A study on skeletal myogenic cell movement in the developing avian limb bud

Summary Quail limb mesenchyme containing myogenic cells of somitic origin were transplanted into chick limb buds to determine whether cell movement might play a role in avian limb myogenesis. In general, cell displacement was not detected 1-day after implantation: all quail cells were found at the graft site. Migration was evident 2-days after implantation but not all cell types were capable of movement; myogenic cells were very invasive while chondrocytes were relatively immobile. The spreading of myogenic cells was discernible up to 4-days after implantation and specifically in a proximodistal direction towards the apex of the limb.     

On the determination of mesodermal tissues in the avian embryonic wing bud

Summary The quail-chick-marker technique has been employed to elucidate the determination of embryonic tissues in the avian wing bud. It was found that myogenic cells of somitic origin are determinated to give rise only to muscular tissue from HH-stage 19 on. Mesenchyme in the cartilage forming regions is determinated to form cartilage from HH-stage 20 on. Tissue from non-cartilage forming regions retains the option to form cartilage at least up to HH-stage 26. Whereas cells of somatopleural lineage do not migrate within the limb bud, cells of somitic origin do so up to at least HH-stage 28.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ch 44/4)     

Translocation of fibronectin-coated and uncoated latex beads in avian embryonic limb buds

Summary Latex beads were implanted into the chick wing bud to determine whether parameters other than active movement, for example matrix-driven translocation and growth of the limb bud, were responsible for the extensive re-allocation of myogenic cells that occurs during limb development. Latex beads were implanted into nine stage 20–24 Hamburger and Hamilton (H.H.) wing buds, and were allowed to develop for 3 days before examination. In all cases, it was found that most of the latex beads (86.57%±11.4%) were confined to the implantation site. A small percentage of beads was observed in the connective and myogenic regions proximal and distal to the graft side. In general the displacement of these beads was relatively short, although in one specimen a few beads were translocated to regions as far as the autopod. The surface of the latex beads was also coated with fibronectin prior to transplantation, to ascertain whether the extracellular matrix can influence the translocation of beads within the limb bud. Ten specimens were examined, and as for uncoated latex beads, most of the fibronectin-coated beads (87.14%±11.67%) were contained within the transplantation site. Again a mall percentage of beads was found in the connective and myogenic but not in the chondrogenic tissues proximal and distal to the graft side. In one specimen fibronectin-coated beads were translocated to regions in the autopod, but in general, bead displacement was relatively short. In sum, latex beads can not move to any great extent within the limb bud, and the coating of these beads with fibronectin did not influence bead translocation.     

The control of directed myogenic cell migration in the avian limb bud

Summary Interspecific grafting experiments between chick and quail embryos were carried out in order to investigate the mechanism controlling myogenic cell migration in the avian limb bud. In six series, various experimental set-ups were prepared involving different age combinations of donor and host. The migration of the myogenic cells contained nor and host. The migration of the myogenic cells contained in the quail donor could be traced due to the prominent perinucleolar heterochromatin of the quail nucleus. Irrespectively of the presence or absence of the apical ectodermal ridge (AER), myogenic cells were found to migrate distally when implanted at a more distal site or into a younger host. They were even found to migrate in the reverse direction when younger host tissue was located proximal to the graft.From these findings, we conclude that the state of differentiation (juvenility) of the limb bud mesenchyme controls the directed migration of myogenic cells.     

Ultrastructural localization of cholinesterase uring chondrogenesis and myogenesis in the chick limb bud

Summary Cholinesterase (ChE) is transiently expressed in undifferentiated embryonic cells. In the chick limb bud ChE-activity was found in the apical ectodermal ridge and in the subridge mesenchyme. The reaction was localized in the perinuclear cisterna, in an extensive network of narrow profiles of endoplasmic reticulum (ER), and in the Golgi complexThe chondroblasts emerging from the subridge mesenenyme, also showed strong ChE-activity. During differentiation the enzyme first disappeared from the Golgi zone. Then, the narrow ChE-positive ER was successively replaced by ChE-negative extended rough ER characteristic for the differentiated chondrocyte.The myoblasts showed weak ChE-activity with the same ultrastructural localization as in other mesenchymal cells. After fusion the myotubes exhibited strong ChE-activity in the perinuclear cisterna and the developing sarcoplasmic reticulum. In later stages of myogenesis the myoblasts were closely attached to the myotubes and had lost their ChE-activity.During mitosis of ChE-positive cells, ChE-activity was retained in fragments of perinuclear cisterna and ER. In ChE-active mesenchymal cells and chondroblasts we observed specialized contact zones between ER and plasma membrane. ChE-active cisternae of ER run parallel to the plasma membrane with a gap of approximately 10–15 nm. We discuss a possible function of a cholinergic system during morphogenesis.     

