Limb Bud Cell Cultures Research Articles

Spatial control of chondrogenesis assessed through micromass cultures of embryonic limb bud cells

Spatial control of chondrogenesis assessed through micromass cultures of embryonic limb bud cells

Reduction in cadmium-induced toxicity by c-Jun modulation in mouse embryo limb bud cells.

While it is known that cadmium-exposed embryonic cells have increased activation of c-Jun N-terminal kinase (JNK), the role of this stress signaling pathway in the embryotoxic response is not clear. Thus, the effects of modification of the transcription factor c-Jun, one of the downstream targets of JNK, on cadmium-induced embryotoxicity were investigated in primary cultures of mouse embryo limb bud cells. Cultures of limb bud cells harvested on day 11 of gestation were pretreated with antisense oligonucleotides (ASO) to c-Jun to reduce its expression, and then incubated with cadmium in the form of cadmium chloride. Toxicity was measured through assessments of cell proliferation and differentiation, while the effectiveness of the ASO in reducing c-Jun was assessed through Western blotting using phosphorylation-specific antibodies. When cells were treated with ASO c-Jun, the total amounts of c-Jun and also cadmium-induced c-Jun activation were diminished. Cadmium-induced cytotoxicity, indicated by reduced cell numbers and differentiation, was found to decrease when cells were exposed to the antisense oligonucleotides to c-Jun. In addition, limb cell numbers and differentiation were also enhanced by exposure to ASO in the absence of cadmium. The JNK pathway, and particularly the downstream effector c-Jun, appears to play an important role in regulating cell survival and differentiation in mouse embryo limb bud cells both in the presence and absence of the toxic metal cadmium.

Novel role for cyclophilin A in regulation of chondrogenic commitment and endochondral ossification.

Recent studies showed that cyclophilin A (CypA) promotes NF-κB/p65 nuclear translocation, resulting in enhanced NF-κB activity and altered expression of its target genes, such as the Sox9 transcriptional factor, which plays a critical role in chondrogenic differentiation and endochondral ossification. In this report, we unveil the role of CypA in signal-induced chondrogenic differentiation and endochondral ossification. Expression levels of the chondrogenic differentiation markers and transcriptional regulators Sox9 and Runx2 were all significantly lower in CypA knockdown chondrogenic cells than in wild-type cells, indicating that CypA plays a functional role in chondrogenic differentiation. In vitro differentiation studies using micromass cultures of mouse limb bud cells further supported the conclusion that CypA is needed for chondrogenic differentiation. Newborn CypA-deficient pups double stained with alcian blue and alizarin red exhibited generalized, pronounced skeletal defects, while high-resolution micro-computed tomography (microCT) analyses of the femurs and lumbar vertebrae revealed delayed or incomplete endochondral ossification. Comparative histology and immunohistochemistry (IHC) analyses further verified the effects of CypA deficiency on chondrogenic differentiation. Our results provide evidence for the important contribution of CypA as a pertinent component acting through NF-κB-Sox9 in regulation of chondrogenesis signaling. These findings are important to better understand signal-induced chondrogenesis of chondrogenic progenitors in physiological and pathophysiological contexts.

Combined developmental toxicity of bisphenol A and genistein in micromass cultures of rat embryonic limb bud and midbrain cells

Bisphenol A (BPA), widely used in industry and dentistry, and genistein (GEN), the predominant component of soy product, are both known environmental estrogen. In the present study, we investigated the developmental toxicity of BPA and GEN and their combined effect using micromass test, which is one of three standard alternative developmental toxicity tests recommended by European Center for the Validation of Alternative Methods (ECVAM). The results showed that IC50-P (cell proliferation) and IC50-D (cell differentiation) of BPA and GEN were approximately 20 and 5 μg/ml, respectively. No observed adverse effect level (NOAEL) of BPA and GEN were 10 and 0.94 μg/ml, respectively. The manifestation of BPA as a teratogen was insufficient, although the “low dose” effect should be paid attention to. While the evidence of GEN as a teratogen was solid, especially with the consideration of “high dose” application in clinical treatment. The combined effect of BPA and GEN was generally additive action except that in MB proliferation.

