Determination of limb bud chondrocytes during a transient block of the cell cycle.

The process of determination is studied by using a cell culture system derived from dissociated chick embryo limb buds. When limb bud cells obtained from embryos younger than stage 25 (undertermined) are temporarily prevented from passing through the cell cycle (either by maintaining the cells on a petri dish or in the presence of high concentrations of cyclic AMP, both of which depress thymidine-H(3) incorporation), some cells subsequently form cartilage colonies. These results support the hypothesis that a temporary block at some stage in the cell cycle causes mesoblasts to acquire the capacity to differentiate into cartilage cells.     

Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro

Cartilage formation in the chick limb follows rapid proliferation, condensation and differentiation of limb mesenchyme. The control of these early events is poorly understood. Platelet-derived growth factor receptor alpha (PDGFR-alpha) is present throughout the mesenchyme of early chick limb buds, while its ligand, PDGF-A, is expressed in the surrounding epithelium. PDGFR-alpha is down-regulated in areas that will not give rise to cartilage and is then lost from cartilage forming areas after they begin to differentiate. PDGF-A increases chondrogenesis in micromass cultures of stage-20-24 limb buds, but not stage 25, where it inhibits chondrogenesis. Ectopic PDGF-A in the chick wing can lead to either a localized increase in cartilage formation, or an inhibition. Inhibition of PDGF signalling in the chick limb results in the loss of cartilage. These data demonstrate that PDGF-A functions to promote chondrogenesis at early stages of limb development and suggest that it inhibits chondrogenesis at later stages.     

In vitro histogenic capacities of limb mesenchyme from various stage mouse embryos

Mesenchyme cells isolated from mouse embryo forelimb buds (stages 15 through 21) and placed in high-density micromass cultures are compared with respect to their in vitro histogenic capacities. Particular emphasis is placed on changes in in vitro chondrogenic capacity. Stage 15 mouse limb cultures form numerous aggregates which uniformly fail to differentiate into cartilage nodules. On the other hand, cartilage nodules are observed in cultures prepared from all subsequent stage limbs, although there is a linear decrease in the size of nodules between stage 16–17 and middle-late stage 21 cultures. This decrease correlates with simultaneous decreases in both the proportion of aggregating cells and the extent of dibutyryl cyclic AMP-stimulated cartilage formation. At the same time, observations indicate that the proportions of nonaggregating and nonchondrogenic mesenchyme, myogenic cells, and, perhaps, fibrogenic mesenchyme are increasing. The only exceptions to these patterns are observed in cultures from middle-late stage 21 limbs, when cartilage differentiation in situ is already extensive. Unlike earlier stage cultures, which form nearly identical numbers of aggregates and nodules, middle-late stage 21 cultures form variable numbers of aggregates, only a few of which differentiate into cartilage nodules. Middle-late stage 21 cultures also contain unexpectedly low numbers of myogenic cells/unit area of culture. Based on changes in the in vitro histogenic capacities, it is concluded that concurrent with a progression of morphogenic events in the limb, there is a progression of changes in the relative proportions of cell subpopulations. Both the existence of the different subpopulations and the changes in their relative proportions can be detected in vitro. Furthermore, it is concluded that cartilage formation in the limbs of both mouse and chick embryos probably occurs according to very similar developmental programs.     

Separation of the myogenic and chondrogenic progenitor cells of undifferentiated limb mesenchyme

Undifferentiated limb bud mesenchyme consists of at least two separate, possibly predetermined, populations of progenitor cells, one derived from somitic mesoderm that gives rise exclusively to skeletal muscle and one derived from somatopleural mesoderm that gives rise to the cartilage and connective tissue of the limb. In the present study, we demonstrate that the inherent migratory capacity of myogenic precursor cells can be used to physically separate the myogenic and chondrogenic progenitor cells of the undifferentiated limb mesenchyme at the earliest stages of limb development. When the undifferentiated mesenchyme of stage 18/19 chick embryo wing buds or from the distal subridge region of stage 22 wing buds is placed intact upon the surface of fibronectin (FN)-coated petri dishes, a large population of cells emigrates out of the explants onto the FN substrates and differentiates into an extensive interlacing network of bipolar spindle-shaped myoblasts and multinucleated myotubes that stain with monoclonal antibody against muscle-specific fast myosin light chain. In contrast, the cells of the explants that remain in place and do not migrate away undergo extensive cartilage differentiation. Significantly, there is no emigration of myogenic cells out of explants of stage 25 distal subridge mesenchyme, which lacks myogenic progenitor cells. Myogenic precursor cells stream out of mesenchyme explants in one or occasionally two discrete locations, suggesting they are spatially segregated in discrete regions of tissue at the time of its explantation. There are subtle overall differences in the morphologies of the myogenic cells that form in stage 18/19 and stage 22 distal subridge mesenchyme explants. Finally, groups of nonmyogenic nonfibroblastic cells which are fusiform-shaped and oriented in distinct parallel arrays characteristically are found along the periphery of stage 18/19 wing mesenchyme explants. Our observations provide support for the concept that undifferentiated limb mesenchyme consists of independent subpopulations of committed precursor cells and provides a system for studying the early determinative and regulatory events involved in myogenesis or chondrogenesis.     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

