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.     

Antero-posterior skeletal patterning is not dependent on continuity of the apical ectodermal ridge in the chick wing bud

The mechanism of antero-posterior specification of limb skeletal pattern is still controversial. If, as proposed by the ZPA model, a diffusible morphogen does exist, its route of passage across the limb field has not been resolved. To investigate the contribution of the apical ectodermal ridge (AER) to the control of anteroposterior pattern formation, we examined the consequences of small wounds made to the AER. The wound response was investigated by means of resin histology and scanning electron microscopy; subsequent limb development and cartilage pattern were examined in wholemount preparations. Although regrowth of the bilaminate dorsal and ventral ectoderm over the wound occurred within 15 h, the more highly differentiated pseudostratified columnar epithelium of the AER did not reform, and there was subsequent retardation of limb outgrowth at the wound site. At 10–11 days of development, the appearance of the limbs allowed them to be placed into one of three categories; presence of supernumerary elements, accentuation of an inter-digital cleft, or normal. The first of these categories included limbs in which digit 3 had bifurcated such that the sum of the parts of the resultant digital skeleton was greater than that which forms in a normal limb. Since in all of the experimental limbs all skeletal elements were present, we propose that continuity of the AER is not a pre-requisite for anteroposterior skeletal pattern formation in the chick wing.     

The distribution of mesenchyme proteoglycan (PG-M) during wing bud outgrowth

Summary This study utilizes immunofluorescence to describe the distribution of several extracellular matrix molecules in the chick embryo during the process of limb outgrowth and the formation of precartilage condensations. A large chondroitin sulfate proteoglycan (PG-M) is detected at the wing level at Hamburger and Hamilton stage 14 in and under the dorsal ectoderm, and is associated with the basement membranes around the neural tube, notochord and pronephros, but not with other basement membranes. The galactose-specific leetin, peanut agglutinin (PNA), has a similar distribution except that it also binds to the dorsal side of the neural tube. PG-M is not detected in the limb mesenchyme until after stage 17, when it is present in the distal region, as is PNA-binding material. With further development of the wing bud, PG-M is present in the subectodermal mesenchyme, the mesenchyme at the distal tip and in the prechondrogenic core. After stage 22 PNA-binding material becomes localized in the prechondrogenic core, the basement membranes under the apical ectodermal ridge, and the ventral sulcus. The distribution of these components (PG-M and PNA binding material) overlaps, but differs from that of type I collagen and fibronectin and basement membrane components, such as laminin, basement membrane heparan sulfate proteoglycan, and type IV collagen. Tenascin, on the other hand, is not detected in the limb bud until stage 25, after the appearance of cartilage matrix components such as type II collagen and cartilage proteoglycan (PG-H). These results are considered in relation to the formation of precartilage aggregates, and indicate that PNA binds to components in precartilage aggregates other than PG-M or tenascin.     

Basal lamina molecules are concentrated in myogenic regions of the mouse limb bud

 Molecular components of basal lamina, such as laminin, stimulate the differentiation of skeletal muscle cells in culture, while interstitial matrix components such as fibronectin are inhibitory. However, the role of extracellular matrix (ECM) molecules in muscle cell differentiation in the embryo is less well understood. As a first step toward understanding the role of the ECM in embryonic myogenesis, the localization of basal lamina molecules in the mouse limb bud before and during muscle cell differentiation was determined by immunofluorescence. Laminin, collagen type IV and nidogen (entactin) were concentrated in myogenic regions of the limb bud both before and during differentiation of skeletal muscle cells. Punctate immunofluorescence for basal lamina molecules was concentrated in dorsal and ventral premuscle and muscle masses, when compared with other regions of limb mesenchyme. In contrast, immunofluorescence for fibronectin, an interstitial extracellular matrix molecule, was decreased in premuscle and muscle masses. These results suggest that basal lamina components play an important stimulatory role in early stages of skeletal muscle differentiation in the developing mouse limb bud. Accepted: 7 May 1998     

Molecular heterogeneity of chondroitin sulphate in the early developing chick wing bud

Proteoglycans are ubiquitous extracellular matrix molecules whose role in development remains poorly understood. In the developing chick limb, the nature and possible roles of a number of extracellular matrix proteins is well documented. Much less is known of the biochemical nature, and more importantly, the roles of proteoglycans. Using a panel of monoclonal antibodies (Mabs) which recognise specifie epitopes on the constituent chondroitin/dermatan sulphate chains, we show that distinct sub-populations of proteoglycans are dynamically expressed within the limb ectoderm, the ectodermal basement membrane and the limb mesenchyme. In particular, prior to chondrogensis, chondroitin-6-sulphate-rich proteoglycans containing over-sulphated domains reside predominantly within the mesenchymal extracellular matrix ECM, whilst chondroitin-4-sulphate (C-4-S) is associated with the ectodermal basement membrane and subjacent mesenchymal ECM. At stage 24, C-4-S is also localized in the prechondrogenic condensation. Concomitantly with overt chondrogenesis, the epitopes recognized by the Mabs become restricted to the chondrifying skeletal elements and the undifferentiated distal mesenchyme. The significance of these findings has yet to be elucidated.     