Reduction in cadmium‐induced toxicity and c‐Jun N‐terminal kinase activation by glutathione in cultured mouse embryonic cells

Cadmium (Cd(2+)) induces limb defects and other malformations in experimental animals. However, the mechanisms of the developmental toxicity of this metal are not fully understood. The ubiquitous intracellular tripeptide glutathione (GSH) protects nonembryonic cells from Cd(2+)-induced cell death and is essential for normal embryonic development. We predicted that pretreatment with GSH would prevent cytotoxicity in cultured mouse embryonic limb bud cells exposed to Cd(2+). Additionally, it was expected that GSH pretreatment would prevent the Cd(2+)-induced activation of the signaling molecule c-Jun N-terminal kinase (JNK), which becomes phosphorylated upon exposure to Cd(2+). Primary micromass cultures of limb bud cells obtained from organogenesis-stage mouse embryos were treated with either Cd(2+) or GSH alone, or both Cd(2+) and GSH. Treatment with GSH commenced 4 hr prior to Cd(2+) treatment. Cell proliferation was inhibited by approximately 50% after exposure to 4 μM Cd(2+) for 5 days. Cd(2+) treatment also resulted in a dose-dependent increase in the intracellular GSH content as measured after 5 days of exposure. Pretreatment with 4 mM GSH for 4 hr prevented the Cd(2+)-induced inhibition of cell proliferation and differentiation and also inhibited a threefold activation of JNK induced by 4 μM Cd(2+) after 24 and 48 hr of exposure. Exogenous GSH protects cultured embryonic limb bud cells from Cd(2+)-induced inhibition of cell proliferation and differentiation, which is associated with the activation of JNK.

Differential induction of MAP kinase signalling pathways by cadmium in primary cultures of mouse embryo limb bud cells

While it is well accepted that mammalian embryos may develop abnormally in response to harmful environmental stresses, the molecular changes that embryonic cells undergo are not always clear. In the present study we examined the induction of mitogen-activated protein kinase (MAPK) pathways by cadmium chloride (CdCl 2) in micromass cultures of limb bud cells from day 11 BALB/c mouse embryos. CdCl 2 exposure was found to cause a marked reduction in both cell survival and differentiation in these cultures. To study stress signalling pathways, cells were lysed and were examined for the phosphorylation of three MAPKs. It was shown that 4 μM CdCl 2 treatment of limb bud cells caused the phosphorylation of c-Jun N-terminal kinase (JNK), but not p38 or extracellular signal-regulated kinase (ERK), over a 120 min period. The results indicate a specific activation of the JNK pathway in response to CdCl 2-induced toxicity in mouse limb bud cells.

Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis

Wnt factors are involved in the regulation of all steps of cartilage development. The activity of Wnt factors is generally regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus and Wnt inhibitory factor 1 (Wif-1). Here we report that Wif-1 is highly expressed at cartilage-mesenchyme interfaces of the early developing skeleton. In fetal and postnatal skeletal development, Wif-1 is expressed in a sharply restricted zone in the upper hyaline layer of epiphyseal and articular cartilage and in trabecular bone. Coimmunoprecipitation and pull-down assays using recombinant Wif-1 and Wnt factors show specific binding of Wif-1 to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11. Moreover, Wif-1 was able to block Wnt3a-mediated activation of the canonical Wnt signalling pathway. Consequently, Wif-1 impaired growth of mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of chondrogenesis in micromass cultures of embryonic chick limb-bud cells. These results identify Wif-1 as a novel extracellular Wnt modulator in cartilage biology.

Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements

SHOX is a homeobox-containing gene, highly conserved among species as diverse as fish, chicken and humans. SHOX gene mutations have been shown to cause idiopathic short stature and skeletal malformations frequently observed in human patients with Turner, Leri–Weill and Langer syndromes. We cloned the chicken orthologue of SHOX, studied its expression pattern and compared this with expression of the highly related Shox2. Shox is expressed in central regions of early chick limb buds and proximal two thirds of later limbs, whereas Shox2 is expressed more posteriorly in the proximal third of the limb bud. Shox expression is inhibited distally by signals from the apical ectodermal ridge, both Fgfs and Bmps, and proximally by retinoic acid signaling. We tested Shox functions by overexpression in embryos and micromass cultures. Shox-infected chick limbs had normal proximo-distal patterning but the length of skeletal elements was consistently increased. Primary chick limb bud cell cultures infected with Shox showed an initial increase in cartilage nodules but these did not enlarge. These results fit well with the proposed role of Shox in cartilage and bone differentiation and suggest chick embryos as a useful model to study further the role of Shox in limb development.

Inhibition of cell differentiation by quinolones in micromass cultures of rat embryonic limb bud and midbrain cells

Micromass cultures (MMC) of rat embryonic limb bud (LB) and midbrain (CNS) cells were applied to compare the developmental toxicity of three quinolone antimicrobials: norfloxacin (Nor), enrofloxacin (Enr) and ciprofloxacin (Cip). Cultures were exposed for 5 days to seven concentrations of drugs. Cytotoxicity was assessed by quantifying neutral red uptake; differentiation—by quantifying alcian blue uptake (LB) or by image analysis of Gill’s haematoxylin stained foci (CNS). Both, LB and CNS cultures showed dose-dependent reduction in total cell number and differentiation. To distinguish specific effect on differentiation, IC 50 for proliferation ( P) and differentiation ( D) were calculated and P/ D ratios were compared. In LB cultures all three drugs were cytotoxic ( P/ D ratios were ⩽1). In CNS cultures P/ D ratios were ⩾1 (up to 2.7 for Nor, up to 4.4 for Enr and up to 16 for Cip) what can suggest specific action on differentiation. Ciprofloxacin was the most toxic and CNS cells were more sensitive than LB. The ranges of IC 50-D values (μg/ml) were as follows: Nor (79–14), Enr (127–179), Cip (91–101) in LB cultures; Nor (22–52), Enr (38–91), Cip (3–17) in CNS cultures. With one exception (Cip in CNS culture) all drugs were classified as weak embryotoxic.

Adaptive response in embryogenesis: IV. Protective and detrimental bystander effects induced by X radiation in cultured limb bud cells of fetal mice.

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

Effects of aliphatic nitriles in micromass cultures of rat embryo limb bud cells

The relative effects of a series of eight saturated (acetonitrile, propionitrile and n-butyronitrile) and unsaturated (acrylonitrile, allylnitrile, methacrylonitrile, cis-2-pentenenitrile, and 2-chloroacrylonitrile) aliphatic nitriles were evaluated using an in vitro test for embryotoxicity, the rat limb bud micromass assay. The concentrations that produced 50% inhibition (IC 50) of viability and differentiation of the cultured embryonic cells were of the same order of magnitude, whatever tested compound. The IC 50 values spread over a wide concentration range from 7–11 μM to 150 mM. Acetonitrile and 2-chloroacrylonitrile were the least and most potent compounds, respectively. The tested chemicals were evaluated using different criteria proposed to identify teratogens in the micromass system, based on either active concentrations or specific inhibition of cell differentiation. A few incorrect classifications were obtained with both nonteratogens and teratogens, when comparing the activity in limb bud cell cultures with the data available on their in vivo teratogenic potential in rats. The concordance between the in vitro and in vivo responses of this set of nitriles was judged insufficient to consider the micromass assay valuable for predicting the in vivo teratogenic outcome of this class of compounds.

The Homeodomain Protein Barx2 Promotes Myogenic Differentiation and Is Regulated by Myogenic Regulatory Factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.

Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediated intercellular communication.

