Brachial Somites Research Articles

The relationship between limb muscle and endothelial cells migrating from single somite.

Somites contribute myogenic and endothelial precursor cells to the limb bud. Transplantations of single somites have shown the pattern of muscle cell distribution from individual somites to individual limb muscles. However, the pattern of the endothelial cell distribution from individual somites to the limb has not been characterized. We have mapped quail muscle and endothelial cell distribution in the distal part of the chick limb after single somite transplantation to determine if there is a spatial relationship between muscle and endothelial cells originating from the same somite. Single brachial somites from quail donor embryos were transplanted into chick embryos, and, following incubation, serial sections were stained with a quail-endothelial cell-specific monoclonal antibody (QH-1), an anti-quail antibody (QCPN) and an anti-desmin antibody to distinguish the quail endothelial and muscle cells from chick cells. Our results show that transplants of somite 16-21 each gave rise to quail endothelial cells in the wing. The anterioposterior position of the blood vessels formed by somitic endothelial cells corresponded to the craniocaudal position of the somite from which they have originated. Endothelial cells were located not only in the peri- and endomysium but also in the subcutaneous, intermuscular, perineural and periost tissues. There was no strict correlation between the distribution of muscle and endothelial cell from a single transplanted somite. Blood vessels formed by grafted quail endothelial cells could invade the muscle that did not contain any quail muscle cells, and conversely a muscle composed of numerous quail muscle cells was lacking any endothelial cells of quail origin. Furthermore, a chimeric limb with very little quail muscle cells was found to contain numerous quail endothelial cells and vice versa. These results suggest that muscle and endothelial cells derived from the same somite migrate on different routes in the developing limb bud.

A comparative study of embryonic development of Japanese quailselected for different patterns of postnatal growth

Patterns of early embryonic development have traditionally been viewed as invariant within vertebrate taxa. It has been argued that the specific differences which are found arise during the later stages of development. These differences may be a result of allometry, heterochrony or changes in relative growth rates. To test whether early embryonic development is indeed invariant, or whether selection of adult characteristics can alter embryonic growth, we compared embryonic development in birds selected for different patterns of postnatal growth. Using quail lines selected for high and low body mass, we compared somite formation, and muscle and feather development. We obtained data that showed changes in the rate of myotome formation in the brachial somites which contribute to muscle formation in the limbs and thorax. We think these observations are connected with intraspecific changes in adult morphology, ie., breast muscle size. Our findings suggest that selection for late ontogenetic/adult stages affects early embryonic development.

Cytochemical localization of calcium in prefusion myoblasts from the chick embryo myotome.

Myoblast fusion is a Ca2+-dependent process. The aim of this report was to study the localization of Ca2+ in prefusion myoblasts from the brachial somites of chick embryos (51-108 h of incubation), using the potassium pyroantimonate cytochemical method. When observed under a transmission electron microscope, electron-dense precipitates of Ca2+-antimonate were found in the basement membrane of the myotome, which separates the myotome from the adjacent mesenchyma. Within myoblasts, triads and sarcoplasmic reticulum associated with the first newly formed sarcomeres were observed, but a T-tubule network was not found. Moreover, Ca2+-antimonate precipitates were not observed in structures resembling T-tubules or sarcoplasmic reticulum. The results suggest that sarcomerogenesis and sarcoplasmic reticulum development occur simultaneously and that prefusion myoblasts have neither a T-tubule network nor Ca2+ deposits on sarcoplasmic reticulum. Small Ca2+ pools were found in the myoblast nuclei, cytoplasmic vesicles and mitochondrias. Ca2+-antimonate precipitates periodically distributed at the cell periphery, close to the cell membrane, were observed. These precipitates could represent internal Ca2+ stores located in the peripheral couplings and it is proposed that these pools of Ca2+ could be mobilized before fusion, leading to the increase in free intracellular Ca2+ that precedes myoblast fusion.

Participation of individual brachial somites in skeletal muscles of the avian distal wing.

In this paper we investigate the somitic origin of the individual muscles of the forearm and hand using quail-chick chimeras. Our results show that only somites 16-21 give rise to wing muscle, but they take part in muscle formation to different extents. Somite 21 does not always participate in the formation of muscle of the forearm and hand. The most cranial somite (16) takes part in the radial muscles and the most caudal somites (20, 21) in the ulnar muscles, reflecting their position with respect to the limb bud. The centrally located somites (17, 18, 19) are involved in all (18) or most (17, 19) muscle primordia. This pattern of distribution is clearest in the forearm, whereas the participation of somites in particular muscle groups is not so distinct in the hand. Hand muscles are mainly made up of cells from somites 18-20. All brachial somites participate in dorsal (extensor) as well as ventral (flexor) muscles of the forearm and hand. Each somite takes part in more than three muscle primordia in a reproducible fashion, and every muscle primordium is derived from at least three somites. Especially the M. ulnimetacarpalis ventralis takes origin from all somites involved in limb muscle formation (16-21). Apart from muscle cells, endothelial cells also and a few fibroblasts of quail origin are found in the limb bud after somite grafting.

