Chondrogenesis in mouse limb bud in vitro. I. Effect of the ectoderm

The role of the ectoderm in the chondrogenesis of mouse limb bud mesoderm was investigated in vitro at several developmental stages by analysis of the evolution of DNA content, the accumulation of sulfated glycosaminoglycans and histochemical procedures. Young limb buds or the undifferentiated apex of older buds (stages 17 and 19 of Theiler's table) from which the ectoderm had been removed with trypsin treatment initiated a large chondrogenesis but not morphogenesis. When the ectoderm was present, these limb buds showed a polarized proximal to distal outgrowth and differentiated skeletal primordia. Mesodermal cells of stage 20 limb bud apex were able to differentiate autopodial skeletons with or without the presence of the ectoderm: cartilaginous areas of the limb skeleton seem determined at this developmental stage. These results, which show the importance of the ectoderm in limb bud morphogenesis, are compared with results obtained using other methods with mouse or bird buds.     

Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.     

Evaluation of the sensitive step of inhibition of chondrogenesis by retinoids in limb mesenchymal cells in vitro

The sensitive step of inhibition of chondrogenesis in vitro by retinoids was investigated in modified micromass cultures of limb bud mesenchymal cells from mouse embryos of day 11 and 12. Evaluation of chondrogenesis was performed after alcian blue staining, using a simple random hit counting of cartilage nodules. All-trans-retinoic acid, 13-cis-retinoic acid, and a newly developed arotinoid, RO 13-6298, were tested for their ability to inhibit chondrogenesis. We found that inhibition of chondrogenesis depended on the dosage and the duration of treatment with the different retinoids. Further analysis showed that chondrogenesis in limb bud mesenchymal cells from the proximal part was irreversibly inhibited after one hour of treatment, whereas distal cells showed a reduction of cartilage development only after a treatment period of 12 and more hours. In respect to the doses of the retinoids, proximal cells were about one magnitude more vulnerable than distal cells. These proximo-distal differences were obtained with 13-cis-retinoic acid at 10 micrograms/ml, with all-trans-retinoic acid at 1 microgram/ml and with arotinoid RO 13-6298 with 10 ng/ml. It is supposed that the late blastemal stage of chondrogenic differentiation before the onset of matrix synthesis is the step which is most vulnerable to retinoid treatment.     

Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid

Embryonic exposure to pharmacologic doses of vitamin A analogs (retinoids) is a well-known cause of limb-skeletal deletions, limb truncation and other skeletal malformations. The exclusively inhibitory effect of retinoic acid (RA) on chondrogenesis in standard serum-containing cultures of limb-bud mesenchymal cells is equally well known and has provided a means to explore the cellular basis for RA-mediated skeletal teratogenesis. Recent studies showing that lower RA concentrations can cause skeletal duplication when applied directly to the anterior border of a developing limb, suggest that RA may have a role in normal limb development as a diffusible morphogen capable of regulating skeletal pattern. While RA treatment causes both, skeletal deletions and duplications are clearly different (if not opposing) effects, the latter of which is difficult to reconcile with RA's heretofore exclusively inhibitory effect on in vitro chondrogenesis. In the present study. RA's effects on chondrogenesis and myogenesis were examined in serum-free cultures of chick limb-bud mesenchymal cells and compared with its effects on similar cultures grown in serum-containing medium. When added to serum-free medium, concentrations of RA known to cause skeletal duplication in vivo dramatically enhanced in vitro chondrogenesis (to over 200% of control values) as judged by both Alcian-blue staining and [35S]sulfate incorporation, while having little effect on myogenesis. Higher concentrations inhibited both chondrogenesis and myogenesis. The results indicate that at physiological concentrations. RA can selectively modulate chondrogenic expression and suggest that at higher concentrations, RA's inhibitory effects are less specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs

