyoD family. Invertebrates such as Drosophila and C. elegans contain only one MyoD homolog in each organism. Earlier observations suggest that factors outside of the MyoD family might function redundantly with MyoD in striated muscle fate specification in these organisms. However, the identity of these factors has remained elusive. This study describes the identification and characterization of FOZI-1, a putative transcription factor that functions redundantly with CeMyoD (HLH-1) in striated body wall muscle (BWM) fate specification in the C. elegans postembryonic mesoderm. fozi-1 encodes a novel nuclear-localized protein with motifs characteristic of both transcription factors and actin-binding proteins. FOZI-1 shares the same expression pattern as CeMyoD in the postembryonic mesodermal lineage, the M lineage, and fozi-1-null mutants exhibit similar M lineage-null defects to those found in animals lacking CeMyoD in the M lineage (e.g. loss of a fraction of M lineage-derived BWMs). Interestingly, fozi-1-null mutants with a reduced level of CeMyoD lack most, if not all, M lineage-derived BWMs. These results indicate that FOZI-1 and the Hox factor MAB-5 function redundantly with CeMyoD in the specification of the striated BWM fate in the C. elegans postembryonic mesoderm, implicating a remarkable level of complexity for the production of a simple striated musculature in C. elegans (Amin, 2007).

A MyoD family gene was identified in the ascidian Ciona intestinalis (Subphylum Urochordata) and designated CiMDF (Ciona intestinalis Muscle Determination Factor). Expression of CiMDF is restricted to the muscle cells of the developing embryo and the body-wall muscle of adults. Two differentially regulated CiMDF transcripts are expressed during development. A 1.8 kb transcript (CiMDFa) appears first and is gradually replaced by a 2.7 kb transcript (CiMDFb). These transcripts encode essentially identical MyoD family proteins with the exception of a 68 amino acid C-terminal sequence present in CiMDFb that is absent from CiMDFa. Although both CiMDFa and CiMDFb contain the cysteine-rich/basic-helix loop helix domain (Cys-rich/bHLH) present in all MyoD family proteins, only CiMDFb contain the region near the C terminus (Domain III) characteristic of this gene family. C. intestinalis has only one MyoD family gene, suggesting that CiMDFa and CiMDFb result from differential processing of primary transcripts. Although gene duplication events result in the appearance of multiple genes encoding distinct MyoD family proteins in vertebrates and amphioxus, these results show that a representative of the evolutionaryily older subphylum, Urochordata, differentially expresses unique MyoD family proteins from a single gene. The existence of two MyoD family proteins that are differentially expressed during ascidian embryogenesis has novel parallels to vertebrate muscle development and may reflect conserved myogenic regulatory mechanisms among chordates (Meedel, 1997).

The expression pattern of CiMDF, the MyoD-family gene of Ciona intestinalis, was analyzed in unmanipulated and microsurgically derived partial embryos. CiMDF encodes two transcripts during development (coding for distinct proteins), the smaller of which, CiMDFa, is detected in maternal RNA. Zygotic activity of CiMDF initiates in cleaving embryos of 32-64 cells. Both CiMDFa and CiMDFb transcripts are detected at this time; however, CiMDFa accumulates more rapidly before declining in abundance such that, by the early tail-formation stage, CiMDFb is more prevalent. Microsurgical isolations of various lineage blastomeres from the eight-cell stage were used to analyze CiMDF expression in the two embryonic lineages that give rise to larval tail muscle-autonomously specified primary cells and conditionally specified secondary cells. CiMDFa and CiMDFb transcripts were detected in both lineages, suggesting that neither functions in a lineage-specific manner. The data also demonstrate that CiMDF expression is autonomous in the primary lineage (i.e., cells derived from the B4.1 blastomeres) and correlates with histospecific differentiation of muscle. In the secondary lineage (i.e., cells derived from the A4.1 and b4.2 blastomeres), CiMDF expression is conditional and, as in the primary lineage, correlates with muscle differentiation. These experiments reveal similar patterns of CiMDF activity in the primary and secondary muscle lineages and imply a requirement for the expression of this gene in both lineages during larval tail muscle development (Meedel, 2002).

