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.     

Inhibition of prostaglandin synthesis reduces cyclic AMP levels and inhibits chondrogenesis in cultured chick limb mesenchyme

The present study investigated effects of inhibiting the synthesis of prostaglandins (PGs) on cyclic AMP concentrations and chondrogenesis in cultured chick limb mesenchyme. Indomethacin produced concentration-dependent inhibition of both PGE₂ synthesis and chondrogenesis over a concentration range of 50--200 M. Half maximal inhibition of PGE₂ was achieved with 50 M concentrations of the drug which also produced visibly reduced amounts of cartilage matrix in cell cultures as evaluated by Alcian green staining on day 6 of culture. The inhibitory effects of indomethacin on chondrogenesis were largely reversed by addition of 1 mM dibutyryl cAMP, indicating that cells could still respond to cyclic AMP stimulation. Endogenous levels of cyclic AMP, which increased by 6 fold during the six days of culture in control cells, did not increase significantly from dissociated cells at the time of plating (day 0) in indomethacin-treated cultures. The results indicate that inhibition of the prechondrogenic rise in PGE₂ concentrations in limb mesenchyme prevents the increase in cyclic AMP levels which occur during this same period resulting in inhibition of chondrogenesis. The data provide further support for the hypothesis that PGE₂, through its effects on the adenylate cyclase-cAMP system, plays an important role in the differentiation of cartilage.     

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.     

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.     

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.     

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.     

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.     

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.     

Cytokinetic studies on the aortic endothelium and limb bud vascularization in avian embryos

Summary Cytokinetic studies on the aortic endothelium using the BrdU/anti-BrdU-method were carried out on 2.5– to 6-day chick and quail embryos. The mitotic activity of the aortic endothelium is related temporally to the age of the avian embryo and spatially to the embryonic region where the aorta originates. The mitotic activity of the aortic endothelium decreases with increasing age of the embryos. In the limb buds, however, the mitotic rate of the aortic endothelial cells increases independently of the age of the embryo. This increase in the mitotic activity of the aortic endothelium at the appropriate levels coincides with the vascularization of the outgrowing limb buds. We concluded therefore that the aortic endothelium probably supplies endothelial cells for the formation of limb vessels at this stage. Thus our results suggest that angiogenesis (sprouting of capillaries from pre-existing vessels) takes place during limb vascularization in avian embryos. On the other hand, immunohistochemical studies with QH-1 or MB-1 antibody show, beside a capillary network in the central core of the wing bud, individual immunolabelled cells of mesenchymal character within the primarily avascular subectodermal region from the onset of vascularization onwards. We suggest that these cells have partly to be regarded as endothelial precursor cells, which have differentiated in situ from the local limb mesenchyme, and which will contribute to the developing vascular plexus. This means that not only angiogenesis, but also vasculogenesis (in situ from mesenchymal precursors differentiated endothelial cells) appears to be involved in limb vessel formation.Supported by the Deutsche Forschungsgemeinschaft (Ch 44/9-1, Ch 44/9-2)This paper is dedicated to Prof. Dr. K.V. Hinrichsen on the occasion of his 65th birthday     

Formation of extra-digits induced by surgical removal of the apical ectodermal ridge of the chick embryo leg bud in the stages previous to the onset of interdigital cell death

Summary In the present work we have studied the mechanism of formation and the possible morphogenetic significance of the process of ectopic chondrogenesis induced by surgical removal of AER of the interdigital spaces of the chick leg bud at stage 28–30 (Hurle and Gañan 1986). Our results show that ridge removal causes condensation and rounding of the underlaying mesenchymal cells followed by chondrogensis. The long-term study of the fate of these ectopic cartilages shows that in a high percentage of the cases the cartilages undergo morphogenesis taking by day 10 of incubation the appearance of the two distal phalanges of an extra-digit. These extra-digits lack tendons and are joined by thin interdigital membranes to the neighboring digits.     

Changes in adenylate cyclase activity during chondrogenesis in cultured limb mesenchyme

An in vitro assay to examine the effects of forskolin on adenylate cyclase (AC) activity and chondrogenesis in micromass cultures of mesenchymal cells derived from the distal tip of stage 25 chick limb buds is described. Forskolin activation of AC increased 6 fold in prechondrogenic cells cultured for 48 hours relative to uncultured cells at the time of cell dissociation (Day 0) and remained elevated and unchanged in differentiated cells during the remaining four days of culture. Equivalent increases occurred in cyclic AMP (cAMP) concentrations over the six day period examined. Forskolin also accelerated chondrogenesis in these cultures. Quantitative analysis of Alcian green binding to cartilage matrix showed approximately 2 fold increases in forskolin-treated cultures relative to controls on each of days 2–4 of culture. These results demonstrate that dramatic increases in the total pool of AC activity occur in prechondrogenic mesenchyme prior to overt chondrogenesis which, through increases in cAMP, may play an important role in cartilage differentiation.     

