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[Demonstration, by means of electron microscopy, of the penetration of somitic cells into the mesoblast of the limb buds of reptile embryos (Anguis fragilis, Lacerta viridis)].

An electron microscopic study of the components of anterior limb buds of the slow-worm (Anguis fragilis) and of the green lizard (Lacerta viridis) (embryos of Anguis whose allantoic bud reach 0,7 to 4 mm of length; embryos of Lacerta 2 to 7 days old) provides data on the cytological characteristics of the components of the limb bud at these early stages. 1. The cells of the distal extremity of the somitic processes extending in the limb bud of Anguis and Lacerta, are elongated cells with ovoid nuclei containing large nucleolus; they possess mitochondria always thin and with dense matrix; they are rich in lipid droplets; they possess cilia; they are devoid of myofilaments; endoplasmic reticulum, free ribosomes and polyribosomes are abundant. Golgi networks display signs of activity. These characteristics are also observed in the cells of the "dermatome" layer of the dermo-myotome; and so, it appears probable that the cells of the "dermatome". Furthermore, in Anguis embryos, the cells of the distal extremities of the somitic processes possess numerous lysosomes and a certain number of cells among them, degenerate early. 2. The somatopleural mesoblastic cells of the limb bud of Anguis and Lacerta embryos keep the characters of the cells of the mesodermic layer of lateral plate from which they originate; they have rounded nuclei, cilia, and their mitochondria are always larger and more transparent to electrons, than the ones of cells of the somitic processes and of cells of the epiblastic apical crest. Golgi networks are well developped, endoplasmic reticulum is abundant, lipid droplets are rare. 3. The processes of somites which extend in the dorsal part of the limb bud of Anguis embryos are cords of cells with thin lumina; at the stage of the allantoic bud of 0,6 to 0,8 mm long, the distal extremity of these processes dislocate in group of cells which afterwards dissociate, releasing individual somitic cells which are integrated among the mesoblastic somatopleural cells. In young lizard embryos (2 to 4 days old) the distal extremity of the somitic processes enlarges into a vesicle from which cells are released and penetrate in the mesoblast of the limb bud. 4. The somitic cells released from the somitic processes of Anguis and Lacerta keep--at least at early stages--the cytological characteristics they displayed when they were still in situ in the somitic processes: grounded on the presence or absence of lipid droplets, on the width and density of the mitochondria, the distinction, at these stages, between the somitic and mesoblastic somatoplerual cells is possible; and it is also possible to observe the integration of the somitic cells into the mesoblast. This study brings the demonstration of the cellular contribution of the somites to the formation of the limb bud in Reptiles. 5...

Allantois

Role of the somitic mesoderm in the development of the thorax in bird embryos. II. Origin of thoracic and appendicular musculature.

The regional embryonic origin of trunk and limb musculature was determined through heterospecific homotopic or heterotopic transplantations of quail somitic or somiticsomatopleural mesoderm into chick hosts, and through localized X-irradiation of the somitic mesoderm. Experiments were performed on 2-day embryos. Results show that the myoblastic component of all truncal and appendicular muscles is of somitic origin. Intra- and perimuscular connective tissue as well as tendons are of somatopleural origin. X-ray destruction of the somitic mesoderm at and beyond the wing level resulted in the complete or almost complete absence of musculature in the wing and corresponding truncal region. The mapping of the cephalocaudal origin of the various muscles was found to be as follows: Intrinsic and extrinsic muscles of the wing and scapular girdle Somites 12-20 Grand pectoral muscle Somites 12-22 Intercostal muscles Somites 19-26 Abdominal muscles Somites 27-29 Intrinsic and extrinsic muscles of the leg and pelvic girdle Somites 26-32 Dorsal and intervertebral muscles Metameric level, specific origin In the heterotopic transplantations, the grafted somitic mesoderm gave rise to site-specific morphogenesis, irrespective of the cephalocaudal level of its origin. This result demonstrates that, at 2 days of incubation, the myogenic somitic cells are not regionalized.