Toxic effects of cyclophosphamide in differentiating chicken limb bud cell culture using rat liver 9,000 g supernatant or rat liver cells as an activation system: an in vitro short-term test for proteratogens

Cells from 4-day chicken embryo limb buds plated as micromass cultures differentiate and form cartilage nodules after a 5- to 6-day growth period. The innate ability of these cells to biotransform compounds, such as cyclophosphamide (CP), into reactive metabolites is apparently inadequate. Protocols used rat liver S9 from Aroclor 1254-pretreated (PCB) rats or hepatocytes from control rats or polychlorinated biphenyls (PCB)-pretreated rats and were added to micromass cultures with CP causing concentration-related toxicity in the cell cultures. Coculturing chicken limb bud cells with PCB-hepatocytes was the most efficient method, yielding an IC50 of 2 micrograms CP per ml. Toxic CP metabolites accumulated in the medium of PCB-hepatocyte cultures and were quite stable. The toxicity of bioactivated CP was nearly identical for both proliferation and cartilage proteoglycan accumulation.     

Changes in the pericellular matrix during differentiation of limb bud mesoderm

Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25-26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20-21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro.     

Regeneration potency of mouse limbs

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.     

Ethanol Exposure Stimulates Cartilage Differentiation by Embryonic Limb Mesenchyme Cells

Studies of neural, hepatic, and other cells have demonstrated thatin vitroethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformitiesin vivo,prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23–25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant ofin vitrochondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in³⁵SO₄incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol onin vitrochondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.     

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

Chondrogenesis from single limb mesenchyme cells

It is believed that cell-cell interaction between mesenchyme cells is involved in the initiation of chondrogenesis, based largely on the inability of limb mesenchyme cells to differentiate into cartilage unless cultures are inoculated at densities greater than confluency. The present study describes a culture situation in which single limb mesenchyme cells either in or on type I collagen gels are shown to differentiate into cartilage, as defined by the appearance of a pericellular alcian blue staining matrix, intracellular type II collagen (demonstrated by indirect immunofluorescence with monoclonal antibody), and clonable cartilage cells. Because the differentiation of cartilage cells from single mesenchyme cells occurs only when the cells are in a round configuration, it is proposed that cell shape changes are one factor that can mediate effects of cell-cell interaction on differentiation.     

Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.     

The patterns on chondrogenesis of cells from facial primordia of chick embryos in micromass culture

Chondrogenesis of mesenchymal cells from the frontonasal mass, mandibles and maxillae of stage-24 chick embryos has been investigated in micromass (high-density) cultures. Distinct differences in the amount and pattern of cartilage differentiation are found. In cultures of frontonasal mass cells, a central sheet of cartilage develops; in cultures of mandible cells, less cartilage differentiates and nodules form; while in cultures of maxillae cells, virtually no chondrogenesis takes place. The same patterns of cartilage are found in cultures established from stage-20 embryos. At stage 28, frontonasal mass cultures form cartilage nodules and the number of nodules in mandible cultures is markedly decreased. There are striking parallels between the chondrogenic patterns of cells from the face and limb buds in micromass culture. The frontonasal mass cell cultures of stage-20 and -24 chick embryos resemble those established from the progress zone of limb buds. The progress zone is an undifferentiated region of the limb in which positional cues operate. Cultures established from the frontonasal mass of stage-28 chick embryos and from the mandibles of all stages resemble cultures of whole limb buds. These contain a mixture of committed and uncommitted cells. Ectoderm from facial primordia locally inhibits chondrogenesis in micromass cultures and this could provide a positional cue. The differences in chondrogenic potential of cells from facial primordia may underlie the specific retinoid effects on the frontonasal mass.     

Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.     

Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation

Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.     

Pre-axonogenesis migration of afferent pioneer cells in the grasshopper embryo

In insects, afferent neurons arise primarily from the ectodermal epithelium in the periphery and differentiate at the site of their precursor mitosis. Here we describe ectodermally derived cells that migrate away from their site of origin and initiate axonogenesis at a distant location. In embryonic grasshopper limb buds, the first two pairs of afferents to differentiate are the pair of Ti1 pioneers at the limb tip and the pair of Cx1 cells found at the base of the limb. While the Ti1 pioneers arise from the mitosis of a pioneer mother cell at the limb tip, the Cx1 cells are shown to emerge from the epithelium at circumferential positions that are approximately 150 degrees apart and that belong to different embryonic compartments. The cells migrate into contact with each other before initiating axonogenesis, and their axons then extend in a new direction that is orthogonal to the route of cell migration.     

Pattern Specification and Pattern Regulation in the Embryonic Chick Limb Bud

SYNOPSIS. The embryonic chick limb bud is a growing organ rudimentwhose undifferentiated cells give rise to a precise spatialpattern of differentiated structures. The establishment of positionalvalues of chick limb bud cells (pattern specification) and theresponse of limb bud cells with established positional valuesto experimental perturbations (pattern regulation) are the majortopics considered in this paper. The results of recent experimentswith developing chick limb buds analyzing pattern specificationand pattern regulation are presented. These studies with thechick limb are described in light of the postulates of a modelthat was originally formulated from experiments performed onregenerating amphibian and insect appendages.     

Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms.

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5'-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.