Somite Morphogenesis Research Articles

A role for N-cadherin in mesodermal morphogenesis during gastrulation

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell–cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.

PCNS: A novel protocadherin required for cranial neural crest migration and somite morphogenesis in Xenopus

Protocadherins (Pcdhs), a major subfamily of cadherins, play an important role in specific intercellular interactions in development. These molecules are characterized by their unique extracellular domain (EC) with more than 5 cadherin-like repeats, a transmembrane domain (TM) and a variable cytoplasmic domain. PCNS ( Proto cadherin in Neural crest and Somites), a novel Pcdh in Xenopus, is initially expressed in the mesoderm during gastrulation, followed by expression in the cranial neural crest (CNC) and somites. PCNS has 65% amino acid identity to Xenopus paraxial protocadherin (PAPC) and 42–49% amino acid identity to Pcdh 8 in human, mouse, and zebrafish genomes. Overexpression of PCNS resulted in gastrulation failure but conferred little if any specific adhesion on ectodermal cells. Loss of function accomplished independently with two non-overlapping antisense morpholino oligonucleotides resulted in failure of CNC migration, leading to severe defects in the craniofacial skeleton. Somites and axial muscles also failed to undergo normal morphogenesis in these embryos. Thus, PCNS has essential functions in these two important developmental processes in Xenopus.

Vertebrate Segmentation: Snail Counts the Time until Morphogenesis

During segmentation of vertebrate embryos, unsegmented mesenchymal mesoderm is divided into epithelial segments called somites. This process is governed by oscillating gene expression of the somite clock. A recent paper identifies the transcription factor Snail as a link between the somite clock and the control of somite morphogenesis.

The evolutionary history of crustacean segmentation: a fossil‐based perspective

The evolution of segmentation in Crustacea, that is, the formation of sclerotized and jointed body somites and arrangement of somites into tagmata, is viewed in light of historical traits and functional constraints. The set of Early to Late Cambrian 'Orsten' arthropods have informed our current views of crustacean evolution considerably. These three-dimensionally preserved fossils document ancient morphologies, as opposed to purely hypothetical models and, because of the unusual preservation of larval stages, provide us with unparalleled insight into the morphogenesis of body somites and their structural equipment. The variety of evolutionary levels represented in the 'Orsten' including lobopodians, tardigrades, and pentastomids also allows phylogenetic interpretations far beyond the Crustacea. The 'Orsten' evidence and data from representatives of the Lower Cambrian Chengjiang biota in southwestern China, including phylogenetically earlier forms, form the major source of our morphology-based review of structural and functional developments that led toward the Crustacea. The principal strategy of arthropods is the simultaneous development of head somites, as expressed in a basal "head larva," and a successive addition of postcephalic somites from a preterminal budding zone with progressive maturation of metameric structures. This can be recognized in the developmental patterns of extant and fossil representatives of several euarthropod taxa, particularly crustaceans, trilobites, and chelicerates (at least basally). The development of these taxa points to an early somite-poor and free-living hatching stage. Embryonic development to a late stage within an egg, as occurring in recent onychophorans and certain in-group euarthropods, is regarded as achieved several times convergently.

Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation.

The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. Here, we analyze the roles of zebrafish Has2 in normal development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, our data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis.

Long-distance cue from emerging dermis stimulates neural crest melanoblast migration.

Neural crest melanoblasts display unique navigational abilities enabling them to colonize the dorsal path between ectoderm and somite. One signal shown here to elicit melanoblast migration is a chemotactic cue supplied by the emerging dermis. Until dermis emerges, melanoblasts fail to enter the dorsal path. The dermis emerges from a site that is too distant to stimulate migration by cell contact. Instead, surgeries show that dermis elicits migration from a distance. When dermis is grafted distally, neural crest cells enter the path precociously. Moreover, large grafts recruit melanoblasts from the control sides (without increasing crest cell numbers) as well as a few crest cells from ventral somite. Because other grafted tissues fail to stimulate migration, the dermis stimulus is specific. This report is the first documentation that trunk neural crest cells can be guided chemotactically. It also extends evidence that migration is exquisitely sensitive to temporal-spatial patterns of somite morphogenesis. Developmental Dynamics 229:99-108, 2004.