A quantitative study of the segmental distribution of somitic cells in the developing chick limb bud using laser-scanning confocal microscopy.

Our aim was to map the segmental distribution of somitic cells in the limb before and after the fusion of these cells into myotubes. Somitic cells of the brachial somites were labeled by injection of DiI and DiO into the somitoceles of embryonic Day 2 (E2) embryos. The quantitative distribution of dye-labeled cells from injected somites was examined in whole mounts of E4 wing buds and in sections of E5 wing buds using confocal microscopy. Dye derived from cells of anterior brachial somites 16 and 17 was highly concentrated in the anterior half of E4 wings, whereas dye from posterior brachial somites 20 and 21 was concentrated in the posterior half of the wing. Not more than 14% (on average) of the dye in the wing was found outside the preferred half. Dye was equally dispersed in the anterior and posterior halves of the wing when the middle two somites contributing to the wing musculature, numbers 18 and 19, were injected. The total amount of dye in the whole limb at E4 was not significantly different after injection of similar amounts of dye into either pair of brachial somites 16/17, 18/19, or 20/21. In E5 embryos the distribution of labeled cells in the newly formed muscle masses, identified with antibody 2H2 to myosin light chains, was examined in cryostat cross-sections of the wing. Dye was more widely distributed in the wing than at E4. As much as 32% (on average) of the dye was found in the muscle mass outside the preferred half after administration of dye into the anterior or posterior pair of somites. We conclude that the branchial somites contribute similar numbers of cells to the wing musculature and that the segmental origin of these cells is rigidly maintained during their lateral migration into the limb. Cells from adjacent segments then "mix" when they fuse to form myotubes at E5. In addition, we found a significantly greater amount of dye per unit of muscle mass in the proximal compared with distal parts of the limb. We argue that this finding is due to division of myogenic cells during the course of their migration in the wing.

Recognition-alignment and adhesion in myoblast fusion

Myoblast fusion results from a sequence of different stages, previously demonstrated “in vitro”. After withdrawal from the cell cycle, myoblasts align forming long chains, in a process termed “recognition-alignment”. This stage is extracellular Ca2+ and N-Cadherin dependent. Alignment is followed by adhesion, defined as the stage prior to membrane fusion when aggegates are resistant to dispersal by EDTA. Adhesion is extracellular Ca2+-independent and N-CAM-dependent. Membrane fusion originates multinucleate myotubes.We have studied these stages of myoblast fusion at the brachial myotome of chick embryo from 51 to 105 h. of incubation. Samples were obtained by embryo microdissection and included the neural tube, the notochord and the brachial somites. These samples were embedded in araldite by conventional methods. Some samples were embedded in Unicryl, a recently formulated GMA derived resin.The first myoblasts clusters were observed in the ventral-lateral region of the brachial myotome in 22-24 Hamburger and Hamilton stages embryos. Clusters of pre-fusion myoblasts are usually surrounded by “electrondense blast cells” within a basal lamina in process of formation.

Initial Axial Level-Dependent Differences in Size of Avian Dorsal Root Ganglia Are Imposed by the Sclerotome

We have recently shown that there is very early variation in dorsal root ganglia (DRG) size based on their axial position. From the time of their condensation at Stage (St.) 20 (Embryonic Day 3), before the onset of apoptosis, the ganglia in brachial segments 14 and 15 are more than 80% larger on the average than those in cervical 5 and 6. This difference in volume is due to increased numbers of cells in the brachial DRG. In addition, the rostrocaudal length was found to be significantly greater for brachial ganglia, and the greater length of the brachial ganglia was found to be correlated with a greater length of brachial than cervical sclerotomes. It was therefore proposed that the difference in DRG size at the time of gangliogenesis is likely to arise from colonization by a larger initial number of neural crest cells of the longer adjacent rostral sclerotomes in brachial somites. In the present work, we have performed two types of experiments to test this hypothesis. First, we have performed heterotopic grafts of segmental plate mesoderm from cervical to brachial levels and vice versa. In all of these grafts, the sclerotomes developed with a rostrocaudal extent (length) corresponding to their level of origin in the donor embryo. DRG that formed in the grafted mesoderm attained a length appropriate to that of ganglia developing in segments of the axial level of the donor mesoderm, not that of the host. Second, we have estimated proliferation of DRG cells at St. 20 using HNK-1/bromodeoxyuridine double-stain immunocytochemistry. The percentage of cells in S-phase at both brachial and cervical levels of the neuraxis in newly formed ganglia was observed to be the same. These two lines of evidence strongly argue that the initial difference in size between DRG at different axial levels is not intrinsic, but rather is imposed by the mesodermal microenvironment in which they develop, as is the case for DRG segmentation. This is in contrast to what may occur in the hindbrain, where determination of rhombomere identity and neural crest segmentation are thought to be intrisic to the nervous system.

