Modeling colorectal cancer: A bio-resource of 50 patient-derived organoid lines.

BACKGROUND AND AIM: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. To improve outcomes for these patients, we need to develop new treatment strategies. Personalized cancer medicine, where patients are treated based on the characteristics of their own tumor, has gained significant interest for its promise to improve outcomes and reduce unnecessary side effects. The purpose of this study was to examine the potential utility of patient-derived colorectal cancer organoids (PDCOs) in a personalized cancer medicine setting. METHODS: Patient-derived colorectal cancer organoids were derived from tissue obtained from treatment-naïve patients undergoing surgical resection for the treatment of CRC. We examined the recapitulation of key histopathological, molecular, and phenotypic characteristics of the primary tumor. RESULTS: We created a bio-resource of PDCOs from primary and metastatic CRCs. Key histopathological features were retained in PDCOs when compared with the primary tumor. Additionally, a cohort of 12 PDCOs, and their corresponding primary tumors and normal sample, were characterized through whole exome sequencing and somatic variant calling. These PDCOs exhibited a high level of concordance in key driver mutations when compared with the primary tumor. CONCLUSIONS: Patient-derived colorectal cancer organoids recapitulate characteristics of the tissue from which they are derived and are a powerful tool for cancer research. Further research will determine their utility for predicting patient outcomes in a personalized cancer medicine setting.

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

Long-Term Culture of Intestinal Cell Progenitors: An Overview of Their Development, Application, and Associated Technologies.

PURPOSE OF REVIEW: Long-term culture of adult progenitor cells in 3D is a recently emerging technology that inhabits the space between 2D cell lines and organ slice culture. RECENT FINDINGS: Adaptations to defined media components in the wake of advances in ES and iPS cell culture has led to the identification of conditions that maintained intestinal cell progenitors in culture. These conditions retain cellular heterogeneity of the normal or tumour tissue, and the cultures have been shown to be genetically stable, such that substantial biobanks are being created from patient derived material. This coupled with advances in analytical tools has generated a field, characterized by the term "organoid culture", that has huge potential for advancing drug discovery, regenerative medicine, and furthering the understanding of fundamental intestinal biology. SUMMARY: In this review, we describe the approaches available for the long-term culture of intestinal cells from normal and diseased tissue, the current challenges, and how the technology is likely to develop further.

Early inhibition of EGFR signaling prevents diabetes-induced up-regulation of multiple gene pathways in the mesenteric vasculature.

Diabetes mellitus is associated with vascular complications including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive hormones. However, the signaling mechanisms leading to vascular dysfunction in diabetes are not fully understood. This microarray-based study was designed to identify differential gene expression between the normal and diabetic mesenteric vasculature and to investigate the effect of inhibiting epidermal growth factor receptor (EGFR) signaling on global gene expression in the mesenteric bed of streptozotocin (STZ)-induced diabetic rats. Transcriptome analysis was performed in triplicate using oligonucleotide microarrays housing 10,000 rat genes on the mesenteric bed of normal, diabetic, and diabetic rats treated with AG1478, a selective inhibitor of EGFR. Four weeks of diabetes led to a profound alteration in gene expression within the mesenteric bed with 1167 of the 3074 annotated genes being up-regulated and 141 genes down-regulated by at least 2-fold. The up-regulated gene ontologies included receptor tyrosine kinases, G-protein coupled receptors and ion channel activity. In particular, significant overexpressions of colipase, phospholipase A2, carboxypeptidases, and receptor tyrosine kinases such as EGFR, erbB2 and fibroblast growth factor receptor were observed in diabetes mesenteric vasculature. A 4-week intraperitoneal treatment of diabetic animals with AG1478 (1.2 mg/kg/alt diem) beginning on the same day as STZ injection prevented up-regulation of the majority (approximately 95%) of the genes associated with STZ diabetes including those apparently "unrelated" to the known EGFR pathway without correction of hyperglycemia. These results suggest that activation of EGFR signaling is a key initiating step that leads to induction of multiple signaling pathways in the development of diabetes-induced vascular dysfunction. Thus, therapeutic targeting of EGFR may represent a novel strategy for the prevention and/or treatment of vascular dysfunction in diabetes.