Effects of prostaglandins (PGs) on accumulation of cyclic AMP (cAMP) in the presence of a phosphodiesterase inhibitor were investigated in cells isolated from avian limb buds at various stages of development. Cells were responsive to PGE2 at the earliest stage investigated (stage 20-21) which was well in advance of specific cytodifferentiation of limb tissues. At three later stages (24-25; 26-28; 30-32), the responsiveness of cells isolated from the developing skeletal anlagen of the limb progressively increased coincident with the differentiation and maturation of the cartilage phenotype. Cells isolated from stage 26-28 cartilage rods were responsive also to prostacyclin (PGI2); however, the response produced was only about 50% of the response to an equivalent concentration of PGE2. Cells were not responsive to either PGF2 alpha or 6-keto PGF1 alpha, at concentrations of 30-33 micrograms/ml demonstrating a degree of specificity for PGE2 and PGI2. In the absence of the phosphodiesterase inhibitor, PGE2 increased cAMP accumulation two-fold over the controls and produced a concentration-dependent response between 0.3-30 micrograms/ml. The results demonstrate that PGs are capable of modulating cAMP levels of undifferentiated limb mesenchymal cells as well as embryonic cartilage cells and suggest a role for these compounds in limb chondrogenesis.     

The expression pattern of the chicken homeobox-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in apical ectodermal ridge-directed outgrowth of limb mesoderm and in programmed cell death

Abstract. The limb buds of the polydactylous mutant embryos, talpid ² and diplopodia -5, possess expanded distal apexes surmounted by prolongated thickened apical ectodermal ridges that promote the outgrowth and formation of digits from both the anterior and posterior mesoderm of the mutant limb buds. The chicken homeobox-containing gene GHox-7 exhibits an expanded domain of expression throughout the expanded subridge mesoderm of the mutant limb buds, providing support for the hypothesis that GHox-7 expression by subridge mesenchymal cells is involved in the outgrowth-promoting effect of the apical ectodermal ridge. During normal limb development GHox-7 is also expressed by the mesoderm in the proximal anterior nonchondrogenic periphery of the limb bud, which includes, but is not limited to the anterior necrotic zone. GHox-7 is also expressed in the posterior necrotic zone at the mid-proximal posterior edge of the limb bud. In contrast, GHox-7 is not expressed in either the proximal anterior or posterior peripheral mesoderm of talpid ² and diplopodia -5 limb buds which lack proximal anterior and posterior necrotic zones. Furthermore, retinoic acid-coated bead implants, which diminish cell death in the anterior necrotic zone, elicit a local inhibition of GHox-7 expression in the proximal anterior peripheral mesoderm. These results support the suggestion that GHox-7 may be involved in defining regions of programmed cell death during limb development. Furthermore, these studies indicate that the distal subridge and proximal anterior nonchondrogenic mesodermal domains of GHox-7 expression are independently regulated.     

Spatial and temporal patterns of cell death in limb bud mesoderm after apical ectodermal ridge removal

A spatiotemporal pattern of cell death occurred in the chick wing and leg bud mesoderm after removal of apical ectodermal ridge at stages 18–20. Cells died in a region extending from the limb bud distal surface to 150–200 μm into the mesoderm. Limb buds from which ridge was removed at later stages in development did not exhibit a spatiotemporal pattern of cell death. In control experiments in which dorsal ectoderm was removed, a pattern of cell death did not occur. Removal of the ridge and part of the 150- to 200-μm zone of prospective cell death resulted in cell death in an area approximately equal to the amount of the zone remaining. After removal of all of the prospective zone of cell death plus the apical ridge, cell death was observed in the remaining limb bud mesoderm. In these limb buds, cell death occurred in a region in which it had not been seen in limb bud with apical ridge alone removed. We conclude that at stages 18–20 the mesodermal cells 150–200 μm beneath the ridge require the apical ridge to survive. More proximal mesodermal cells do not die after ridge removal alone, but apparently require the presence of the more distal mesoderm to survive. Whether this is a requirement for something intrinsic to the distal mesoderm or something it possesses by way of the ridge is unknown. After stage 23, the limb mesoderm cells do not die when the apical ridge is removed. Nevertheless, at the later stages, ridge continues to be required for limb bud proximal-distal elongation and the differentiation of distal limb elements.     

