Cell-cell interaction by mouse limb cells during in vitro chondrogenesis: Analysis of the brachypod mutation

This paper contains observations and experiments which collectively demonstrate a requirement for cell-cell interactions among limb bud mesenchyme cells during chondrogenic differentiation. Limb bud cells isolated from brachypodism^H (bp^H) and wild-type mouse embyros between Thieler stage 16–17 and midstage 21 were compared with respect to their abilities to undergo chondrogenic differentiation in high-density micromass cultures. Nodules formed by dissociated Day 12 (stage 20) bp^H limb bud cells have been reported previously to be abnormally reduced in size and number, and delayed in formation. We corroborate these results, but find that bp^H cultures prepared from earlier-stage limb buds (between stages 16–17 and early stage 21) are progressively more like wild-type cultures. Stage 16–17 bp^H cultures at 72 hr actually contain normal numbers of and size nodules, while stage 18 bp^H cultures are intermediate between stages 16–17 and stage 21 in nodule formation. On the other hand, we also find that the initial rate of aggregate formation is normal even in bp^H cultures prepared from stage 20 cultures in which nodule formation is not normal. Preparation of cultures composed primarily of early stage 21 bp^H limb bud cells mixed with small quantities (e.g., 5%) of stage 16–17 wild-type limb bud cells showed significant increases in cartilage nodule formation over control cultures composed only of early stage 21 bp^H cells. Greater proportions of wild-type cells obtained from embryos older than stages 16–17 were required for the same degree of normalization, supporting the hypothesis that a specific cell type, whose proportion decreases normally in the limb bud over time, is required to increase in vitro chondrogenesis by bp^H cells. Additionally, cultures containing stage 23 chick limb cells and early stage 21 bp^H cells at a ratio of 1:20 contained wild-type levels of nodules per square millimeter of culture. Thus, bp^H cells appear to respond to chondrogenic inductive signals from normal limb mesenchyme cells. In order to test for the ability of bp^H limb bud mesenchyme to induce chondrogenesis, stage 16–17 bp^H and wild-type limb bud cells, which form identical numbers of aggregates and nodules in culture, were each mixed with early stage 21 bp^H cells at ratios of 1:20, 1:10, and 1:3. Although low proportions of wild-type stage 17 cells significantly increased the number of aggregates and nodules in these mixed cultures, low proportions of bp^H stage 16–17 cells did not. It is, therefore, concluded that the primary defect of the bp^H mutation is likely to reside in the reduced ability of a specific mesenchyme cell subpopulation to provide an inductive stimulus for 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.     

Evidence for histogenic interactions during in vitro limb chondrogenesis

The requirement for homotypic cell interaction was studied by making chimeric micromass cultures containing various proportions of chick and quail limb mesenchyme. Cultures made from limb mesenchyme from embryos of Hamburger and Hamilton stages 23–24 produce large clumps of cartilage cells, identified by the accumulation of an extracellular matrix which stains with alcian blue at pH 1 and by the ability of cells to take up ³⁵SO₄ rapidly, as demonstrated autoradiographically. Dissociated mesenchyme from stage 19 embryos did not produce cartilage in micromass cultures, but only precartilage cell aggregates. Micromass cultures prepared from mixtures of mesenchyme cells obtained from stage 19 and stages 23–24 embryos contained decreasing numbers of cartilage nodules as the proportion of stage 19-derived mesenchyme increased. At the same time the number of aggregates was not affected. When the ratio of stage 19- to stage 24-derived cells was 3:1 or greater, no nodules were detected. The actual number of cells from each stage was verified by using mixtures of quail and chick cells, which are microscopically distinguishable. Additional evidence suggests that the stage 19-derived mesenchyme inhibits chondrogenesis by passively preventing stage 24-derived cells from interacting. The results presented are consistent with the suggestions that (1) homotypic cell interaction plays a role in limb chondrogenesis and (2) the capacity to interact in the required manner is acquired after the embryos have reached stage 19. These phenomena might be involved in the normal histogenesis of cartilage tissue.     