Undifferentiated mesenchymal cells in the limb bud integrate a complex array of local and systemic signals during the process of cell condensation and chondrogenic differentiation. To address the relationship between bone morphogenetic protein (BMP) signaling and gap junction-mediated intercellular communication, we examined the effects of BMP-2 and a gap junction blocker 18 alpha glycyrrhetinic acid (18alpha-GCA) on mesenchymal cell condensation and chondrogenic differentiation in an in vitro chondrogenic model. We find that connexin43 protein expression significantly correlates with early mesenchymal cellular condensation and chondrogenesis in high-density limb bud cell culture. The level of connexin43 mRNA is maximally upregulated 48 h after treatment with recombinant human BMP-2 with corresponding changes in protein expression. Inhibition of gap junction-mediated intercellular communication with 2.5 microM 18alpha-GCA decreases chondrogenic differentiation by 50% at 96 h without effects on housekeeping genes. Exposure to 18alpha-GCA for only the first 24-48 h after plating does not affect condensation or later chondrogenic differentiation suggesting that gap junction-mediated intercellular communication is not critical for the initial phase of condensation but is important for the onset of differentiation. 18alpha-GCA can also block the chondrogenic effects of BMP-2 without effects on cell number or connexin43 expression. These observations demonstrate 18alpha-GCA-sensitive regulation of intercellular communication in limb mesenchymal cells undergoing chondrogenic differentiation and suggest that BMP-2 induced chondrogenic differentiation may be mediated in part through the modulation of connexin43 expression and gap junction-mediated intercellular communication.

Conditioned medium of mechanically compressed chick limb bud cells promotes chondrocyte differentiation

AbstractPrevious studies have demonstrated that chondrocyte differentiation can be stimulated by cyclic mechanical compression of chick limb bud cell-agarose constructs. This study reveals that medium collected from these compressed cultures promotes chondrocyte differentiation of noncompressed cells to approximately the same extent as compression alone. In micromass cultures of chick limb bud cells, incubation with medium from compressed cells significantly enhanced cell proliferation and the average rate of proteoglycan synthesis in a dose-dependent manner. These findings indicate that the response of undifferentiated chick limb bud cells to mechanical loading involves the secretion of one or more soluble differentiation factors. The chondrogenic activity of the conditioned medium was substantially inhibited by passage through a filter with a nominal molecular-weight cutoff (MWCO) of 10kDa but not inhibited when using a MWCO of 30kDa, suggesting that at least one of these factors has a molecular mass between 10 and 30kDa.

Ectodermal FGFs Induce Perinodular Inhibition of Limb Chondrogenesis in Vitro and in Vivo via FGF Receptor 2

The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm formed continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produced one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substituted for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, rendered FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides blocked appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis and, when introduced into the limb buds of developing embryos, led to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements.

Liarozole Markedly Increases all trans-Retinoic Acid Toxicity in Mouse Limb Bud Cell Cultures: A Model to Explain the Potency of the Aromatic Retinoid (E)-4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] Benzoic Acid

The remarkable toxicity of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB) compared to all trans-retinoic acid (tRA) is due to multiple factors, including reduced affinities for cytosolic binding proteins (CRABPs), resistance to metabolism, and prolonged nuclear receptor activation. To further investigate the role of half-life in retinoid toxicity, experiments were performed to determine whether, and to what extent, inhibition of tRA metabolism by liarozole increased its toxicity comparable to that of TTNPB in the mouse limb bud system. Liarozole is a known inhibitor of tRA 4-hydroxylation (CYP26). In the absence of liarozole, the IC50 for inhibition of chondrogenesis by tRA was 140 nM compared to 0.3 nM for TTNPB, a 467-fold difference. Following the addition of liarozole (10−6 M) to limb bud cultures, the potency of tRA to inhibit chondrogenesis was increased approximately 14-fold (IC50 of 9.8 nM). Although liarozole markedly increased toxicity of tRA in mouse limb bud micromass cultures, tRA metabolism was inhibited only about 10%. These results indicate that a relatively minor decrease in the metabolism of tRA in the mouse limb bud system is associated with a marked enhancement of toxicity that is likely related to the prolongation of tRA half-life during a critical period of development. Thus, the prolonged half-life of TTNPB is the most significant factor contributing to the remarkable teratogenicity of this synthetic aromatic retinoid.