Body wall morphogenesis: Limb-genesis interferes with body wall-genesis via its influence on the abaxial somite derivatives

The vertebrate body wall is regionalized into thoracic and lumbosacral/abdominal regions that differ in their morphology and developmental origin. The thoracic body wall has ribs and intercostal muscles, which develops from thoracic somites, whereas the abdominal wall has abdominal muscles, which develops from lumbosacral somites without ribs cage. To examine whether limb-genesis interferes with body wall-genesis, and to test the possibility that limb generation leads to the regional differentiation, an ectopic limb was induced in the thoracic region by transplanting prospective limb somatopleural mesoderm of Japanese quail between the ectoderm and somatopleural mesoderm of the chick prospective thoracic region. This ectopic limb generation induced the somitic cells to migrate into the ectopic limb mesenchyme to become its muscles and caused the loss of distal thoracic body wall (sterno-distal rib and distal intercostal muscle), without causing any significant effect on the more proximal region (proximal rib, vertebro-distal rib and proximal intercostal muscle). According to a new primaxial–abaxial classification, the proximal region is classified as primaxial and the distal region, as well as limb, is classified as abaxial. We demonstrated that ectopic limb development interfered with body wall development via its influence on the abaxial somite derivatives. The present study supports the idea that the somitic cells give rise to the primaxial derivatives keeping their own identity and fate, whereas they produce the abaxial derivatives responding to the lateral plate mesoderm.     

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.     

Inhibition of limb chondrogenesis by fibronectin

Abstract. This study compares the chondrogenic capacity of high density cultures prepared from either the develop-mentally younger, distal region or more advanced proximal region of stage 23/24 limb mesenchyme in high density cultures. Distal cultures undergo extensive chondrogenesis whether in F₁₂ medium supplemented with 10% fetal calf serum, 5% fetal calf serum, or fibronectin. On the other hand, proximal cultures fail to undergo chondrogenesis in medium containing 10% fetal calf serum or fibronectin, but do form cartilage in medium containing a decreased serum concentration or no serum. Furthermore, if the cells are cultured at low densities in native type I collagen gels, proximal cells have a reduced chondrogenic capacity in the presence of fibronectin, while chondrogenesis by distal cells is unaffected by the addition of fibronectin. The results demonstrate that proximal and distal cells respond differentially to serum and to fibronectin, and they suggest that the response of the cell to prevalent components of the extracellular matrix might change with development.     

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05–1.0 μg/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 μg/ml totally inhibiting cartilage formation. Concentrations of PGE₂ and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 μg/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE₂ levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE₂ and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.     

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.     

Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.     

Hyaluronan in limb morphogenesis

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.     

Characterization of bone-derived chondrogenesis-stimulating activity on embryonic limb mesenchymal cells in vitro

Demineralized bone matrix contains factors which stimulate chondrogenesis and osteogenesis in vivo. A water-soluble extract of bone has been shown to stimulate chondrogenesis in vitro in embryonic limb mesenchymal cells (Syftestad, Lucas & Caplan, 1985). The aim of this study was to analyse the cellular mechanism of the bone-derived chondrogenesis-stimulating activity, with particular attention on how normal requirements for chondrogenesis may be altered. The effects of bovine bone extract (BBE) on chondrogenesis in vitro were studied using micromass cultures of chick limb bud mesenchyme isolated from embryos at Hamburger-Hamilton (HH) stage 23/24, an experimental system which is capable of undergoing chondrogenic differentiation. Bovine diaphyseal long bones were demineralized and extracted with guanidine-HCl to prepare BBE (Syftestad & Caplan, 1984). High-density mesenchyme cultures (30 x 10(6) cells/ml) were exposed to different doses of BBE (0.01-1.0 mg ml-1) and chondrogenesis was quantified based on cartilage nodule number and [35S]sulphate incorporation. BBE was tested on micromass cultures of varying plating densities (2-30 x 10(6) cells/ml), on cultures of 'young' limb bud cells (HH stage 17/18), and on cultures enriched with chondroprogenitor cells obtained from subridge mesoderm. Since poly-L-lysine (PL) has recently been shown (San Antonio & Tuan, 1986) to promote chondrogensis, PL and BBE were introduced together in different doses, in the culture medium, to determine if their actions were synergistic. Our results show that BBE stimulates chondrogenesis in a dose-dependent manner and by a specific, direct action on the chondroprogenitor cells but not in normally non-chondrogenic, low density or 'young' limb bud cell cultures. The effects of PL and BBE are additive and these agents appear to act by separate mechanisms to stimulate chondrogenesis; PL primarily enhances nodule formation, and BBE appears to promote nodule growth.     