Inactivation of Patched1 in the Mouse Limb Has Novel Inhibitory Effects on the Chondrogenic Program

The bones of the vertebrate limb form by the process of endochondral ossification, whereby limb mesenchyme condenses to form an intermediate cartilage scaffold that is then replaced by bone. Although Indian hedgehog (IHH) is known to control hypertophic differentiation of chondrocytes during this process, the role of hedgehog signaling in the earlier stages of chondrogenesis is less clear. We have conditionally inactivated the hedgehog receptor Ptc1 in undifferentiated limb mesenchyme of the mouse limb using Prx1-Cre, thus inducing constitutively active ligand-independent hedgehog signaling. In addition to major patterning defects, we observed a marked disruption to the cartilage elements in the limbs of Prx1-Cre:Ptc1^c/c embryos. Using an in vitro micromass culture system we show that this defect lies downstream of mesenchymal cell condensation and likely upstream of chondrocyte differentiation. Despite early increases in levels of chondrogenic genes, soon after mesenchymal condensation the stromal layer of Prx1-Cre:Ptc1^c/c-derived micromass cultures is characterized by a loss of cell integrity, which is associated with increased cell death and a striking decrease in Alcian blue staining cartilage nodules. Furthermore, inhibition of the hedgehog pathway activation using cyclopamine was sufficient to essentially overcome this chondrogenic defect in both micromass and ex vivo explant assays of Prx1-Cre:Ptc1^c/c limbs. These data demonstrate for the first time the inhibitory effect of cell autonomously activated hedgehog signaling on chondrogenesis, and stress the importance of PTC1 in maintaining strict control of signaling levels during this phase of skeletal development.     

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.     

Nicotinamide adenine dinucleotide levels in cells of developing chick limbs: possible control of muscle and cartilage development

The studies reported here show that NAD⁺ levels are low in chick limbs which have not yet attained the stage of cellular commitment, that these low levels persist during a time period when major chondrogenic commitment and expression occur, that beyond this stage the NAD⁺ levels in chick limbs rise dramatically and continuously, corresponding to the period of major myogenic development, and that developing cultures of stage 24 mesodermal cells seem to mimic these in vivo events in that myogenic cells are observed when NAD⁺ levels are high and chondrogenic cells are observed when NAD⁺ levels are low. These observations are consistent with the hypothesis that pyridine nucleotides may play some role in the control of muscle and cartilage development in embryonic chick limbs.     

Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.     

A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes

Demineralized adult bone contains factors which stimulate nonskeletal mesenchymal cells to undergo a developmental progression resulting in de novo endochondral ossification. In this study, isolated embryonic stage 24 chick limb bud mesenchymal cells maintained in culture were utilized as an in vitro assay system for detection of specific bioactive components solubilized from adult chicken bone matrix. Guanidinium chloride extracts (4 M) of demineralized-defatted bone were fractionated and tested in limb mesenchymal cell cultures for possible effects upon growth and chondrogenesis. Two low-molecular-weight fractions were found to be active in these cultures. A cold water-insoluble, but warm Trisbuffered saline-soluble fraction provoked a dose-dependent increase in the amount of cartilage formed after 7 days of continuous exposure as evidenced by an increased number of chondrocytes observed in living cultures, elevated cell-layer-associated ³⁵S incorporation per microgram DNA, and greater numbers of toluidine blue-staining foci (i.e., cartilage nodules). Growth inhibitory substances were detected in a low-molecular-weight, water-soluble fraction; 7 days of continuous exposure to this material resulted in less cartilage formation and reduced cell numbers (accumulated DNA) on each plate. These observations demonstrate the usefulness of stage 24 chick limb bud cell cultures for identifying bioactive factors extracted from adult bone matrix. In addition, the action of these factors on mesenchymal cells may now be studied in a cell culture system.     

Formation of chondrous and osseous tissues in micromass cultures of rat frontonasal and mandibular ectomesenchyme

