Etude de la migration des cellules somitiques dans le mésoderme somatopleural de l'ébauche de l'aile

Summary In chick embryos, observations were made on serial semithin transverse sections of the wing level. In addition homo- or heterotopic replacements of the wing or leg somitic mesoderm by labelled somitic or nonsomitic mesoderm were made in 2-to 2.5-day embryos. The nuclear label used was either natural (quail donor embryos in heterotopic transplantations) or isotopic (chick donors labelled with tritiated thymidine).Histological examination revealed that the first somitic cells to leave somite 15 apparently did so at the 20 to 22 somite stage, while the last ones to leave somite 20 apparently did so shortly before the 36 somite stage.Transplantation experiments with labelled donor cells revealed the routes of migratory somitic cells and the time-course of their invasion into the outgrowing limb bud (non-somitic graft cells did not noticeably invade the limb anlage). They showed furthermore that the somitic mesoderm is not regionalized with respect to its limb myogenic properties.These results are compared with those obtained in other classes of vertebrates.

Ce travail a été subventionné en partie par la D.G.R.S.T. et le C.N.R.S.     

Expression of lbx1 involved in the hypaxial musculature formation of the mouse embryo

Somites are the source of hypaxial musculature including skeletal muscles of the limb, tongue, and trunk. To get insight into the function of mouse Lbx1 homeobox gene in early somitic mesoderm differentiation, in situ hybridization analyses were performed. At the 4-6 somite stage (8 dpc), Lbx1 was first expressed in the lateral portion of the epithelial somite and dermomyotomal epithelium. This was in contrast to the expression of myf-5 in the medial region of the somite. The lateral expression of Lbx1 in somitic mesoderm then occurred regionally along the anterior-posterior body axis. Later, at 10 dpc (stage 1 of limb bud development), Lbx1-positive migrating cells originated in the lateral dermomyotomal lips at occipital, forelimb, and hindlimb levels. They also expressed Pax-3 and c-met, known as markers of the migrating limb muscle precursor cells. In stage 4 hindlimb bud (11.5 dpc), the dorsal and ventral muscle precursor populations expressed Lbx1. In stage 8 forelimb buds (12.5 dpc), Lbx1 expression was reduced in the proximal muscle masses, where the high expression of myogenin accompanying muscle differentiation was detected. These results suggest that mouse Lbx1 might be involved in the commitment or determination of a muscle cell subpopulation during hypaxial musculature development. J. Exp. Zool. 286:270-279, 2000.     

Attempt to produce a chick/mouse heteroclass musculature

Summary Heteroclass chick/mouse chimaeras were prepared by transplanting somitic presumptive myogenic cells or limb bud myoblasts from donor mouse embryos into chick hosts, to replace (1) previously extirpated brachial somitic mesoderm or (2) experimentally deleted limb premuscular masses. Since mouse and chick cells can be distinguished by differential staining affinities, this parameter was used to verify the viability of the implant and to assess its fate. Our analyses showed that transplanted mouse somitic myogenic stem cells or limb bud myoblasts did not participate in the host brachial musculature, whatever the experimental conditions.     

Vital labelling of somite-derived myogenic cells in the chicken limb bud

Summary In order to understand how myogenic cells migrate in the limb bud, it is indispensable to distinguish undifferentiated myogenic cells from other mesenchymal cells. Thus, a suitable method for this purpose has been sought. A method to exchange the somites of a chicken and a quail microsurgically has widely been used, since the nuclei of the two species are morphologically distinguishable. However, microsurgery is accompanied by disturbances at the operated locus, and introducing cells of different species might induce unexpected effects. We report a new method for labelling chicken myogenic cells without transplantational operations, and describe their migration pattern in limb buds. Injection of a fluorescent carbocyanine dye into the somite lumen intensely labelled the somitic cells. Myogenic cells derived from the somite were clearly detected in limb buds. Before stage 20, the labelled cells were diffusely distributed in the proximal region of the limb bud. At about stage 21 in both wing and leg buds, labelled cells began to form dorsal and ventral masses. The label was followed until the cells differentiated and expressed myosin. This vital labelling method has advantages over the somite transplantation method: it does not include surgical operations that may disturb the normal development, and the cells are labelled intensely enough to be detected in a whole mount preparation. Offprint requests to: K. Hayashi     

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...     

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.     