Zebrafish MyoD family: Differential requirements for myogenic regulatory factors distinguish medial and lateral somitic, cranial and fin muscle fiber populations

Myogenic regulatory factors of the Myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. However, the roles of each gene in myogenesis remain unclear, owing partly to genetic linkage at the Myf5/Mrf4 locus and to rapid morphogenetic movements in the amniote somite. In mice, Myf5 is essential for the earliest epaxial myogenesis, whereas Myod is required for timely differentiation of hypaxially derived muscle. A second major subdivision of the somite is between primaxial muscle of the somite proper and abaxial somite-derived migratory muscle precursors. This study used a combination of mutant and morphant analysis to ablate the function of each of the four conserved MRF genes in zebrafish, an organism that has retained a more ancestral bodyplan. A fundamental distinction in somite myogenesis is shown to be into medial versus lateral compartments, which correspond to neither epaxial/hypaxial nor primaxial/abaxial subdivisions. In the medial compartment, Myf5 and/or Myod drive adaxial slow fibre and medial fast fibre differentiation. Myod-driven Myogenin activity alone is sufficient for lateral fast somitic and pectoral fin fibre formation from the lateral compartment, as well as for cranial myogenesis. Myogenin activity is a significant contributor to fast fibre differentiation. Mrf4 does not contribute to early myogenesis in zebrafish. It is suggested that the differential use of duplicated MRF paralogues in this novel two-component myogenic system facilitated the diversification of vertebrates (Hinits, 2009).

Previous work in C. elegans has shown that posterior embryonic bodywall muscle lineages are regulated through a genetically defined transcriptional cascade that includes PAL-1/Caudal-mediated activation of muscle-specific transcription factors, including HLH-1/MRF and UNC-120/SRF, which together orchestrate specification and differentiation. Using chromatin immunoprecipitation (ChIP) in embryos, direct binding of PAL-1 in vivo to an hlh-1 enhancer element has been detected. Through mutational analysis of the evolutionarily conserved sequences within this enhancer, two cis-acting elements and their associated transacting factors (PAL-1 and HLH-1) were identified that are crucial for the temporal-spatial expression of hlh-1 and proper myogenesis. The data demonstrate that hlh-1 is indeed a direct target of PAL-1 in the posterior embryonic C. elegans muscle lineages, defining a novel in vivo binding site for this crucial developmental regulator. The same enhancer element is also a target of HLH-1 positive auto regulation, underlying (at least in part) the sustained high levels of CeMyoD in bodywall muscle throughout development. Together, these results provide a molecular framework for the gene regulatory network activating the muscle module during embryogenesis (Lei, 2009).

Xenopus Nautilus homologs

Tenascin (Drosophila homolog: Tenascin major) is a large glycoprotein which is expressed in a restricted pattern in the extracellular matrix (ECM) of vertebrate embryos. Tenascin interferes with cell-fibronectin interactions in vitro, and may play a role in the control of cell migration and differentiation during development. In Xenopus, tenascin immunoreactivity is first detected at the early tailbud stage in the ECM of the most anterior somite. Thereafter, it is distributed dorsally along neural crest cell migration pathways. Tenascin mRNA is most abundant in dorsal mesoderm at the neurula stage and in somites at the early tailbud stage, indicating that the initial accumulation of tenascin in the ECM is due to secretion from paraxial mesoderm. To understand how tenascin expression in somitic mesoderm is controlled, Xbra and the myogenic factors XMyoD and XMyf5 were expressed in blastula animal cap tissue. The tenascin gene is activated by all three transcription factors. Interestingly, expression of tenascin mRNA, and accumulation of the protein in the ECM, can occur without formation of muscle. These results suggest that tenascin regionalization in early Xenopus embryos depends on tenascin RNA expression by somitic mesoderm, where it is likely to be activated by myogenic factors (Umbhauer, 1994).