Eph/Ephrin Signaling Regulates the Mesenchymal-to-Epithelial Transition of the Paraxial Mesoderm during Somite Morphogenesis

Background: During somitogenesis, segmental patterns of gene activity provide the instructions by which mesenchymal cells epithelialize and form somites. Various members of the Eph family of transmembrane receptor tyrosine kinases and their Ephrin ligands are expressed in a segmental pattern in the rostral presomitic mesoderm. This pattern establishes a receptor/ligand interface at each site of somite furrow formation. In the fused somites (fss/tbx24) mutant, lack of intersomitic boundaries and epithelial somites is accompanied by a lack of Eph receptor/Ephrin signaling interfaces. These observations suggest a role for Eph/Ephrin signaling in the regulation of somite epithelialization.Results: We show that restoration of Eph/Ephrin signaling in the paraxial mesoderm of fss mutants rescues most aspects of somite morphogenesis. First, restoration of bidirectional or unidirectional EphA4/Ephrin signaling results in the formation and maintenance of morphologically distinct boundaries. Second, activation of EphA4 leads to the cell-autonomous acquisition of a columnar morphology and apical redistribution of β-catenin, aspects of epithelialization characteristic of cells at somite boundaries. Third, activation of EphA4 leads to nonautonomous acquisition of columnar morphology and polarized relocalization of the centrosome and nucleus in cells on the opposite side of the forming boundary. These nonautonomous aspects of epithelialization may involve interplay of EphA4 with other intercellular signaling molecules.Conclusions: Our results demonstrate that Eph/Ephrin signaling is an important component of the molecular mechanisms driving somite morphogenesis. We propose a new role for Eph receptors and Ephrins as intercellular signaling molecules that establish cell polarity during mesenchymal-to-epithelial transition of the paraxial mesoderm.

The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation, patterning and differentiation of somites.

The paraxial mesoderm of the somites of the vertebrate embryo contains the precursors of the axial skeleton, skeletal muscles and dermis. The Meox1 and Meox2 homeobox genes are expressed in the somites and their derivatives during embryogenesis. Mice homozygous for a null mutation in Meox1 display relatively mild defects in sclerotome derived vertebral and rib bones, whereas absence of Meox2 function leads to defective differentiation and morphogenesis of the limb muscles. By contrast, mice carrying null mutations for both Meox genes display a dramatic and wide-ranging synthetic phenotype associated with extremely disrupted somite morphogenesis, patterning and differentiation. Mutant animals lack an axial skeleton and skeletal muscles are severely deficient. Our results demonstrate that Meox1 and Meox2 genes function together and upstream of several genetic hierarchies that are required for the development of somites. In particular, our studies place Meox gene function upstream of Pax genes in the regulation of chondrogenic and myogenic differentiation of paraxial mesoderm.

Roles for Zebrafish Focal Adhesion Kinase in Notochord and Somite Morphogenesis

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord–somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord–somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.

The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos.

One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary. Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM. Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that establishes the segmental boundaries between posterior and anterior cells in adjacent segments.

Control of her1 expression during zebrafish somitogenesis by a Delta-dependent oscillator and an independent wave-front activity

Somitogenesis has been linked both to a molecular clock that controls the oscillation of gene expression in the presomitic mesoderm (PSM) and to Notch pathway signaling. The oscillator, or clock, is thought to create a prepattern of stripes of gene expression that regulates the activity of the Notch pathway that subsequently directs somite border formation. Here, we report that the zebrafish gene after eight (aei) that is required for both somitogenesis and neurogenesis encodes the Notch ligand DeltaD. Additional analysis revealed that stripes of her1 expression oscillate within the PSM and that aei/DeltaD signaling is required for this oscillation. aei/DeltaD expression does not oscillate, indicating that the activity of the Notch pathway upstream of her1 may function within the oscillator itself. Moreover, we found that her1 stripes are expressed in the anlage of consecutive somites, indicating that its expression pattern is not pair-rule. Analysis of her1 expression in aei/DeltaD, fused somites (fss), and aei;fss embryos uncovered a wave-front activity that is capable of continually inducing her1 expression de novo in the anterior PSM in the absence of the oscillation of her1. The wave-front activity, in reference to the clock and wave-front model, is defined as such because it interacts with the oscillator-derived pattern in the anterior PSM and is required for somite morphogenesis. This wave-front activity is blocked in embryos mutant for fss but not aei/DeltaD. Thus, our analysis indicates that the smooth sequence of formation, refinement, and fading of her1 stripes in the PSM is governed by two separate activities.