Rat frontonasal and mandibular mesenchyme was isolated from day-12 1/2 (stage-22) rat embryos and cultured at high density for up to 12 days. The stage chosen was based on the observation that mandibular mesenchyme at this stage became independent of its epithelium with respect to the production of both cartilage and bone. Frontonasal cultures developed aggregates of anastomosing columns of cells within 2 days. These grew as the cells enlarged, laying down an Alcian-blue-positive matrix by day 3 of culture. Significant mineral was detected by von Kossa staining by day 5 at which time the aggregates covered a large portion of the culture, eventually covering the entire micromass by day 10-12. Mandibular cultures developed centrally located nodular aggregates by 3 days of culture. These nodules increased in number, spreading outwards as the cells enlarged, laying down an Alcian-blue-positive matrix by day 4 and mineral by days 6-7. At this time the nodules began to elongate and coalesce, but never covered the entire culture over the 12-day period. Antibody staining revealed that in both cultures the cells were initially positive for type I collagen. Subsequently, the aggregates began expressing type II collagen, followed by type X, which coincided with the onset of mineralization. At this time some cells were negative for these cartilage markers, but positive for osteoblast markers, bone sialoprotein II, osteocalcin and type I collagen. In addition osteonectin and alkaline phosphatase were demonstrable in all of the aggregate cells late in the culture period. This provided clear evidence that chondroblast and osteoblast differentiation was proceeding within these cultures. The culture of rat facial mesenchyme should prove very useful, not only for the analysis of bone and cartilage induction and lineage relationships, but also in furthering our knowledge of craniofacial differentiation, growth and pattern formation by extending our analysis to a mammalian system.     

Neural Tissue Co-Culture with Mesenchyme to Investigate Patterningof Peripheral Nerve During Murine Embryonic Limb Development

Lateral plate mesoderm is native to the developing limb while other cells such as neurons extend migratory axonal processes from the neural tube. Questions regarding how axons migrate to their proper location in the developing limb remain unanswered. Extracellular matrix molecules expressed in developing limb cartilages, such as the versican proteoglycan, may function as inhibitory cues to nerve migration, thus facilitating its proper patterning. In the present study, a method is described for co-culture of neural tissue with high density micromass preparations of mouse limb mesenchyme in order to investigate neurite patterning during limb chondrogenesis in vitro. Comparison of hdf (heart defect) mouse limb mesenchyme, which bears an insertional mutation in the versican proteoglycan core protein, with wild type demonstrated that the described technique provides a useful method for transgenic analysis in studies of chondrogenic regulation of neurite patterning. Differentiating wild type limb mesenchyme expressed cartilage characteristic Type II collagen and versican at 1 day and exhibited numerous well defined cartilage foci by 3 days. Wild type neurites extended into central regions of host cultures between 3 and 6 days and consistently avoided versican positive chondrogenic aggregates. Wild type neural tubes cultured with hdf limb mesenchyme, which does not undergo cartilage differentiation in a wild type pattern, showed that axons exhibited no avoidance characteristics within the host culture. Results suggest that differentiating limb cartilages may limit migration of axons thus aiding in the ultimate patterning of peripheral nerve in the developing limb.     

Cell sorting and chondrogenic aggregate formation in micromass culture

A fundamental feature of cartilage differentiation in the developing limb is the formation of a prechondrogenic cell condensation. An apparently similar process of prechondrogenic cell aggregation occurs in micromass cultures of limb bud mesenchyme with the formation of cellular aggregates which often differentiate into cartilage nodules. We have investigated the process of aggregate formation in micromass culture using chimaeric mixtures of potentially chondrogenic and nonchondrogenic cell types. Two systems were studied: mixtures of distal and proximal limb mesenchyme cells and mixtures of distal limb cells with avian tendon fibroblasts. In both cases cultures of varying proportions of each cell type have been prepared. The results demonstrate that aggregate formation in vitro is the consequence of a cell sorting process which can involve prechondrogenic cells of widely different spatial origins within the developing limb. This contrasts with in vivo prechondrogenic condensation in which there is no evidence of cell sorting (Searls, R.L. (1967), J. Exp. Zool. 166, 39-50). However, our findings do indicate that cell surface differences occur in apparently undifferentiated limb mesenchyme. The results also suggest that mesenchymal cell aggregates must achieve a threshold size before chondrogenesis can proceed. In addition, the results show that under some culture conditions nonchondrogenic cells will form aggregates.     

Position-related capacity for differentiation of limb mesenchyme in cell culture.

A quantitative comparison (i.e., number of cartilage nodules) of cartilage differentiation was made between micromass cell cultures prepared with cells from different locations (core vs periphery) within prechondrogenic chick wing buds. Wing bud core cells in micromass culture exhibit a greater developmental bias toward cartilage differentiation than periphery cells from the same limbs. In addition, myogenic cells appear more frequently in cultures prepared from wing bud periphery than in those prepared from core tissue. Therefore a stage 23–24 wing bud is not a homogeneous population of multipotential mesenchymal cells. Instead, a stage 23–24 wing bud contains two classes of cells, each characterized by a bias for either cartilage or muscle differentiation, and a third class of uncharacterized mesenchymal cells.     

Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.     

A tissue culture analysis of the steps in limb chondrogenesis

Summary Tissue-culture methods can be used to test the developmental capacity of embryonic cells. In micro-mass cultures, derived from wing cells of stages 21 through 24 chick embryos, aggregates of cells form and then differentiate into cartilage nodules, as judged by the presence of an Alcian blue staining extracellular matrix. Wing cells derived from embryos as young as stage 17 can form aggregates. However, unless they are treated with db cyclic AMP and theophylline, it is not until stage 20 that these aggregates can produce cartilage in culture. In clonal cell culture, cartilage colonies are not produced by primary cell suspensions of limb cells until stage 25 when overt cartilage differentiation is occurring in vivo. It is possible to obtain clonable cartilage cells from limb cells from embryos between stages 20 and 24 if the cells are either treated with db cyclic AMP and theophylline or maintained in suspension culture for 12 to 48 hr. On the basis of these in vitro results a multiple step model for the conversion of limb mesenchyme into cartilage cells is proposed. The model involves the appearance of cells with a predisposition to form aggregates, development of the capacity to form cartilage in response to elevated levels of cyclic AMP, the appearance of receptors that translate changes in either cell shape or cell cycle parameters into elevated levels of cyclic AMP, aggregation, elevated levels of cyclic AMP, cartilage cell determination, and differentiation. This model can serve as the basis for further tests. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by USPHS Training Grant HD00152 from the National Institute of Child Health and Human Development, while P.B.A. was a postdoctoral trainee, and by NIH Grant HD05505 to M.S.     

Characterization of a bone-specific alkaline phosphatase in chick limb mesenchymal cell cultures

Previous morphometric and biochemical studies suggested that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. Others have shown that osteoblast expression is marked by an increase in bone-specific alkaline phosphatase activity. Our results indicate that chick limb mesenchymal cells develop alkaline phosphatase activity that is identical to that of the chick embryonic bone-specific isoenzyme. The alkaline phosphatase isozymes were partially purified from samples of chick intestine, liver, stage 38 embryonic limbs, and cultures of stage 24 limb mesenchymal cells. These tissues were separately extracted with butanol, acetone precipitated, redissolved, and passed over a DEAE-Sephacel ion-exchange column and ion-filtration column (Sephadex A-25). From the data obtained during this purification scheme, we conclude that the alkaline phosphatase from stage 38 limbs (bones) and Day 4 cultures are identical, and this activity is different from the enzyme purified from intestine and liver. The cell culture isozyme has an apparent K_m, heat lability, response to specific inhibitors, electrophoretic mobility, and molecular weight similar to those of bone-specific alkaline phosphatase. These observations support the view that osteoblastic progenitor cells are present in the stage 24 limb mesenchyme and that under specific culture conditions, bone development can be uniquely observed in vitro.     

The differentiation of normal and muscle-free distal chick limb bud mesenchyme in micromass culture

Distal chick wing bud mesenchyme from stages 19 to 27 embryos has been grown in micromass culture. The behavior of cultures comprising mesenchyme located within 350 microns of the apical ectodermal ridge (distal zone mesenchyme) was compared to that of cultures of the immediately proximal mesenchyme (subdistal zone cultures). In cultures of the distal mesenchyme from stages 21-24 limbs, all of the cells stained immunocytochemically for type II collagen within 3 days, indicating ubiquitous chondrogenic differentiation. At stage 19 and 20, this behavior was only observed in cultures of the distal most 50-100 microns of the limb bud mesenchyme. Between stages 25 and 27, distal zone cultures failed to become entirely chondrogenic. At all stages, subdistal zone cultures always contained substantial areas of nonchondrogenic cells. The different behavior observed between distal zone and corresponding subdistal zone cultures appears to be a consequence of the presence of somite-derived presumptive muscle cells in the latter, since no such difference was observed in analagous cultures prepared from muscle-free wing buds. The high capacity of the distal zone for cartilage differentiation supports a view of pattern formation in which inhibition of cartilage is an important component. However, its consistent behavior in vitro indicates that micromass cultures do not reflect the in vivo differences between the distal zones at different stages. The subdistal region retains a high capacity of cartilage differentiation and the observed behavior in micromass reflects interactions with a different cell population.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Osteogenesis in cultures of limb mesenchymal cells

The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 10⁶ cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 10⁶ cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.