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 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.     

Macromolecular permeability of chick wing microvessels: an intravital confocal study

The development of the vertebrate limb requires the formation of a normal vasculature to nurture the soft and hard tissue phenotypes. The pattern of embryonic limb bud vessels has been extensively studied, but little is known about the permeability characteristics of the developing circulation. In the present study, the microvascular endothelial cell phenotype was examined by in vivo confocal microscopy following the systemic injection of a graded series of fluorescent dextrans (40,000, 70,000, 150,000 molecular weight) into chick embryos at stages 21–23 in order to determine how selective is the endothelial lining of microvessels as a partition between the blood vessels and the interstitium. Videodensitometry, over a gray scale range of 0–255, was used to quantitate the amount of tracer found within the interstitial compartment of the limb. The tracers of larger molecular weight (70,000, 150,000) were confined exclusively to the vascular lumina, whereas that of smaller molecular weight (40,000) was found to cause perivascular brightening due to extravasation into the surrounding interstitium. The reported differences in permeability were not dependent upon the stage of the embryo used in this study, but were due to the size of the tracer. These data indicate that embryonic wing microvessels demonstrate permselectivity to macromolecular efflux across the endothelium. The present results provide a basis for additional studies concerned with the dynamic characteristics of the limb microvasculature and challenge our concepts about the role of diffusible morphogens in vertebrate limb development.     

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.     

Extracellular matrix modifications in the interdigital spaces of the chick embryo leg bud during the formation of ectopic digits

In previous studies we have observed that the interdigital mesenchyme of the chick leg bud, in the stages preceding the onset of cell death, retains a significant regulatory potential, forming ectopic extra digits under a variety of surgical manipulations. Most evidence suggests that interdigital extra digits are caused by the abolition of local antichondrogenic effects operating in the interdigital spaces under normal conditions rather than by modifications of the signalling mechanisms accounting for the normal patterning of the digits in early stages of development. The interdigital spaces exhibit a complex scaffold of extracellular matrix with well-defined domains of spatial distribution of type I and type VI collagens, tenascin, fibronectin, laminin and elastic matrix components that have been proposed to play a role in the establishment of the non-chondrogenic fate of the interdigital tissue in situ. In an attempt to analyze this possible role of the interdigital extracellular matrix (ECM), in the present work we have studied changes in the pattern of ECM distribution associated with the formation of extra digits. Extra digits were induced by making a T-cut in the third interdigital space of the leg but of stage 29 HH chick embryos. Subsequent modifications of the ECM were detected immunohistochemically in whole-mount specimens using laser confocal microscopy. Our results reveal that in the first hours after the operation, changes in the ECM apparently related to the healing of the wound cause a significant reorganization of the normal ECM scaffold of the interdigit. In addition, chondrogenesis of the interdigital tissue is preceded by disappearance of elastin fibers in the interdigital mesenchyme subjacent to the wound and by an intense deposition of tenascin. Tenascin deposition and loss of the elastin fibrillar scaffold were also observed preceding chondrogenesis in fragments of interdigital tissue explanted to culture conditions. The significance of these observations in relation to the establishment of the skeletal elements of the autopodium is discussed.     

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.     

The talpid2 chick limb has weak polarizing activity and can respond to retinoic acid and polarizing zone signal.