Surface ectoderm is necessary for the morphogenesis of somites

The paraxial mesoderm of the neck and trunk of mouse embryos undergoes extensive morphogenesis in forming somites. Paraxial mesoderm is divided into segments, it elongates along its anterior posterior axis, and its cells organize into epithelia. Experiments were performed to determine if these processes are autonomous to the mesoderm that gives rise to the somites. Presomitic mesoderm at the tailbud stage was cultured in the presence and absence of its adjacent tissues. Somite segmentation occurred in the absence of neural tube, notochord, gut and surface ectoderm, and occurred in posterior fragments in the absence of anterior presomitic mesoderm. Mesodermal expression of Dll1 and Notch1, genes with roles in segmentation, was largely independent of other tissues, consistent with autonomous segmentation. However, surface ectoderm was found to be necessary for elongation of the mesoderm along the anterior–posterior axis and for somite epithelialization. To determine if there is specificity in the interaction between ectoderm and mesoderm, ectoderm from different sources was recombined with presomitic mesoderm. Surface ectoderm from only certain parts of the embryo supported somite epithelialization and elongation. Somite epithelialization induced by ectoderm was correlated with expression of the basic-helix-loop-helix gene Paraxis in the mesoderm. This is consistent with the genetically defined requirement for Paraxis in somite epithelialization. However, trunk ectoderm was able to induce somite epithelialization in the absence of strong Paraxis expression. We conclude that somitogenesis consists of autonomous segmentation patterned by Notch signaling and nonautonomous induction of elongation and epithelialization by surface ectoderm.

Xl erg: expression pattern and overexpression during development plead for a role in endothelial cell differentiation.

The ets gene family encodes transcription factors related to the proto-oncogene c-ets-1 and involved in cell proliferation, differentiation, and oncogenic transformation. We have characterized the Xenopus homologue of the human erg gene, an ets-related-gene, and its expression has been examined throughout early embryonic development. Xl erg encodes at least two proteins, resulting from alternative splicing events. The transcripts are restricted to the forming endocardium, the endothelial cells of the blood vessels and to the neural crest-derived mesenchyme cells of the pharyngial arches. When Xl ERG is expressed ectopically in Xenopus embryos by microinjection of synthetic mRNA, multiple developmental defects are observed. Dorsally injected embryos have their AP axis shortened and present severe defects in eye and somite morphogenesis. Ventrally injected embryos show a posteriorization of the cells having received the message together with ectopic endothelial cell differentiation as revealed by the accumulation of X-msr transcripts. In both cases, accumulation of erythrocytes in structures not connected with the blood circulatory system can be observed. Our data suggest that Xl erg may be involved in cell motility and in the development of the circulatory system.

Adhesive Subdivisions Intrinsic to the Epithelial Somites

Developing somites express two subtypes of classic cadherin adhesion receptors, N-cadherin and cadherin-11 (cad11). To investigate the role of these adhesion molecules in somite morphogenesis, we analyzed the somites of mice whose N-cadherin and cad11 genes were disrupted. The epithelial somites of N-cadherin null mutant mice were fragmented as reported, whereas those of cad11(−/−) mice showed no structural anomaly. In mice double homozygous for N-cadherin and cad11 mutation, however, somites were further fragmented into smaller clusters than in the N-cadherin-deficient mice, suggesting that these two cadherins cooperate in the maintenance of epithelial somites. Despite the disorganization of epithelial structures, dorsoventral polarity markers were expressed in their correct patterns in all of these mutant somites. Uncx4.1, whose expression is localized only in the caudal region of each somite, was also expressed in a normal pattern in the mutant somites. However, the staining for Uncx4.1 revealed that, in the N-cadherin mutants, each somite tended to be cleaved at the border between the Uncx4.1-positive and -negative regions and that the cleaved subunits maintained the clustered state, often exhibiting epithelioid morphology. This separation of the rostral and caudal regions was observed as soon as the epithelial somites had been formed. In the N-cadherin/cad11 double-homozygous mutants, this tendency was also observed, although each half of the somite further disintegrated into randomly arranged cell clusters. These results suggest that cells of the rostral and caudal regions of each epithelial somite have an activity to aggregate independently or separate from one another and that one role of N-cadherin and cad11 is to connect the two halves into a single unit.