Pax1 and QmyoD are early sclerotome and myotome-specific genes, activated in epithelial somites of quail embryos in response to axial notochord/neural tube signals. pax1 is a member of the paired box family of transcription factors (see Drosophila Paired), but pax1 has no close Drosophila homolog. In situ hybridization experiments reveal that the developmental kinetics of activation for pax1 and QmyoD differ greatly, suggesting that myotome and sclerotome specification are controlled by distinct developmental mechanisms. pax1 activation first occurs in in somite IV, indicating that pax1 regulation is tightly coordinated with early steps in somite maturation. In contrast, QmyoD is delayed and does not occur until embryos have 12-14 somites. At this time, QmyoD is the first of the myogenic regulatory factor (MRF) genes to be activated in preexisting somites in a rapid, anterior to posterior progression until the 22 somite stage. Experiments involving transplantation of newly formed somites to ectopic sites along the anterior to posterior embryonic axis were performed to distinguish the contributions of axial signals and somite response pathways to the developmental regulation of pax1 and QmyoD. These studies show that pax1 activation is regulated by somite formation and maturation, not by the availability of axial signals, which are expressed prior to somite formation. In contrast, the delayed activation of QmyoD is controlled by developmental regulation of the production of axial signals as well as by the competence of somites to respond to these signals. These somite transplantation studies, therefore, provide a basis for understanding the different developmental kinetics of activation of pax1 and QmyoD during sclerotome and myotome specification, and suggest specific molecular models for the developmental regulation of myotome and sclerotome formation in somites through distinct signal/response pathways. In contrast to pax1 activation, which is regulated strictly by control of the capacity of somites to respond to Sonic hedgehog, QmyoD activation is controlled by two regulatory mechanisms: the first involves developmental regulation of the production of signals from the axial tissues (notochord), and the second involves compentence of the somites to respond to these axial signals. It is possible the Shh may be is a signaling molecule for both QmyoD and pax1 and Wnt4 may be a neural tube maintenance factor (Borycki, 1997).

Activated Notch-Delta signaling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. The implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split [E(spl)] family that may function as a nuclear effector of the Notch signaling pathway in regulating XMyoD gene expression at the initial step of myogenesis was examined. Xhairy-1 transcripts are expressed soon after mid-blastula transition and Xhairy-1 exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. Overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signaling through hairy-related genes may specifically regulate XMyoD (Umbhauer, 2001).

In amphibian development, muscle is specified in the dorsal lateral marginal zone (DLMZ) of the gastrula embryo. Two critical events specify the formation of skeletal muscle: the expression of the myogenic transcription factor, XMyoD, and the secretion of bone morphogenetic protein (BMP) antagonists by the adjacent Spemann organizer. Inhibition of BMP signaling during early gastrula stages converts XMyoD protein into an instructive differentiation factor in the DLMZ. Yet, the intracellular signaling factors connecting BMP antagonism and activation of XMyoD remain unknown. BMP antagonism induces the activity of mitogen-activated protein kinase (MAPK), and the activity of MAPK is necessary for muscle-specific differentiation. Treatment of gastrula-stage DLMZ explants with MAPK pathway inhibitors ventralizes mesoderm and prevents muscle differentiation. Expression of XMyoD in ventral mesoderm weakly induces muscle formation; however, the coexpression of a constitutively active MEK1 with XMyoD efficiently induces muscle differentiation. Activation of the MAPK pathway does not induce the transcription of XMyoD, but increases its protein levels and transcriptional activity. Thus, MAPK activation is subsequent to BMP antagonism, and participates in the dorsalization of mesoderm by converting the XMyoD protein into a potent differentiation factor (Zetser, 2001).

The myogenic transcription factor Xmyf-5 of Xenopus is the earliest known gene to be expressed specifically in the dorsolateral mesoderm of the gastrula, a domain that is established by the interaction of dorsal and ventral signals. A 7.28 kb Xenopus tropicalis Xmyf-5 (Xtmyf-5) genomic DNA fragment has been identified that accurately recapitulates the expression of the endogenous gene. Deletion and mutational analysis has identified the homeobox motif approximately 1.2 kb upstream from the start of transcription, which is necessary for both activation and repression of Xtmyf-5 expression, implying that positional information is integrated at this site. HBX2 is a complex site, consisting of 4 TAAT motifs, two of which overlap. Electrophoretic mobility shift assays demonstrate that HBX2 specifically interacts with gastrula stage embryonic extracts and that in vitro translated Xvent-1 protein, a transcriptional repressor, binds to one of its functional motifs. Combined with gain- and loss-of-function experiments, the promoter analysis described here suggests that Xvent-1 functions to repress Xmyf-5 expression in the ventral domain of the marginal zone. Furthermore, the identification of HBX2 provides a tool with which to identify other molecules involved in the regulation of Xmyf-5 expression during gastrulation (Plli, 2002).

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, myogenesis has been examined 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. 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. A model is proposed for the evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk (Grimaldi, 2004).