Animals

The mechanism of somite segmentation in the chick embryo.

The segmentation of somites in the chick embryo has been studied by transmission and scanning electron microscopy (stages 8-14). The segmental plate mesoderm consists of loosely arranged mesenchymal cells, whereas the newly formed somites are composed of elongated, spindle-shaped cells arranged radially around a lumen, the myocoele. The diamter of each somite is thus two cells plus the myocoele. Two major factors appear to be responsible for the change in cell shape at segmentation: (1) Each prospective somite cell becomes anchored at one end to the adjacent epithelia (i.e. the neural tube, the notochord, the ectoderm, the endoderm or the aorta) by means of collagen fibrils. These fibrils are already present in the segmental plate before the somites begin to form. (2) A change in cell-to-cell adhesiveness causes the free ends of these cells to adhere to one another. (Bellairs, Curtis & Sanders, 1978). This adhesion is then supplemented by the development of tight junctions proximally in the somite. Because it is anchored at both ends, each somite cell is under tension in much the same way as a fibroblast cell in tissue culture is under tension. Each somite cell therefore becomes elongated and the somite as a whole accommodates its general shape to that of the space available between the adjacent tissues. The arrangement of the cells in the more differentiated somites (stages 17-18) has also been examined and it has been found that the chick resembles Xenopus in that the myotome cells undergo rotation and become orientated in an anteroposterior direction.

Animals

Inhibition of "spontaneous," notochord-induced, and collagen-induced in vitro somite chondrogenesis by the calcium lonophore, A23187.

The present study represents a first step in investigating the possible involvement of calcium (Ca2+) in the stimulation of somite chondrogenesis elicited by extracellular matrix components produced by the embryonic notochord. The ionophore, A23187, a drug that facilitates Ca2+ uptake leading to elevation of cytoplasmic Ca2+ levels, at concentrations of 0.25-1.0 microgram/ml severely impairs "spontaneous" somite chondrogenesis, i.e., inhibits the formation of the small amount of cartilaginous matrix normally formed by embryonic somites in vitro in the absence of inducing tissues. This inhibition is reflected in a considerable reduction in sulfated glycosaminoglycan (GAG) accumulation by A23187-treated somite explants. Furthermore, A23187 inhibits the striking stimulation of cartilaginous matrix formation and sulfated GAG accumulation normally elicited by the embryonic notochord and collagen substrates. In fact, 1.0 microgram/ml of A23187 reduces sulfated GAG accumulation by somites cultured in association with notochord or on collagen to a level even below that accumulated by somites cultured in the absence of these inductive agents. Although these results must be interpreted with caution, they provide incentive for considering a possible regulatory role for Ca2+ in the chondrogenic response of somites to extracellular matrix components produced by the embryonic notochord.

Animals

Differentiation of the musculature of the teleost Brachydanio rerio. II. Effects of immobilization on the shape and structure of somites.

The development of the shape and structure of somites in the teleost Brachydanio rerio was studied in embryos under normal conditions and in immobilized embryos. Three different immobilization methods were applied: enclosure in agar, a glass rod in the neural tube and anaesthesia in MS-222. When the performance of the lateral body movements is prevented, the shape development of the somites in embryos and young larvae becomes reversed. When the agar-immobilization is terminated, the larvae resume their normal movements. In about 10 days, the shape of the somites is again as in control larvae. We conclude, that the lateral body movements have both a shape-determining and a shape-stabilizing role during the early stages of somite morphogenesis. It is suggested that in normal embryos differences in shortening between lateral and medial muscle fibres, cause differences in longitudinal growth of the muscle fibres and that the oblique muscle fibre arrangement is a consequence of these differences in growth. In immobilized embryos and larvae, the longitudinal growth of the muscle fibres is decreased. Also the difference in the longitudinal growth rate between lateral and medial muscle fibres diminishes in all somites. We conclude that for the normal morphogenesis of the somites the performance of the specific function, that is to bring about lateral body movements, is required. We suggest, that the impact of the lateral body movements on the development of the structure of the somites is mediated through adaptive growth of the muscle fibres. The suggestion may also apply to the development of the pinnate structure of muscles of higher vertebrates.