Role of the brachial somites in the development of the appendicular musculature in rat embryos.

DiI, a fluorescent lipophilic dye, was micro-injected into the brachial somites of 10.5 day rat embryos to determine whether these somites can contribute cells to the development of the fore-limb bud. The injected embryos were cultured and harvested at the 20-25-somite stage. The dye did not interfere with somitogenesis because, at the injection site, the DiI-labelled somites were able to differentiate into dermomyotome and sclerotome. We have analyzed cryo-sections of 20-21-somite stage embryos and were unable detect the presence of DiI-labelled cells in the fore-limb buds. However, at the 22-somite stage, a few DiI-positive cells were found in the proximal region of the limb bud. These labelled cells had migrated into the limb from the lateral border of the dermomyotome. From the 23-somite stage onwards, there were even more DiI-positive cells inside the limb. We have performed an additional set of experiments to confirm that the somitic cells do have the ability to invade and colonize the limb bud. This was achieved by first labelling newly formed somites isolated from the caudal region of 10.5 day embryos with DiI and then grafting them into corresponding regions in 8-11-somite stage hosts. The donor somites were not orientated when they were implanted into the host. However, this did not disrupt their ability to undergo normal somitogenesis. We have detected the presence of DiI-positive cells in the limb buds of approximately 71% of the 19-30-somite stage embryos that have been examined. This is similar to what we obtained for the injected embryos. Nevertheless, there is one slight difference and that is the stage the somitic cells begin their invasion of the limb. For the injected embryos, migration began at the 22-somite stage but in the transplanted embryos, it commenced as early as the 18-somite stage. We have also investigated the myogenic potential of the fore-limb bud at various stages of development to ascertain whether there is a correlation between the stage the somitic cells first appear in the limb bud and the stage the bud acquires the capacity to form skeletal muscles. This was realized by culturing fore-limb buds excised from 18-30-somite stage embryos conventionally and in the kidney capsules of adult rats. In both methods, bone and cartilage were present in all of the cultures whereas skeletal muscles were only present in cultured explants older than the 21-22-somite stage.(ABSTRACT TRUNCATED AT 400 WORDS)

Delayed somite formation in a quail line exhibiting myofiber hyperplasia is accompanied by delayed expression of myogenic regulatory factors and myosin heavy chain

A myofiber hyperplastic quail line P has been developed through selection for heavy body weight. Since the number of muscle fibers is determined early in development and skeletal muscle originates from somites, we compared somite formation and muscle-specific gene expression in P- and control C-line quail embryos. At 47 hours of incubation, C embryos had 18 somite pairs and P embryos had 14.3. By 72 and 120 hours, both lines appeared to be at the same stage of somite development. To determine whether the delay in the formation of the brachial somites was accompanied by alterations in muscle-specific gene expression, we conducted whole-mount in situ hybridization and immunofluorescence studies. At 47 hours of incubation, C embryos were expressing qmf1 in the first 12 somites, while in P embryos only the first 7 somites showed qmf1 activation. Delays in expression were also observed for qmf3 at 43 hours and for all three myogenic factors (qmf1, qmf2 and qmf3) at 60 hours. At 65 hours, C embryos expressed myosin heavy chain in the first 15 somite pairs and P embryos in the first 7. At 72 hours, the transient delay in somite formation had disappeared and there was no lag in myosin heavy chain expression between the lines. The phase delay in brachial somite formation, myogenic factors and myosin heavy chain expression may be associated with the observed myofiber hyperplasia in P-line quail by allowing an increase in the muscle stem cell population.

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.