Regulation and Function of SF/HGF during Migration of Limb Muscle Precursor Cells in Chicken

Limb muscles of vertebrates are derived from migratory dermomyotomal cells which emanate from a limited number of somites located adjacent to the developing limb buds. We have generated additional limb buds in chicken embryos by implantation of FGF-beads into the interlimb region in order to analyze whether these somites can be programmed to supply ectopic limbs with myogenic precursor cells. We show that migrating myogenic precursor cells are released from somites at the level of the newly formed limb, even when cell migration into the natural limb has been completed. The implantation of FGF beads in the lateral plate mesoderm rapidly induces SF/HGF expression. FGF beads implanted between HH stages 10 and 12 inhibit limb bud formation or shift the normal limb position. When an additional FGF bead was implanted at the original limb position at HH stage 15, SF/HGF expression was transiently induced to low levels without inducing a new limb. This demonstrates that the initial induction of SF/HGF by FGF does not require limb formation. Expression of SF/HGF during early limb bud stages was found in the entire developing bud and the adjacent lateral plate mesoderm with direct contacts to the lateral edge of the dermomyotome. Later, the SF/HGF expression domain retracts to a distal region below the apical ectodermal ridge. To investigate the role of SF/HGF in the migratory process, we implanted beads soaked in SF/HGF-alone or together with FGF into different locations of the developing chick embryo. In the experiments SF/HGF caused delamination of migratory cells from the dermomyotomal epithelium but no chemotactic attraction of migrating cells toward the SF/HGF source.     

Spatial and temporal contribution of somitic myoblasts to avian hind limb muscles

Skeletal muscles of the avian limb are derived from mononucleated myogenic precursor cells (myoblasts) that migrate into the somatopleural mesoderm of the developing limb bud from the ventrolateral dermomyotome of limb adjacent somites. In the present study, we utilized replication-deficient lacZ-encoding retroviruses to elucidate the source of myoblasts for all hind limb muscles in the chick and define the distinct patterns of myoblast distribution within the limb. We also examined, using the same marker, whether the time of migration from the somites into the limb dictates the spatial contribution the myoblasts make to the developing musculature, particularly in relation to the proximodistal and dorsovental axes. Finally, we used these investigations to examine whether the precursors of both primary and secondary myotubes are derived from somitic mesoderm, a presumption, which up until now, has not been demonstrated in vivo. Overall, the results of our studies demonstrate that individual somites have a selective spatial pattern of participation in the development of the avian hind limb musculature and contribute to both primary and secondary myotubes. We also show that both early and later migrating myoblasts can contribute fully to the formation of the appendicular muscles.     

On the role of the connective tissue in the patterning of the chick limb musculature

Summary By modifying the temporal relationship between connective tissue and myogenic cell invasion during early limb bud development new evidence of the organizing role of the connective tissue was obtained.Muscle cell-deprived wing buds were allowed to grow up to stages 22 to 27 of Hamburger and Hamilton, when they received a transplant of quail myogenic cells (somitic mesoderm or wing premuscular mass) into the dorsal face of their presumptive upper arm. Muscular arrangement in forearm and hand was analyzed 4 days later. In 8 out of 14 of those cases which had received a graft of premuscular mass before stage 25 of Hamburger and Hamilton, muscle development took place distally to the graft-site in accordance with the wing segment.     

The migration of myogenic cells from the somites at the wing level in avian embryos

This study is concerned with establishing a morphological basis for the initiation of migration of putative myogenic cells from the somites into the presumptive wing bud in avian embryos. At the 22 somite stage (stage 14) vasculogenesis is a prevalent activity. By use of a quail specific monoclonal antibody to vascular endothelial cells, vascular cells are recognized in the lateral plate, on the intermediate mesoderm, and on somite surfaces. Cells that are found between the lateral plate mesoderm and somites are shown to be vascular endothelial cells. The lateral body folds progressively bring the lateral plate mesoderm close to the lateral margin of the somites and vascular elements disappear from surface view. It is not until the 24 somite stage (stage 15) that some cells in the ventral lateral margin of somites at the wing level can be seen in scanning electron micrographs to extend basal cell processes toward adjacent vascular tubes. These results provide a morphological basis for the early migratory behavior of myogenic cells and demonstrate their close proximity to the prepatterned vascular network.     

Tbx18 and boundary formation in chick somite and wing development

The chicken Tbx gene, Tbx18, is expressed in lateral plate mesoderm, limb, and developing somites. Here we show that Tbx18 is expressed transiently in axial mesenchyme during somite segmentation. We present evidence from overexpression and transplantation experiments that Tbx18 controls fissure formation in the late stages of somite maturation. In presumptive wing lateral plate mesoderm, ectopic Tbx18 expression leads to anterior extension of the wing bud. These results suggest that Tbx18 is involved in producing mesodermal boundaries, generating in paraxial mesoderm morphological boundaries between somites and in lateral plate mesoderm a wing- or non-wing-forming boundary.     

The somitogenetic potential of cells in the primitive streak and the tail bud of the organogenesis-stage mouse embryo.