Requirement of the paraxis gene for somite formation and musculoskeletal patterning.

The segmental organization of the vertebrate embryo is first apparent when somites form in a rostrocaudal progression from the paraxial mesoderm adjacent to the neural tube. Newly formed somites appear as paired epithelial spheres that become patterned to form vertebrae, ribs, skeletal muscle and dermis. Paraxis is a basic helix-loop-helix transcription factor expressed in paraxial mesoderm and somites. Here we show that in mice homozygous for a paraxis null mutation, cells from the paraxial mesoderm are unable to form epithelia and so somite formation is disrupted. In the absence of normal somites, the axial skeleton and skeletal muscle form but are improperly patterned. Unexpectedly, however, we found that formation of epithelial somites was not required for segmentation of the embryo or for the establishment of somitic cell lineages. These results demonstrate that paraxis regulates somite morphogenesis, and that the function of somites is to pattern the axial skeleton and skeletal muscles.

Combinatorial signals from the neural tube, floor plate and notochord induce myogenic bHLH gene expression in the somite.

The neural tube, floor plate and notochord are axial tissues in the vertebrate embryo which have been demonstrated to play a role in somite morphogenesis. Using in vitro coculture of tissue explants, we have monitored inductive interactions of these axial tissues with the adjacent somitic mesoderm in chick embryos. We have found that signals from the neural tube and floor plate/notochord are necessary for expression of the myogenic bHLH regulators MyoD, Myf5 and myogenin in the somite. Eventually somitic expression of the myogenic bHLH genes is maintained in the absence of the axial tissues. In organ culture, at early developmental stages (HH 11-), induction of myogenesis in the three most recently formed somites can be mediated by the neural tube together with the floor plate/notochord, while in more rostral somites (stages IV-IX) the neural tube without the floor plate/notochord is sufficient. By recombining somites and neural tubes from different axial levels of the embryo, we have found that a second signal is necessary to promote competence of the somite to respond to inducing signals from the neural tube. Thus, we propose that at least two signals from axial tissues work in combination to induce myogenic bHLH gene expression; one signal derives from the floor plate/notochord and the other signal derives from regions of the neural tube other than the floor plate.

Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos

We report the cloning of two new quail myogenic cDNAs, quail myogenic factor 2 (qmf2) and qmf3, which encode helix-loop-helix proteins homologous to mammalian myogenic factors myogenin and myf-5. In situ hybridization has been used to investigate the developmental expression of qmf2 and qmf3, as well as qmf1, the quail homologue to mammalian MyoD1, during the formation of the brachial somites. These studies show that qmf1 and qmf3 are activated sequentially in medially localized somite cells, immediately following somite formation but prior to myotome formation. qmf1, qmf2, and qmf3 are expressed in the myotome of compartmentalized somites. These findings suggest that determination of the myogenic cell lineage in quail somites is a progressive process controlled by influences of the neural tube on the expression of the qmf regulatory genes in newly forming somites.

Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos

The structural similarity between Drosophila and vertebrate homeobox genes begs the question of whether the vertebrate gene products affect cell fate and pattern formation. To study the function of the Xenopus homeobox protein, Xhox-1A, we microinjected fertilized Xenopus eggs with an excess of synthetic Xhox-RNA and assayed for effects on development. The predominant phenotype is a disturbance in somite formation. When embryos are injected with Xhox-1A mRNA, but not with control mRNAs, morphogenesis of somites occurs chaotically and individual segments are lost. Histological staining, in situ hybridization, and immunohistochemistry indicate that the disorganized somitic tissue has differentiated into muscle cells. Overall, these results suggest that correct regulation of the Xhox-1A gene may be important for the normal development of the segmented somite pattern in early embryos. Moreover, the inferred role of Xhox-1A in somite formation indicates that there may be molecular parallels between mechanisms of segmentation in flies and vertebrates.