Positional signals: evidence for a possible role in muscle fibre-type patterning of the embryonic avian limb

Abstract Current evidence favours the hypothesis that avian muscle fibre-type differentiation is intrinsically programmed during early embryogenesis and proceeds independent of neuronal influences (for review, Miller & Stockdale, 1987; Sanes, 1987). Previous interspecific (chick/quail) and intraspecific (chick/chick) chimaeric studies, involving the heterotopic transplantation of unsegmented somitic mesoderm, indicate that the ability of premyogenic cells to form specific limb muscles is determined by positional cues operative in the host embryo. To determine if similar influences are associated with embryonic fibre-type differentiation, the limb musculature of intraspecific (chick/chick) chimaeras was assessed using differential myosin-ATPase activity. The limb musculature analysed histochemically was derived from donor unsegmented thoracic somitic mesoderm transplanted to replace extirpated brachial somites of a host embryo at 48 –52 h in ovo. Our results demonstrated that (1) the donor tissue formed, on schedule, an appropriate brachial musculature and (2) the experimental muscles duplicated the fibre-type patterning characteristic of control muscles. Thus, our data suggest that an extramyogenic, non-neural factor(s) operative in the host limb-forming region is associated with both muscle pattern formation and muscle fibre-type pattern formation. Whether or not the same putative factor(s) influences both processes remains to be determined.

Autonomy of differentiation in avian branchial somites and the influence of adjacent tissues.

This study investigates the differentiative abilities of avian brachial somites at stages of development before, during and after the migration of somitic cells into the wing primordium. These somites are the source of cells that migrate into the forelimbs and there give rise exclusively, and totally, to the skeletal muscle lineage of the wing and yet show no morphological evidence of commitment to that fate when they leave the somites. The aim of the study was to see if the brachial somitic cells are committed to particular developmental pathways at these stages. The brachial somites were isolated from HH stage-12, -15 and -18 chick embryos, either by microdissection or enzymatic dissociation, and grown in organ culture, in explant culture on different substrata or on the chorioallantoic membrane (CAM) of host chicks, either alone or in combination with adjacent tissues. Myogenesis and chondrogenesis occurred in all stage-18 enzymatically separated somites, regardless of the growth environment. Myogenesis was reduced in stage-15 somites and unobservable in stage-12 somites; however, recombination of stage-12 somites with epithelium or neural tube increased the incidence of myogenesis at this stage. The incidence of chondrogenesis was also less in the younger explants. Unlike its effect on myogenic expression, recombination with epithelium resulted in a dramatic decrease in chondrogenesis in both stage-12 and -15 somites. The recombination experiments suggest that conditions that maintain the normal spatial relationships within isolated somites permit expression of a preexisting specification to a particular fate. They also show that the overlying epithelium can inhibit chondrogenesis in these somites. Overall, the results suggest that by the time migration of somitic cells into wing regions is finishing, brachial somitic cells have become stabilized in their ability to undergo both myogenesis and chondrogenesis for they will do so under a variety of growth conditions and independently of adjacent tissues. However, immediately before (stage 12) and shortly after (stage 15) the onset of migration, both myogenic and chondrogenic expression by brachial somitic cells are still under the influence of interactions with adjacent tissues.

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles.

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.

Myogenic differentiation in early chick wing mesenchyme in the absence of the brachial somites

A controversy exists in the literature over the ability of wing mesenchyme of somatopleural origin to form skeletal muscle. Experimental approaches used in such studies leave open the possibility of postoperative accessibility of the experimental wings to somitic cell invasion. In the present study wing somatopleural tissue was isolated from HH stage-12 to -21 chick embryos and grown either in organ culture (OC) or on the chorioallantoic membrane (CAM) of host chicks, conditions under which postoperative entry of somitic cells is impossible. In the presence of axial and somitic tissues of the brachial region, the wing territories from all four stages underwent comparable growth and tissue differentiation. However, isolated wing regions showed a stage-dependency in the differentiation of skeletal muscle but not of other limb tissues. The incidence and amount of skeletal muscle was markedly reduced in HH stage-12 and -15 isolated wing regions while myogenesis in HH stage-18 and -21 wing buds was not affected by the absence of the somitic tissues. These results are consistent with reported stages of somitic cell migration into wings with the exception of HH stage-12 explants which should have been muscleless if somitic cells are the sole source of wing myofibres. The possibility that somitic cells had been included in these explants was investigated by testing the myogenic potential of lateral plate tissue adjacent to the wing, altering the dissection procedure for isolating wing territories and using antibodies to skeletal muscle myosin and actin to detect myotubes. The results from this series of experiments illustrate the need for extraordinary care in the isolation of wing regions when investigating limb-somite relationships and suggest that the myogenic capacity attributed to wing somatopleural cells in the past can be accounted for by either postoperative entry of somitic cells into experimental wings or inadvertent inclusion of somitic cells in the primordia when dissected. Overall, the results show that somitic cells are the sole source of wing myofibres for, in their absence, somatopleural cells from all mesodermally-derived wing cell types except skeletal myofibres.