Inhibition of cell differentiation by manganese chloride in micromass cultures of mouse embryonic limb bud cells

Although manganese is an essential element, in excess it has proven to be toxic to a number of organ systems. In several studies, exposure to manganese compounds during embryological development has been linked to skeletal abnormalities. Micromass cultures of dissociated mouse embryonic limb bud cells were used to investigate whether manganese could interfere with the process of chondrogenesis. Cell viability was significantly reduced in cultures grown in the presence of 1 and 10 m m MnCl 2, whereas as little as 10 μ m MnCl 2 inhibited differentiation of limb cells into proteoglycan-producing nodules. Because the concentration of MnCl 2 needed to interfere with differentiation is so much lower than the cytotoxic concentration, it is suggested that manganese has the potential to be teratogenic, and to specifically interfere with the development of a cartilage matrix, although by an unknown mechanism.

Effect of Compressive Loading on Chondrocyte Differentiation in Agarose Cultures of Chick Limb-Bud Cells

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Effect of Compressive Loading on Chondrocyte Differentiation in Agarose Cultures of Chick Limb-Bud Cells

It is well established that mechanical loading is important to homeostasis of cartilage tissue, and growing evidence suggests that it influences cartilage differentiation as well. Whereas the effect of mechanical forces on chondrocyte biosynthesis and gene expression has been vigorously investigated, the effect of the mechanical environment on chondrocyte differentiation has received little attention. The long-term objective of this research is to investigate the regulatory role of mechanical loading in cell differentiation. The goal of this study was to determine if mechanical compression could modulate chondrocyte differentiation in vitro. Stage 23/24 chick limb-bud cells, embedded in agarose gel, were subjected to either static (constant 4.5-kPa stress) or cyclic (9.0-kPa peak stress at 0.33 Hz) loading in unconfined compression during the initial phase of commitment to a phenotypic lineage. Compared with nonloaded controls, cyclic compressive loading roughly doubled the number of cartilage nodules and the amount of sulfate incorporation on day 8, whereas static compression had little effect on these two measures. Neither compression protocol significantly affected overall cell viability or the proliferation of cells within nodules. Since limb-bud mesenchymal cells were seeded directly into agarose, an assessment of cartilage nodules in the agarose reflects the proportion of the original cells that had given rise to chondrocytes. Thus, the results indicate that about twice as many mesenchymal cells were induced to enter the chondrogenic pathway by cyclic mechanical compression. The coincidence of the increase in sulfate incorporation and nodule density indicates that the primary effect of mechanical compression on mesenchymal cells was on cellular differentiation and not on their subsequent metabolism. Further studies are needed to identify the primary chondrogenic signal associated with cyclic compressive loading and to determine the mechanism by which it influences commitment to or progression through the chondrogenic lineage, or both.

A novel method for analysis of the periodicity of chondrogenic patterns in limb bud cell culture: correlation of in vitro pattern formation with theoretical models.

To experimentally examine whether the pattern changes predicted by theoretical models of pattern formation actually occur in a limb bud cell culture system, we developed a practical method to automatically measure the periodicity of chondrogenic patterns in vitro. The method utilizes binary image processing to quantify the total number of peak and valley pixels in a pattern to obtain the average interval between stripes in the chondrogenic pattern, and we named it the peak length method. The reliability of the peak length method was examined by using computer simulation results. The peak length method enabled us quantitatively obtain the average interval between chondrogenic islands, and the values obtained by this method were closely correlated with the average intervals obtained by manual measurement and two-dimensional Fourier transformation. The average intervals obtained by the peak length method were shown to be stable over a wide range of pattern variations that are frequently observed in actual experiments. By applying the peak length method to actual experimental data, we compared the validity of two theoretical models of pattern formation (cell sorting model and reaction-diffusion model) and it was concluded that the peak-length method is a useful tool to quantitatively analyze chondrogenic patterns in limb micromass culture and to relate theoretical predictions and experimental results of pattern formation.