Development of wing-bud-derived muscles in normal and wingless chick embryos: a computer-assisted three-dimensional reconstruction study of muscle pattern formation in the absence of skeletal elements.

The mechanisms whereby the normal pattern of muscles within the developing chick limb bud is generated are largely unexplored. It has been proposed that the muscle pattern is established independently of the pattern for the limb skeletal elements to which the muscles normally attach (Shellswell and Wolpert: "The Pattern of Muscle and Tendon Development in the Chick Wing."In: Vertebrate Limb and Somite Morphogenesis. Cambridge University Press, Cambridge, pp. 71-86, 1977). To further examine this possibility we studied the formation of the proximal wing muscles in normal and wingless chick embryos. The muscles of the shoulder region (including the pectoralis) arise as part of the dorsal and ventral premuscle masses of the developing limb bud. These secondarily migrate out of the limb to take origin from the pectoral girdle while inserting onto the humerus (Sullivan: Aust. J. Zool., 10:458-516, 1962). With rare exceptions, wingless embryos have complete absence of wing skeletal elements, but they may possess more than 40% of the normal volume of wing-bud-derived muscles. The muscles that remain in wingless embryos are primarily shoulder muscles, and to a varying extent, the pectoralis. The question we sought to answer was whether in wingless embryos the proximal wing muscles could form a normal pattern in the absence of the humerus and distal wing skeletal elements. By examining three-dimensional reconstructions of the proximal wing region in normal and wingless embryos, we found that the initial subdivision of the dorsal and ventral premuscle masses proceeded normally in the absence of the wing skeleton. This resulted in a grossly normal pattern of proximal wing muscles despite the absence of wing skeletal elements. However, some subsequent cleavages of individual muscles within premuscle mass divisions did not occur in wingless embryos. This suggests that the skeleton may be required for this step in muscle morphogenesis to occur. We also observed that the wing-bud-derived muscles in wingless embryos were nearly always anchored to the pectoral girdle at both ends. Sometimes this resulted in muscles making abnormal tendonous fusions with other muscles derived from the opposite (i.e., dorsal or ventral) premuscle mass. Therefore, attachment to the skeleton may be important for some facet of muscle development. Finally, the supracoracoideus muscle was absent in all but one wingless embryo we examined in the present study. In that one, it was substantially reduced in volume compared to normal. absence of this muscle, the space normally occupied by the supracoracoideus was maintained beneath the pectoralis.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative analysis of amphibian somite morphogenesis: cell rearrangement patterns during rosette formation and myoblast fusion

Detailed SEM observations of the changes in cellular morphology, arrangements, and contacts that occur during the process of somite formation were made in two species of urodele amphibians, Ambystoma mexicanum and Pleurodeles waltlii, and one species of anuran amphibian, Rana sphenocephala. After fixation, embryos were fractured transversely, horizontally, and parasagittally, and the intrasomitic cellular arrangement pattern was examined with the SEM. It was found that Ambystoma and Pleurodeles embryos followed exactly the same development sequence in rosette formation and myoblast fusion. Rana somites did not, however, appear to form rosettes. Those myotomal cells underwent fusion immediately after a few segmentations occurred. Patterns of cellular rearrangement were also described during urodele rosette formation at the time of somite segmentation and during myoblast fusion. Extensive changes in cell shape and orientation appeared to occur during those processes. When cells changed their orientation, they often exhibited a triangular configuration. Probable roles of these triangular-shaped cells in rosette formation and myoblast fusion are discussed. During the initial period of myoblast or myotomal cell fusion, cells first send out specialized cell processes and then establish their cell-cell contacts. The establishment of such contacts eventually leads to tight membrane appositions and fusion. Since myoblast fusion appeared to occur between two cells which were tandemly arranged in a rosette, the origin of multi-nuclearity in the fused cells is discussed. Finally, comparative analyses of the pattern of somite formation and subsequent muscle development were made between different species of amphibians. The possibility is discussed that patterns of somitogenesis may provide useful indicators for determining how different families of amphibians evolved.