Limb development in chick embryos: cyclic AMP-dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20-23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25-28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP-dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80-85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane-enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP-dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP-protein phosphorylation system in the differentiation of limb tissues.     

Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis

Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.     

Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMP1

Effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on chondrogenesis and concentrations of prostaglandin E2 (PGE2) and cyclic AMP (cAMP) were investigated in micromass cultures of chick limb mesenchyme derived from the distal tip of stage 25 limb buds. TPA completely inhibited chondrogenesis during the first 4 days of culture; however, a few small cartilage nodules formed by day 6. Relative to control cultures, both PGE2 and cAMP concentrations were altered by TPA treatment during the 6-day period of cell culture. Concentrations of both compounds increased in control cells during the first 24 h of culture and then declined during the remaining 5 days. In TPA-treated cells both PGE2 and cAMP levels increased progressively during the 6 days of days of cell culture, each being elevated at day 6 by twofold over control cells. The results suggest the presence of regulatory pathways important in chondrogenesis which occur independent of those initiated by PGE2 and the cAMP system.     

Characterization of bone-derived chondrogenesis-stimulating activity on embryonic limb mesenchymal cells in vitro

Abstract. Demineralized bone matrix contains factors which stimulate chondrogenesis and osteogenesis in vivo. A water-soluble extract of bone has been shown to stimulate chondrogenesis in vitro in embryonic limb mesenchymal cells (Syftestad, Lucas & Caplan, 1985). The aim of this study was to analyse the cellular mechanism of the bone-derived chondrogenesis-stimulating activity, with particular attention on how normal requirements for chondrogenesis may be altered. The effects of bovine bone extract (BBE) on chondrogenesis in vitro were studied using micromass cultures of chick limb bud mesenchyme isolated from embryos at Hamburger-Hamilton (HH) stage 23/24, an experimental system which is capable of undergoing chondrogenic differentiation. Bovine diaphyseal long bones were demineralized and extracted with guanidine-HCl to prepare BBE (Syftestad & Caplan, 1984). High-density mesenchyme cultures (30 times 10⁶ cells/ml) were exposed to different doses of BBE (0–01-1-0 mg ml^-1) and chondrogenesis was quantified based on cartilage nodule number and [³⁵S]sulphate incorporation. BBE was tested on micromass cultures of varying plating densities (2–30 times 10⁶ cells/ml), on cultures of ‘young’ limb bud cells (HH stage 17/18), and on cultures enriched with chondroprogenitor cells obtained from subridge mesoderm. Since poly-L-lysine (PL) has recently been shown (San Antonio & Tuan, 1986) to promote chondrogensis, PL and BBE were introduced together in different doses, in the culture medium, to determine if their actions were synergistic. Our results show that BBE stimulates chondrogenesis in a dose-dependent manner and by a specific, direct action on the chondroprogenitor cells but not in normally non-chondrogenic, low density or ‘young’ limb bud cell cultures. The effects of PL and BBE are additive and these agents appear to act by separate mechanisms to stimulate chondrogenesis; PL primarily enhances nodule formation, and BBE appears to promote nodule growth.