Agar

Somitogenesis in amphibian embryos. I. Experimental evidence for an interaction between two temporal factors in the specification of somite pattern.

Somitogenesis is described in two species of anuran amphibians, Xenopus laevis and Rana temporaria, in which the cellular mechanics of somite formation are distinctly different. Heat shocks are employed to demonstrate a wave of cellular change which precedes somite formation down the body axis. This prior wave is shown to be kinematic. It is not a propagated wave. It is a consequence of the temporal activities of the cells laid out in space, but there is no evidence that these activities depend upon an interpretation of their position. Heat shocks cause characteristic segmental abnormalities over a zone of somites which is formed several hours after the shock. Evidence from double heat shock experiments suggests that the pattern of abnormality is the result of (i) a disturbance of co-ordination between pre-somitic cells, and (ii) the time available to those cells for recovery before they are recruited into a segmental pre-pattern at the time of passage of the prior wave. It is a temporal co-ordination that is disturbed and subsequently recovered following a heat shock. This temporal co-ordination of pre-somitic cells does not depend upon position along the axis. The evidence for two physiologically independent temporal patterns of cellular processes, which interact to specify the segmental pattern of somites (their size, shape and number), gives experimental support for the theoretical account of somitogenesis proposed by Cooke & Zeeman (1976).

Animals

Retention during embryonic life of the ability of avian spinal cord to induce somitic chondrogenesis in vitro.

It is well established that the spinal cord of embryonic vertebrates induces sclerotomal somitic mesoderm to chondrify. We have investigated whether the spinal cord retains this inductive ability for the duration of the life of the avian embryo. Somites were isolated from embryos of H.H. stages 16 to 18 and either cultured alone in a medium which would not allow spontaneous chondrogenesis or cultured in direct contact with the spinal cord from embryos ranging in age between H.H. stages 33 and 44 (7 1/2--18 days of incubation). Somites cultured alone did not chondrify. Somites cultured in contact with either the ventral surface of the spinal cord or with the ependyma of the spinal cord chondrified in virtually 100% of all cultures--irrespective of the age of the donor embryo providing the spinal cord. The somites which were cultured in contact with the dorsal surface of the spinal cord did not undergo chondrogenesis. We conclude that the ventral spinal cord and the ependyma retain inductive ability through embryonic life and discuss the possible reasons for this.

Animals

The effect of collagen on the cyclic AMP content of embryonic somites.

Previous studies have demonstrated that collagen substrates stimulate in vitro somite chondrogenesis, and that agents that elevate intracellular cyclic AMP levels in hibit the ability of somites to respond to the inductive influence of collagen. In the present investigation, radiommunoassay was utilized to compare the cyclic AMP content of somite explants cultured on purified Type I collagen substrates with control explants cultured on Millipore filters. During the period of culture, the cyclic AMP content of collagen-treated explants is significantly lower than the cyclic AMP content of control explants. The cyclic AMP content of collagen-treated explants is 66% of control values as early as one hour following the initiation of culture, and the cyclic AMP content of collagen-treated explants remains lower than controls throughout the 3-day cultured period. The greatest difference in the cyclic AMP content of collagen-treated and control explants is observed at the seventeenth hour of culture, at which time the cyclic AMP content of collagen-treated explants is 56% of controls. These results combined with previous studies provides support for the hypothesis that collagen elicits a reduction in the cyclic AMP content of embroyic somites and that this reduction is necessary to trigger chondrogenic differentiation.