Ultrastructure of developing muscle in the upper limbs of the human embryo and fetus

Background: The ultrastructure of the myogenesis, which proceeds along with the appearance of muscle-specific proteins and isozymes, has not been fully described in the upper limb of staged human embryos. Methods: Eight human embryos (Carnegie stage 14–22) and two fetuses (11 and 12 weeks of gestation) were fixed with 5% glutaraldehyde, 4% paraformaldehyde, and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.2. The upper limbs were dissected out and processed for transmission electron microscopy, and sections of the biceps brachii muscle were cut and examined. Results: At stage 14, the myoblasts were loosely scattered in the ventral proximal region of the upper limb bud and had a small amount of cytoplasm with a few intracellular organelles. At stage 16, the myoblasts were spindle shaped and oriented parallel to the axis of the upper limb bud. These cells had irregularly shaped nuclei with prominent nucleoli, rough endoplasmic reticulum (ER), and mitochondria, but no myofilaments were observed. At stages 17–19, rough ER, free ribosomes, and mitochondria increased in number and thick and thin filaments with faint Z-lines appeared in the peripheral cytoplasm of the myotube. The plasma membranes of some neighboring myotubes were continuous, suggesting that these cells were in the initial stages of the fusion process. At stage 22, the striated pattern of the myofilaments became evident and tubular structures appeared around them and near the plasma membrane. In the fetus at the 11th week, the basal lamina began to surround the myotubes, and T-tubules with sarcoplasmic reticulum were observed. Dyads and triads were observed in the myotube of the 12th week fetus. Conclusion: These findings suggest that rapid myogenesis occurs during the late embryonic period in human upper limbs and that the ultrastructural characteristics of mature myotubes are established during the early fetal period. © 1995 Wiley-Liss, Inc.     

The formation of premuscle masses during chick wing bud development

Summary The skeletal musculature of chick limb buds is derived from somitic cells that migrate into the somatopleure of the future limb regions. These cells become organized into the earliest muscle primordia, the dorsal and ventral premuscle masses, prior to myogenic differentiation. Therefore, skeletal-muscle specific markers cannot be used to observe myogenic cells during the process of premuscle mass formation. In this study, an alternative marking method was used to determine the specific stages during which this process occurs. Quail somite strips were fluorescently labeled and implanted into chick hosts. Paraffin sections of the resulting chimeric wing buds were stained with the monoclonal antibody QH1 in order to identify graft-derived endothelium. Non-endothelial graft-derived cells present in the wing mesenchyme were assumed to be myogenic. At Hamburger and Hamilton stage 20, myogenic cells were distributed throughout the central region of the limb, including the future dorsal and ventral premuscle mass regions and the prechondrogenic core region. By stage 21, the myogenic cells were present at greater density in dorsal and ventral regions than in the core. By stage 23, nearly all myogenic cells were located in the dorsal and ventral premuscle masses. Therefore, the two premuscle masses become established by stage 21 and premuscle mass formation is not complete until stage 23 or later. Premuscle mass formation occurs concurrently with early chondrogenic events, as observed with the marker peanut agglutinin. To facilitate the investigation of possible underlying mechanisms of premuscle mass formation, the micromass culture system was evaluated, to determine whether or not it can serve as an accurate in vitro model system. The initially randomly distributed myogenic cells were observed to segregate from prechondrogenic regions prior to myogenic differentiation. This is similar to myogenic patterning in vivo.     

An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos

The present study describes a simple, rapid protocol for culture for limb tissue from individual 10.5-day post coitum mouse embryos that supports cartilage differentiation over a six-day period. This technique differs from other commonly used methods utilizing pooled limb tissue in that: 1) forelimbs from individual embryos were used as donor tissue; 2) limb tissue was dissociated by very gentle enzymatic digest (0.1% trypsin, 5 min); and, 3) cell suspensions were plated at a lower density (1 × 10⁷ vs. 2 × 10⁷ cells/ml) in a reduced volume of 3–5 l. Under these modified conditions to increase limb cell yield from each embryo, histochemical and immunohistochemical analyses demonstrated reproducible for-mation of precartilage aggregates and subsequent overt chondrogenesis over a predictable time course. Using this culture protocol, analysis of limb mesenchyme from heterozygous hdf embryos, which bear an insertional mutation of the Cspg2 gene encoding the core protein of the chondroitin sulfate proteoglycan, versican, revealed an overall similar chondrogenic potential to that observed for wild-type littermates. This technique readily enables in vitro culture of limb bud mesenchyme from individual mouse embryos at this developmental stage and may be utilized by investigators to study the effects of the hdf and other transgenic mutations on mammalian limb development in vitro.     