Sarcomere formation in dystrophic skeletal muscle.

Early aspects of in vivo sarcomere organization were studied using normal and dystrophic New Hampshire chick embryos. Brachial somites 17-22 were removed from stages 16 through 28 embryos, processed for electron microscopy, and analyzed. Comparisons of normal and dystrophic material disclosed that thick filaments appeared later in dystrophic myotomal cells. This was correlated with a similar delay in the appearance of long polyribosomes. By stage 28, normal myotomal cells contained well-defined sarcomeres, whereas dystrophic sarcomeres were frequently unorganized, with myofilaments in poor longitudinal alignment. Periodic tubules were poorly developed or lacking.

Chimaera studies of the origin and formation of the pectoral musculature of the avian embryo.

Cervical, brachial, and thoracic somites and brachial somatopleure were transplanted from quail into 2-day chick embryos to determine their contribution to the formation of the pectoralis major muscle. The results show that the musculature of the pectoralis major and pectoralis minor is derived entirely from brachial somites (16-20) while the brachial somatopleure provides much of the connective tissue of these muscles. Migration of somitic cells into the somatopleure appears to begin before the somites are fully formed. The primordium of the pectoralis muscle forms at the base of the wing bud and extends ventro-caudally into the thoracic area to attach to the sternum.

Experimental analysis of the origin of the wing musculature in avian embryos.

Interspecific grafts of somites, as well as parts of the somatic plate mesoderm, have been made between quail and chick embryos (stages 12--14 H. H.) at the level of the prospective wing bud in order to examine the relationship between somites and wing bud myogenesis. The stability of the natural quail nuclear labelling makes it possible to follow the developmental fate of grafted mesodermal cells in the host embryo. Embryos examined after subsequent incubation periods of 3--7 days show the following distribution of somatic and somitic cells within the wing bud: as soon as the three zones of different cell density within the mesoderm can be distinguished, cells of somitic origin are limited to the prospective myogenic area which is made up of mixed population of somatic and somitic cells, whereas the prospective chondrogenic area as well as the subectodermal zone only consists of cells originated from the somatic plate mesoderm. After further incubation, single muscle blastema are present which were also seen to be a mixture of somatic and somitic cells. The cells of muscular bundles are of somitic origin, while the muscle connective tissue cells are derived from the somatic plate mesoderm. After grafting into the coelomic cavity or on the chorio-allantoic membrane, fragments of the somatic plate mesoderm previously isolated from quail embryos (stage 14 H.H.) at the level of the prospective wing bud exhibit well developed skeletal elements, but fail to differentiate any musculature. These experimental investigations support previous evidence for a somitic origin of wing bud myogenic cells. Histological and scanning electron microscopic studies of the brachial somites and the adjacent somatic plate mesoderm of chick embryo (stages 13--15 H.H.) reveal that migration of still undifferentiated somitic cells into the brachial somatic plate mesoderm begins to take place in embryos at stage 14.

Differentiation of myoblasts and the relationship between somites and the wing bud of the chick embryo.

Electron microscope studies were performed of the differentiation of myoblasts in the brachial somites and in the wing bud of chick embryos at stages 17 to 27 according to Hamburger and Hamilton. Concurrent optical inspection of short series of stained thick sections enabled the following statement concerning the relationship between the dermomyotome and the wing bud as well as the ingrowing nerve fibers.

Substitution of limbs for brachial somites

Journal of Experimental ZoologyVolume 83, Issue 3 p. 445-456 Article Substitution of limbs for brachial somites S. R. Detwiler, S. R. Detwiler Department of Anatomy, College of Physicians and Surgeons, Columbia University, and Department of Biological Sciences, Hunter College, New York CitySearch for more papers by this authorB. L. Maclean, B. L. Maclean Department of Anatomy, College of Physicians and Surgeons, Columbia University, and Department of Biological Sciences, Hunter College, New York CitySearch for more papers by this author S. R. Detwiler, S. R. Detwiler Department of Anatomy, College of Physicians and Surgeons, Columbia University, and Department of Biological Sciences, Hunter College, New York CitySearch for more papers by this authorB. L. Maclean, B. L. Maclean Department of Anatomy, College of Physicians and Surgeons, Columbia University, and Department of Biological Sciences, Hunter College, New York CitySearch for more papers by this author First published: April 1940 https://doi.org/10.1002/jez.1400830309Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume83, Issue3April 1940Pages 445-456 RelatedInformation