Animals

The MURCS association: Müllerian duct aplasia, renal aplasia, and cervicothoracic somite dysplasia.

Two patients and 28 others in the literature were ascertained because of congenital vaginal agenesis associated with clinical and/or radiographic evidence of malformations derived from the cervicothoracic somites. In these patients, there was a high incidence of Müllerian duct aplasia/hypoplasia (96%), renal agenesis and/or ectopy (80%), and abnormalities related to cervicothoracic somite dysplasia, particularly 2 to 4 anomalous vertebrae located between C5-T1 (80%). These consistent findings suggest a distinctive non-random association of malformations: Müllerian duct (MU) aplasia, renal (R) aplasia, and cervicothoracic somite (CS) dysplasia (MURCS). Identification of one component of the MURCS association suggests the presence of the other associated anomalies. A hypothesis for the embryogenic pathogenesis of the MURCS association is proposed which attributes the malformations to an alteration of the blastemas of the lower cervical-upper thoracic somites, arm buds, and pronephric ducts, all of which have an intimate spatial relationship at the end of the fourth week of fetal life. A presently unidentified teratogen may be one of the possible causes of the MURCS association on the basis of a lack of familial transmission, normal chromosomal studies, and the similar effects of a known teratogen (thalidomide) on the developing genitourinary tract.

Abnormalities, Multiple

From stem cells to somites: Revealing genetic and exogenous factors of human embryogenesis.

Stem-cell-based human embryo models offer an ethically tractable platform for studying early human development. This study employs somitoids, three-dimensional models of human somitogenesis, to investigate how transcriptional programs and culture conditions influence somite formation and segmentation. We show that pre-differentiation culture medium impacts the developmental potential of induced pluripotent stem cells (iPSCs), with StemFit medium and Matrigel embedding outperforming mTeSR Plus medium in generating robust somite-like structures. Strikingly, these differences arise despite only subtle changes in transcriptomic and time-resolved proteomic profiles. P300-based proximity labeling also reveals a largely overlapping set of chromatin-associated regulators across iPSC conditions. In somitoids, enhancer-associated profiling highlights factors linked to somitogenesis, including MESP2 and TBX6. Knockout of three identified regulators, BPTF, RBPJ, and CITED2, demonstrate their essential roles in somite formation. Together, these findings highlight how culture conditions and enhancer-associated networks influence early human development and demonstrate somitoids as a scalable system for functional genomics.

Humans

[Electron microscopic study of the penetration and distribution of somitic cells in the mesoblast of the limb buds of reptiles (Anguis fragilis and Lacerta viridis)].

Based on characteristics of mitochondria and on the amount of lipid inclusions, a distinction between somitic cells and mesoblastic somatopleural cells is possible, at the early stages of the development of the limb bud in Reptiles (Anguis fragilis and Lacerta viridis). The dislocation of the ventral processes of the somites and the localisation of the somitic cells in the mesoblast of the anterior limb buds could be studied.

Allantois

Somitogenesis in amphibia. II. Origins in early embryogenesis of two factors involved in somite specification.

A somite pre-pattern is established shortly before visible segmentation. The pre-pattern results from the interaction of two components: a wave of cell behavioural change that passes along the axis, and, an underlying co-ordination of the cells that is the basis for their association into large somite-sized groupings. The evidence is derived from studies of the zones of abnormal segmentation that follow temperature shocks delivered between the neurula and tail-bud stages (Pearson & Elsdale, 1979). Temperature shock given earlier at the mid-gastrula stage is however ineffective in inducing abnormalities in somitogenesis. Shocks given before the mid-gastrula stage reveal a prior period of sensitivity stretching back into the blastula. Thus early and late sensitive periods can be defined separated by a short refactory period. Quite different patterns in the distribution of somite abnormalities characterize the results of shock during the two sensitive periods, suggesting different aetiologies. It is concluded that the wave of rapid cell change is set up early in embryogenesis during the blastula stage, and each cell of the prospective paraxial mesoderm carries a determination to change after a specific length of time, i.e. a countdown is set in each cell. As a result of the movements of gastrulation, the prospective paraxial mesoderm cells become laid out along the axis of the neurula in the order (antero-posterior sequence) in which they will change. The achievement of the correct redistribution of the cells depends crucially on the conservation of the sequence in the blastula by the maintenance of topological integrity throughout gastrulation. It is suggested that early shock disturbs gastrulation movements, causing some mixing up of the cells resulting in incoherence of the wavefront. Whereas early shocks are thus assumed to affect the wave, the evidence suggests that late shock undergoes co-ordination. It is concluded therefore that co-ordination is established later, after the refractory period, around the late gastrula stage.