The regulative potential of the limb region in 11.5-day rat embryos following the amputation of the fore-limb bud

Summary The regulative potential of the fore-limb region following the removal of the limb bud was investigated in 11.5-day rat embryos. Fore-limb buds were amputated from a total of 54 embryos. Five embryos were immediately examined via scanning electron microscopy (SEM) to assess the quality of the operation and the reproducibility of the technique. In all cases, the forelimb bud and adjacent tissues extending down to the celomic cavity at the same level were completely removed. The remaining 49 operated embryos were cultured in vitro and examined at different time intervals. Gross inspection of embryos which had been cultured for 24 h, revealed that 24 out of 37 had developed a pair of limb bud-like protrusions at the operation site. Twelve formed only a single protrusion, while nothing was found in the remaining embryo. These protrusions were examined in greater detail under SEM and light microscopy. SEM observations showed that these protrusions were covered with an intact layer of ectoderm. In embryos with a pair of protrusions, these outgrowths developed opposite somites 7 to 13. The failure of either one of these two outgrowths to form, produced our second type of experimental embryo, those which had just a single protrusion. Histological examination revealed that an apical ectodermal ridge (AER) was discernible on the protrusions of 36% of the embryos. Finally, we have established how these protrusions were constructed from SEM observations of operated embryos cultured for 6 h and 10 h.     

Relationships between ectoderm and skeletal morphogenesis in the chick embryo limb bud

Summary When in chick embryos (H.-H. stages 22 to 25) a variously large area of ectoderm with the subjacent mesodermal layer external to the superficial vessel network, loosened from the dorsal face of the wing bud is rotated 180° in situ, or a similar ecto- and mesodermal sheet isolated from the dorsal face of the leg bud is grafted, in normal of 180° reversed orientation, onto the dorsal face of the wing bud, no changes in the normal developmental pattern of the wing skeleton ensue. As the grafted tissue, which apparently does not contain prospective chondrogenic cells, develops as a flat implant, the normal geometry of the ectodermal hull is not altered: therefore, the biomechanical conditions and the polarized growth of the skeletogenous mesenchyme of the wing bud, which seem to be controlled by the enveloping epithelium, remain practically unchanged.Morphological alterations of the skeletal pieces of the wing and formation of ectopic cartilage follow instead the implantation on the dorsal face of the wing bud, in normal or 180° reversed orientationm of an ecto- and mesodermal sheet similar to the one mentioned above but containing also a varying amount of the mesenchyme lying beneath the superficial vessel network.     

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

Summary 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 are which is made up of a 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.     

Myogenic potential of chick limb bud mesenchyme in micromass culture

Summary The myogenic potential of chick limb mesenchyme from stages 18–25 was assessed by micromass culture under conditions conductive to myogenesis, and was measured as the proportion of differentiated (muscle myosin-positive) mononucleated cells detected. It was found that similar myogenic potentials existed in mesenchyme from whole limbs between stages 18 and 19, but this potential was halved by stage 20. At stage 21, proximal mesenchyme showed significantly more myogenesis than distal mesenchyme, but this difference was abolished by stage 22. Thereafter, myogenesis was increasingly restricted from the distal mesenchyme, whilst the potential in more proximal regions did not significantly increase after stage 23. When the ratio between total limb myoblasts which differentiated on days 1 and 4 of culture was analysed, it was found that two distinct peaks existed at stages 20 and 23. The significance of these ratio peaks is unclear, but may be related to different proliferative potentials of the pre-myoblasts at these stages.     