In vitro production of monoclonal antibodies to cultured embryonic chick limb mesenchyme

A simple, rapid protocol for the in vitro production of monoclonal antibodies (MAbs) that recognize native antigens in cultured chick limb mesenchyme during chondrogenic differentiation is described. Murine lymphocytes were stimulated by direct exposure to methanol-fixed micromass cultures of limb mesenchyme derived from the distal tip of stage 25 chick limb buds. Initial immunohistochemical characterization of two antibodies (DIDI and DIIA5) produced by this method showed preferential localization of reactivity with antigens in developing cartilage nodules during chondrogenesis in cultured chick limb mesenchyme. This study demonstrates the utility of in vitroimmunization of lymphocytes for the production of MAbs to native antigens expressed by differentiating embryonic limb cells in culture. Immunohistochemical data provided by DIDI and DIIA5 suggest that antigens bearing these epitopes may be important in early morphogenetic events during limb skeletal development.     

The development of alkaline phosphatase activity in limb buds of mouse embryos in vitro and its relation to chondrogenesis

Summary The development of alkaline phosphatase (aPh) activity and chondrogenesis were studied in the limb buds of mouse embryos (day 11 p.c.) that had been grown in an organ culture. During a 12-day culture period an increase in aPh activity to more than 40 mU/limb bud was measured from day 2 in vitro onward. Depending on the time of application, aPh formation can be inhibited by certain substances. Cytosine-arabinoside inhibits aPh activity when the substance is added on day 2, 3, or 4. Chondrogenesis, on the other hand, is affected on days 1, 2, and 3 and to a lesser degree on day 4. Actinomycin D interferes with aPh activity after its addition on day 1, 2, 3, or 4. Chondrogenesis is only inhibited when the drug is applied on the 1st, 2nd, or to a lesser degree on the 3rd day. Cycloheximide inhibits aPh formation on all days of treatment, but to a lesser degree on days 5 and 6; chondrogenesis is most influenced on days 2, 3, and 4.On day 6 of the culture period, aPh activity can be demonstrated histochemically only in the region of humerus and proximal parts of radius and ulna. Alterations in the distal cartilage skeleton, therefore, do not influence the activity data. A prerequisite for an increase in aPh activity is cartilage growth in the proximal part of the limb buds and subsequent induction of a perichondral cell population to proliferation and differentiation.This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonal-Pharmakologie.     

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.     

N-CAM, polysialic acid and chick tail bud development

Summary We have previously shown that the binding of the lectin wheat germ agglutinin (WGA) to developing tail buds results in a range of caudal axial defects, which were most likely due to the affinity of the lectin for sialic acid residues. In the present study, we examined the distribution and role of a sialic acid-containing glycoprotein, N-CAM, in chick tail bud development. In the early tail bud, anti N-CAM, staining was found in the medullary cord. However, there was no uptake of an antibody specific to N-CAM containing moderate to long chains of polysialic acid (5A5 monoclonal antibody). At later stages, while N-CAM localized throughout the neural tube, staining with the 5A5 antibody was restricted to the floor plate. Sub-blastodermal injection of the anti N-CAM antibody beneath the tail bud region of HH stages 13–14 embryos produced caudal axial malformations. These malformations included the presence of accessory segments of neural tube and/or notochord, and fusion between the neural tube and underlying segment of notochord. Our results suggest that N-CAM is present during the development of the secondary neuraxis from the tail bud, although the highly sialylated form of this molecule could not be visualized until relatively late stages. N-CAM probably plays a role in the normal course of tail bud development, since perturbation of the molecule with an antibody resulted in malformations. Since these malformations were similar to those we have previously reported when we treated similarly staged chick embryos with WGA, there is a possibility that the sialic acid residues recognized and bound by the lectin are those associated with the N-CAM molecule.     

Mathematical modelling of the growth processes in the developing chick wing bud

This paper illustrates how a simple geometric model resembling the shape of the chick wing bud at an early growth stage can be mathematically expanded to simulate subsequent growth characteristics of the developing bud. The model was tested against several sets of experimental data and gave an acceptable representation of growth over the range considered. Representing growth patterns in this form enables the determination of differential growth characteristics in different parts of the bud and provides boundary constraints which will play an important part in the eventual evaluation of internal growth mechanisms.