Animals

Muscle-forming potential of the non-somitic cells of the early avian limb bud.

It has recently been shown that the musculature of the chick wing arises by migration of cells from the somites, and that on morphological grounds this process begins at about stage 14. We have carried out grafts of wing anlagen separate from the somites from quail donors to the extra-embryonic coelom of chicks, and find that anlagen from as early as stage 10 (11 pairs of somites) can give rise to muscle. We discuss the possible reasons for this finding, and conclude that in the absence of the cells normally giving rise to the musculature, mesodermal cells themselves can give rise to muscle.

Animals

Researches on the formation of axial organs in the chick embryo. IX. On the development of somites in axial-paraaxial segments explanted to the zona pellucida.

Axial-paraaxial segments (neural tube, chorda, unilateral meso- and endoderm) excized from explanted 36--40-hour-incubated chick embryos at the level of unsegmented mesoderm, after removal of the ectoderm, were grafted onto subectodermal pockets of the zona pellucida. Under these conditions somites develop and differentiate normally. Paraaxial segments (unilateral meso- and endoderm) grafted under the same conditions show (retarded) somitogenesis only in 15% of the cases. Pure paraaxial unsegmented mesoderm grafted under the same conditions develops somites in 14% of the cases. Since in situ, the removal of the axial organs and of the endo- and ectoderm does not inhibit somitogenesis, the above-mentioned results prove that under conditions of grafting, some additionary "factors of realization" necessary for normal somitogenesis are lacking.

Animals

Somite formation in the early chick embryo following grafts of Hensen's node.

Quail grafts of Hensen's node were examined for their potential to induce somites in chick blastoderms. The origin of the structures induced depended on the distance of the graft from the host's midline. Nodes placed at the margin of the area pellucida resulted in structures differentiated from the cells of the graft, whereas medially the graft organized host cells to form rows of somites. The results are discussed in terms of competence of graft and host mesenchyme and a positional signal from the node.

Animals

The migration of myogenic cells from the somites into the leg region of avian embryos. An ultrastructural study.

The migration of myogenic stem cells into the leg anlagen of chick embryos between stages 16--20 of Hamburger and Hamilton was examined. SEM and TEM studies reveal that cell migration starts at stage 16 from the just-formed somites 26-28. The migrating myogenic cells are elongated and oriented in a medio-lateral direction. The leading ends branch into filopodia which contact a fibrillar network. At first, single cells migrate; later on the cells leaving the ventro-lateral edge of the dermatome migrate in strands and have specialized contacts between them. After reaction with ruthenium red and concanavalin A the migrating cells show a thick surface coat to which ruthenium red-positive particles are attached. The surface coat may be important in the interactions among the migrating cells as well as between the cells and the substrate. The migration of myogenic stem cells was found to take place in a matrix of collagenous fibrils and ruthenium red-positive particles, probably containing glycosaminoglycans. At the onset of migration the fibrillar network exhibits a preferred medio-lateral orientation. Therefore, it may be concluded that this alignment of the fibrils influences the direction of cell migration.

Animals

Culture of early somite mouse embryos during organogenesis.

Early somite (2-4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum and Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.

Animals