The orientation of the wing mesenchyme influences the direction of the migration of myoblasts in the avian embryonic wing bud

Summary In order to analyze the influence of the orientation of the wing bud mesenchyme on the proximodistal direction of the migration of myoblasts in the avian embryonic wing bud, blocks of wing-bud mesenchyme were cut out and rotated around a dorso-palmar axis through 90° or 180°. Tissues originating from the quail wing bud and containing myoblasts were grafted into the space between the wing mesenchyme and the rotated blocks of mesenchyme proximal to the latter. In all experiments the donor-embryos were older than the acceptor-embryos. From HH stage 24 on, the rotation of the block of mesenchyme inhibited the migration of the myoblasts in a distal direction. We therefore propose that the orientation of the wing bud mesenchyme has an influence on the migratory behavior of myoblasts. This influence could provide “directional information” for the migrating myoblasts, allowing the migration of myoblasts in a distal direction only.     

Ca2+ channel activities in the limb bud of early embryonic chick

Ca²⁺ channel activities were recorded in the limb bud of embryonic day 4 chick with Ca²⁺ sensitive fluorescence (Fura-2) measurements and patch clamp techniques. Rises in intracellular Ca²⁺ concentrations were evoked by depolarization with the application of 100 mM K⁺ and this Ca²⁺ response was abolished by removing extracellular Ca²⁺. The Ca²⁺ response was blocked by 10 M nifedipine and enhanced by 5 M Bay K 8644. Long-lasting inward currents were revealed by whole-cell patch clamp recordings from dissociated cells of the limb bud. The inward current was also blocked by 10 M nifedipine. Our study suggested the presence of L-type Ca²⁺ channels in the limb bud cells.     

A study on the regenerative potential of partially excised mouse embryonic fore-limb bud

Summary The ability of day E10 mouse fore-limb bud to regulate following the removal of a portion of limb tissue was investigated. A longitudinal strip of tissue, two to three somites in width and extending from the base of the limb bud to its distal tip, was excised. The embryos were then maintained in a roller culture system for periods of 6 h, 12 h or 24 h post-operatively prior to fixation and subsequent examination. The embryos were examined with scanning electron microscopy (SEM) and light microscopy. SEM revealed that about two thirds of the operated limbs grossly restored their overall morphology. The sequence of morphological changes involved in the restoration process is described. The ability of the restored limb bud to develop an apical ectodermal ridge (AER) is shown in histological sections.     

An autoradiography study of myogenic cell movement in avian limb buds following heterospecific and homospecific transplantation

Summary Species specificity and the use of quail cells as a marker in the study of myogenic cell movement in the developing avian limb was investigated. In order to establish whether or not observed myogenic cell movement in quail/chick limb transplantation experiments might be an artefact produced by cellular interaction between these cell types a series of homospecific and heterospecific transplantations was performed. Chick wing fragments (staged 20–25 H.H.) were labelled with tritiated thymidine and inserted into unlabelled chick wing bud (homospecific) in ovo. In addition, quail wing fragments were also labelled with tritiated thymidine and transplanted in the same manner into chick (heterospecific), so that the effectiveness of tritium as a marker could be assessed. After 4 days post-incubation, myogenic cell movement was detected in eight out of the ten homospecific transplantions performed. Myogenic cell movement in avian limbs is therefore not produced by interaction between chick and quail cells, as migration was also detected in the chick/chick transplants. Nonetheless, heterospecific transplantation results revealed that autoradiographic methods failed to reveal completely the true extent to which myogenic cell movement occurred, because tritiated thymidine was subject to dilution.     

The migration of myogenic cells from the somites into the leg region of avian embryos

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.     

Somitomeres in the chick tail bud: an SEM study

Summary In the chick embryo the final number of somites is achieved at about stage 22 of Hamburger and Hamilton. By this time the neural tube and notochord have reached the tip of the tail bud but some paraxial mesoderm remains unsegmented. In this study using scanning electron microscopy we show that somitomeres are present in this mesoderm. This indicates that the terminal paraxial mesoderm of the tail bud may have the potential to form supplementary somites, though we do not as yet know what prohibits the completion of segmentation to the tip.     