The developmental potency of cells isolated from the primitive streak and the tail bud of 8.5- to 13.5-day-old mouse embryos was examined by analyzing the pattern of tissue colonization after transplanting these cells to the primitive streak of 8.5-day embryos. Cells derived from these progenitor tissues contributed predominantly to tissues of the paraxial and lateral mesoderm. Cells isolated from older embryos could alter their segmental fate and participated in the formation of anterior somites after transplantation to the primitive streak of 8.5-day host embryo. There was, however, a developmental lag in the recruitment of the transplanted cells to the paraxial mesoderm and this lag increased with the extent of mismatch of developmental ages between donor and host embryos. It is postulated that certain forms of cell-cell or cell-matrix interaction are involved in the specification of segmental units and that there may be age-related variations in the interactive capability of the somitic progenitor cells during development. Tail bud mesenchyme isolated from 13.5-day embryos, in which somite formation will shortly cease, was still capable of somite formation after transplantation to 8.5-day embryos. The cessation of somite formation is therefore likely to result from a change in the tissue environment in the tail bud rather than a loss of cellular somitogenetic potency.     

Determination of epithelial half-somites in skeletal morphogenesis.

The segmental body plan of vertebrates arises from the metameric organization of the paraxial mesoderm into somites. Each mesodermal somite is subdivided into at least two distinct domains: rostral and caudal. The segmental pattern of dorsal root ganglia, sympathetic ganglia and nerves is imposed by differential properties of either somitic domain. In the present work, we have extended these studies by investigating the contribution of rostral or caudal-half somites to vertebral development using grafts of multiple somite halves. In both rostral and caudal somitic implants, the grafted mesoderm dissociates normally into sclerotome and dermomyotome, and the sclerotome further develops into vertebrae. However, the morphogenetic capabilities of each somitic half differ. The pedicle of the vertebral arch is almost continuous in caudal half-somite grafts and is virtually absent in rostral half-somite implants. Similarly, the intervertebral disk is present in rostral half-somite chimeras, and much reduced or virtually absent in caudal somite chimeras. Thus, only the caudal half cells are committed to give rise to the vertebral pedicle, and only the rostral half cells are committed to give rise to the fibrocartilage of the intervertebral disk. Each vertebra is therefore composed of a pedicle-containing area, apparently formed by the caudal half-somite, followed by a pedicle-free zone, the intervertebral foramen, derived from the rostral somite. These data directly support the hypothesis of resegmentation, in which vertebrae arise by fusion of the caudal and rostral halves of two consecutive somites.     

Combinations of monoclonal antibodies distinguish mesenchymal, myogenic, and chondrogenic precursors of the developing chick embryo

Monoclonal antibodies (MAbs) were used as probes for molecular differences in the surfaces of nonterminally differentiated cells of the developing chick limb. The specificity of the MAbs was determined by immunofluorescent localization performed on cultured breast muscle and limb bud cells and cryosections of a variety of embryonic (stages 15-37) and neonatal tissues. Subpopulations of MAb-positive and -negative cells were isolated by fluorescence-activated cell sorting and their developmental potential was assessed in vitro. Cells of the compacted somite, lateral plate mesoderm, and early limb bud were labeled with the CSAT MAb. Myogenic precursors of the dermatome and limb bud were labeled with the CSAT and L4 MAbs. Chondrogenic precursors of the sclerotome and limb bud were labeled with the CSAT, L4, and C5 MAbs. These precursors were distinguished from fibroblasts which were labeled with the CSAT and C1 MAbs. The differentiation and maturation of muscle and cartilage were accompanied by alterations in the labeling patterns of the MAbs. These results indicate that combinations of these MAbs can be used to distinguish mesenchymal, myogenic, and chondrogenic precursors, identify their site of origin during development, and isolate subpopulations of embryonic cells.     

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.     

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.     

Spatial and temporal patterns of cell death in limb bud mesoderm after apical ectodermal ridge removal

A spatiotemporal pattern of cell death occurred in the chick wing and leg bud mesoderm after removal of apical ectodermal ridge at stages 18–20. Cells died in a region extending from the limb bud distal surface to 150–200 μm into the mesoderm. Limb buds from which ridge was removed at later stages in development did not exhibit a spatiotemporal pattern of cell death. In control experiments in which dorsal ectoderm was removed, a pattern of cell death did not occur. Removal of the ridge and part of the 150- to 200-μm zone of prospective cell death resulted in cell death in an area approximately equal to the amount of the zone remaining. After removal of all of the prospective zone of cell death plus the apical ridge, cell death was observed in the remaining limb bud mesoderm. In these limb buds, cell death occurred in a region in which it had not been seen in limb bud with apical ridge alone removed. We conclude that at stages 18–20 the mesodermal cells 150–200 μm beneath the ridge require the apical ridge to survive. More proximal mesodermal cells do not die after ridge removal alone, but apparently require the presence of the more distal mesoderm to survive. Whether this is a requirement for something intrinsic to the distal mesoderm or something it possesses by way of the ridge is unknown. After stage 23, the limb mesoderm cells do not die when the apical ridge is removed. Nevertheless, at the later stages, ridge continues to be required for limb bud proximal-distal elongation and the differentiation of distal limb elements.