The talpid2 (ta2) chick mutant has wide, polydactylous wings and legs. Talpid2 limb cartilages have abnormal morphology and a very subtle anteroposterior polarity. Specifically, posterior ta2 limb structures are identifiable, while more anterior cartilages are less distinctive. Here, we investigate the development of anteroposterior limb pattern in the ta2 embryo. We show that ta2 posterior limb bud mesoderm is capable of respecifying the anteroposterior axis of a normal wing. However, the average duplication obtained after grafting a ta2 polarizing region was significantly less than the average duplication formed after a graft of normal wing bud polarizing zone. Thus, polarizing activity appears to be weak in ta2. Grafts of normal polarizing zone to the posterior edge of ta2 wing buds had no effect on the ta2 phenotype. This result suggests that a weakly functioning polarizing signal does not account for the altered anteroposterior polarity in ta2 limbs, and that normal polarizing zone activity is not sufficient for formation of normal limb bud cartilages. We demonstrated that transmission of a polarizing signal through the ta2 limb mesoderm was normal. In addition, ta2 anterior border mesoderm had no polarizing activity. We also assessed the ability of ta2 limb bud mesoderm to respond to a polarizing signal. Either normal polarizing zone tissue or a bead containing retinoic acid was placed at the anterior edge of ta2 wing buds at stages 18-23. Both polarizing zone and retinoic acid caused respecification of the ta2 wing anteroposterior axis. The result was that a ta2 ulna replaced the radius, and the most posterior digit was duplicated anteriorly. Limb cartilages with normal morphology never formed. When a bead containing retinoic acid was placed at the posterior edge of ta2 wing buds, there was no effect on anteroposterior pattern. However, beads with retinoic acid always caused a reduction in the number of ta2 wing digits which formed, whether the beads were placed at the anterior or posterior edge of the developing ta2 limb.     

Diffusion or autocatalysis of retinoic acid cannot explain pattern formation in the chick wing bud.

We have collected several experimental data of pattern duplications due to the ZPA transplantation or application of retinoic acid on the developing chick limb bud. We have compared these data with the predictions of models based on diffusion or autocatalysis of retinoids. It turns out that these models cannot comprehensively explain the data. More specifically, retinoic acid cannot be either diffusing from a ZPA source or participate in an autocatalytic gradient formation.     

Pathway formation and the terminal distribution pattern of the spinocerebellar projection in the chick embryo

Summary Pathway formation and the terminal distribution pattern of spinocerebellar fibers in the chick embryo were examined by means of an anterograde labelling technique with wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP).Spinocerebellar fibers, which originate in the lumbar spinal cord and are located in the marginal layer of the spinal cord, reach the corsal part of the cerebellar plate on embryonic day (E)8. On the way to the cerebellum the fibers form one distinct bundle, that suggests that gross projection errors probably do not occur during the formation of the spinocerebellar pathway.On E10, labelled fibers are located mostly in the medullary zone of the anterior lobe. By E12, the number of labelled fibers increases greatly in the inner granular and molecular layers. In transverse sections labelling was distributed throughout the mediolateral extent of the medullary zone. By E14, sagittal strips of labelling were clearly recognized in lobules II–IV; however, labelled terminals were present throughout lobule I. Although the adult pattern of terminal distribution is attained by E14, the mossy fiber terminals are still quite immature. The density of labelling decreased greatly by E16, and small terminal varicosities were first recognized. Structural differentiation of mossy fiber terminals continues to the end of the embryonic or the newly posthatched period.     

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.     

Differential regional expression of multiple ADAMs during feather bud formation

The expression of seven members of the ADAM family was investigated by in situ hybridization in the developing feather buds of chicken. The expression profiles of the ADAMs in the cells and tissues of the feather buds differ from each other. ADAM9, ADAM10, and ADAM17 are expressed in the epidermis of the feather bud, whereas ADAM23 expression is restricted to the bud crest, with a distribution similar to that of sonic hedgehog. ADAM13 is not only expressed in the epidermis, but also in restricted regions of the dermis. Both ADAM12 and ADAM22 are expressed in the dermis of the feather bud, with an opposite mediolateral and anteroposterior polarity. Furthermore, the mRNAs of all investigated ADAMs show regional differences in their expression, for example, in the neck and in the roots of the leg and wing. These results suggest that ADAMs play a variety of roles during avian feather bud formation. Developmental Dynamics 240:2142–2152, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Expression of bone morphogenetic protein‐5 gene during chick heart development: Possible roles in valvuloseptal endocardial cushion formation

The bone morphogenetic protein (BMP) family, comprising multifunctional peptide growth factors, regulates many developmental processes in a variety of tissues. We examined the spatiotemporal expression of BMP5 by in situ hybridization in chick embryonic hearts from stages 5 to 33. The BMP5 gene was first expressed in the endoderm underlying the precardiac mesoderm at stages 5 to 8. Thereafter, BMP5 expression was restricted to the myocardium of the atrioventricular (AV) canal and outflow tract (OT) regions, where the valvuloseptal endocardial cushion tissue is induced. These results suggest that BMP5 may play important roles not only in myocardial differentiation, but also in the formation and maintenance of endocardial cushion tissue. Anat Rec 264:313–316, 2001. © 2001 Wiley‐Liss, Inc.