It is suggested that Hh signalling from ventral midline acts on medial somitic cells to promote XMyf5 expression and early slow myogenesis. These cells rapidly differentiate, express XMyoD and move to the superficial somite surface where they elongate anteroposteriorly to make superficial slow fibres. Simultaneously, most somitic cells differentiate into fast fibres, also elongating anteroposteriorly to form the bulk of somitic muscle. Undifferentiated cells form a dermomyotome. At later stages, a second population of slow muscle fibres is generated from dermomyotome, probably at dorsomedial and ventrolateral lips, independent of Hh signalling. In anterior somites, despite early notochord-dependent XMyf5 expression, a block on slow muscle formation prevents appearance of the first wave of slow fibres. Fast fibre formation is abundant, and precocious compared with zebrafish. However, some cells remain undifferentiated to form the superficial dermomyotome. Dorsal and ventral dermomyotomal lips continue to express XMyf5 and XMyoD, reflecting their continued role as myogenic centres. Slow fibre formation is initiated from dermomyotome independently of Hh signalling. Extra fast fibres probably also arise from dermomyotome at all anteroposterior levels. At even later stages Hh signalling is again required for XMyoD expression, somite growth and third wave slow fibre formation at dermomyotomal lips throughout the axis (Grimaldi, 2004).

Chicken Nautilus homologs

In the avian embryo, previous work has demonstrated that the notochord provides inductive signals to activate myoD and pax1 regulatory genes, which are expressed in the dorsal and ventral somite cells that give rise to myotomal and sclerotomal lineages. Bead implantation and antisense inhibition experiments have been carried out that show that Sonic hedgehog is both a sufficient and essential notochord signal molecule for myoD and pax1 activation in somites. Genes of the Sonic hedgehog signal response pathway [specifically patched (the Sonic hedgehog receptor) and gli and gli2/4, (two zinc-finger transcription factors)] are activated in coordination with somite formation, establishing that Sonic hedgehog response genes play a regulatory role in coordinating the response of somites to the constitutive notochord Sonic hedgehog signal. The expression of patched, gli and gli2/4 is differentially patterned in the somite, providing mechanisms for differentially transducing the Sonic hedgehog signal to the myotomal and sclerotomal lineages. The activation of gli2/4 is controlled by the process of somite formation and signals from the surface ectoderm, whereas upregulation of patched and activation of gli is controlled by the process of somite formation and a Sonic hedgehog signal. Therefore, the Sonic hedgehog signal response genes carry out important functions in regulating the initiation of the Sonic hedgehog response in newly forming somites and in regulating the patterned expression of myoD and pax1 in the myotomal and sclerotomal lineages following somite formation (Borycki, 1998).

Cells of the cranial paraxial mesoderm give rise to parts of the skull and muscles of the head. Some mesoderm cells migrate from locations close to the hindbrain into the branchial arches where they undergo muscle differentiation. In spite of a lack of overt subdivision, the cranial paraxial mesoderm gives rise to separate cell lineages, including craniofacial muscles and some bones of the chordal skull (e.g. supra occipital, sphenoid, pars canalicularis and cochlearis of the otic capsule). The migratory pathways of the cranial paraxial mesoderm have been characterized in chick embryos either by DiI-labelling cells before migration or by grafting quail cranial paraxial mesoderm orthotopically. These experiments demonstrate that depending on their initial rostrocaudal position, cranial paraxial mesoderm cells migrate in streams to fill the core of the nearest branchial arch. A survey of the expression of myogenic genes has shown that the myogenic markers Myf5, MyoD and myogenin are expressed in branchial arch muscle, but at comparatively late stages, as compared with their expression in the somites. Pax3 is not expressed by myogenic cells that migrate into the branchial arches, despite its expression in migrating precursors of limb muscles. In order to test whether segmental plate or somitic mesoderm has the ability to migrate in a cranial location, quail trunk mesoderm was grafted into the cranial paraxial mesoderm region. While segmental plate mesoderm cells do not migrate into the branchial arches, somitic cells are capable of migrating and are incorporated into the branchial arch muscle mass. Grafted somitic cells in the vicinity of the neural tube maintain expression of the somitic markers Pax3, MyoD and Pax1. By contrast, ectopic somitic cells located distal to the neural tube and in the branchial arches do not express Pax3. These data imply that signals in the vicinity of the hindbrain and branchial arches act on migrating myogenic cells to influence their gene expression and developmental pathways (Hacker, 1998).

The myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signaling is known to prevent differentiation in different systems and species, attempts were made to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. It is concluded that activation of Notch signaling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stage (Delfini, 2000).

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