Posterior half amputation of the chick wing bud: the response of the developing vasculature, and subsequent wound healing

Summary Experimental analyses examining pattern formation in the developing chick limb have concentrated on the skeleton, muscles and nerves, and have rarely considered blood vessels. To investigate the relationship between the vasculature and limb development, posterior amputations were performed on 3.5–4 day chick limb-buds. It has been shown that the removal of the posterior half alters the developmental fate of the anterior tissue: it becomes necrotic and fails to differentiate into the complement of skeletal parts predicted by fate maps. The possibility that this developmental failure results from interference with the future arterial supply was examined by Indian ink injection between 3–48 h after operation. Scanning electron microscopy (SEM) and resin histology were used to examine the wound repair at similar post-operative intervals. Results from the Indian ink injections showed that within 6 h of operation a collateral circulation was established by means of a branch from the truncated primary subclavian artery. The capillary density in the operated limbs appeared normal when compared to the contralateral limb. The results support the view that the poor developmental performance of the anterior half is due to removal of the zone of polarising activity (ZPA) rather than to experimentally-induced alteration to the vascular supply. Histological and SEM examination of the wound healing process showed that epithelialization of the cut surface occurred within 24 h, and that the peridermal cells of the bilayered ectoderm appeared to initiate the regrowth. The wound site was not visible 48 h after operation, showing that wound healing at these developmental ages occurs quickly, with no scar tissue formation. These results show that the vasculature in the developing limb is labile, and that the cell death resulting from posterior-half amputation is not due to vascular insufficiency or ischaemia. In addition, this study of wound healing demonstrates the role of the ectoderm in establishing an avascular margin in the subjacent mesenchyme.     

Non-invasive determination of instantaneous heart rate in developing avian embryos by means of acoustocardiogram

Previous noninvasive studies of the mean heart rate of embryonic birds have prompted an investigation into the instantaneous heart rate (IHR), which may be informative in developmental studies of cardiac rhythm. Using the acoustocardiogram (ACG), a noninvasive, long-term measuring system for embryonic IHR is developed, and the IHR in chickens during the last half of embryonic development is determined. The system, which uses a micro-computer, samples the ACG at a frequency of 50 Hz, restores the ACG wave by sinc function and calculates the IHR with an error in accuracy of less than 1 beat min⁻¹. It was found that characteristic, transient bradycardia begins to appear late in the second week of incubation, and, with the additional development of transient tachycardia, the embryonic cardiac rhythm becomes more arrhythmic towards hatching. Simultaneous measurements of IHR with somatic movements showed no relationship between arrhythmia and embryonic activities. This system is useful, providing new evidence on long-term IHR developmental patterns.     

Interdigital chondrogenesis and extra digit formation in the duck leg bud subjected to local ectoderm removal

Summary In the chick embryo the interdigital tissue in the stages previous to cell death exhibits in vitro a high chondrogenic potential, and forms extra digits when subjected in vivo to local ectodermal removal. In the present work we have analyzed the chondrogenic potential both in vivo and in vitro of the interdigital mesenchyme of the duck leg bud. As distinct from the chick, the interdigital mesenchyme of the duck leg bud exhibits a low degree of degeneration, resulting in the formation of webbed digits. Our results show that duck interdigital mesenchyme exhibits also a high chondrogenic potential in vitro until the stages in which cell death starts. Once cell death is finished chondrogenesis becomes negative and the interdigital mesenchyme forms a fibroblastic tissue. In vivo the interdigital mesenchyme of the duck leg bud subjected to ectoderm removal forms ectopic foci of chondrogenesis with a range of incidence similar to that in the chick. Unlike those of the chick the ectopic cartilages of the duck are rounded and smaller, and appear to be located at the distal margin of the interdigital mesenchyme. Formation of extra digits in the duck occurs with a lower incidence than in the chick. It is concluded that ectopic chondrogenesis and formation of extra digits is related to the intensity of interdigital cell death. The non-degenerating interdigital mesenchymal cells destined to form the interdigital webs of the duck appear to contribute very little to the formation of interdigital cartilages.     

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

Summary 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.At stages 17 and 18, myoblasts are present only in the middle part of the myotome. The ventral part of the myotome consists of cells not yet specifically differentiated and passes directly into the dermatome. The borderline between the ventral edge of the dermomyotome and the adjacent mesenchyme is indistinct, and a few cells are released from the ventral edge of the dermomyotome. These cells are integrated into the adjacent mesenchyme. We can offer no observations indicating that they participate in wing myogenesis.From stage 19 onwards the myotomic ventral edge consists of myoblasts and is distinctly demarcated. No myoblasts are released from it, nor do they migrate into the wing bud. The nerve fibers growing into the wing bud have no contact with the somite cells. In the wing bud myoblasts begin to differentiate during stages 24 and 25. They are irregularly scattered among the muscle blastema cells. From stage 26 onwards the myotubes present are oriented in parallel with the longitudinal axis of the limb.