A missense mutation in the murine Opa3 gene models human Costeff syndrome.

Opa3 mRNA is expressed in all tissues examined to date, but currently the function of the OPA3 protein is unknown. Intriguingly, various mutations in the OPA3 gene lead to two similar diseases in humans: autosomal dominant inherited optic atrophy and cataract (ADOAC) and a metabolic condition; type 3-methylglutaconic aciduria (MGA). Early onset bilateral optic atrophy is a common characteristic of both disorders; retinal ganglion cells are lost and visual acuity is impaired from an early age. In order to investigate the function of the OPA3 protein, we have generated a novel ENU-induced mutant mouse carrying a missense mutation in the OPA3 gene. The heterozygous mutation in exon 2, causes an amino acid change p.L122P (c.365T>C), which is predicted to alter tertiary protein structure. In the heterozygous state, the mice appear uncompromised however; in the homozygous state mice display some of the features of MGA. Visual function is severely reduced, consistent with significant loss of retinal ganglion cells and degeneration of axons in the optic nerve. In the homozygous optic nerve, there was evidence of increased mitochondrial activity, as demonstrated by the increased presence of mitochondrial marker Cytochrome C Oxidase (COX) histochemistry. Mice homozygous for the opa3(L122P) mutation also display a severe multi-systemic disease characterized by reduced lifespan (majority dying before 4 months), decreased weight, dilated cardiomyopathy, extrapyramidal dysfunction and gross neuro-muscular defects. All of these defects are synonymous with the phenotypic characteristics of Type III MGA found in humans. This model will be of major importance for future studies of the specific function of the OPA3 gene.

Cell selective glucocorticoid induction of caveolin-1 and caveolae in differentiating pulmonary alveolar epithelial cell cultures.

Increased caveolin-1 expression is a marker of the differentiation of lung alveolar epithelial type II cells into a type I phenotype. Here, we show in both a primary differentiating rat alveolar culture, and a human alveolar cell line (A549) that caveolae formation and caveolin-1 expression are dependent upon dexamethasone Dex, and is inhibited by the glucocorticoid receptor (GR) antagonist, mifepristone. Study of a panel of 20 different cell types showed the effect of (Dex) upon caveolin-1 expression to be highly cell selective for lung alveolar epithelial cells. The actions of glucocorticoid upon caveolin-1 appear indirect acting via intermediary genes as evidenced by cycloheximide (CHX) abolition of Dex-induced increases in caveolin-1 mRNA and by recombinant transfection studies using the caveolin-1 promoter cloned upstream of a reporter gene. Treatment with actinomycin D (ACD) revealed that the effects of Dex are also, at least in part, mediated by stabilisation of caveolin-1 mRNA. Collectively, these results indicate that glucocorticoids modulate the expression of caveolin-1 and caveolae biogenesis within alveolar epithelial cells via both transcriptional and translational modifications. The cell-selective effects of glucocorticoid upon caveolin may represent a previously unrecognised mechanism by which glucocorticoids affect lung development.

Opa1 deficiency in a mouse model of autosomal dominant optic atrophy impairs mitochondrial morphology, optic nerve structure and visual function.

OPA1 is a ubiquitously expressed, nuclear dynamin-related GTPase, targeted to the inner mitochondrial membrane, which plays a role in mitochondrial fusion. Mutations in the OPA1 gene on chromosome 3q28-qter are associated with autosomal dominant optic atrophy (ADOA), the most common inherited optic neuropathy, in which retinal ganglion cells (RGCs) are lost and visual acuity is impaired from an early age. We have generated a novel ENU-induced mutant mouse carrying a protein-truncating nonsense mutation in opa1 in order to explore the pathophysiology of ADOA. The heterozygous mutation, B6; C3-Opa1(Q285STOP), located in exon 8 immediately before the central dynamin-GTPase, leads to approximately 50% reduction in opa1 protein in retina and all tissues on western analysis. The homozygous mutation is embryonic lethal by 13.5 days post coitum, demonstrating the importance of Opa1 during early development. Fibroblasts taken from adult heterozygous mutant mice show an apparent alteration in morphology, with an increase in mitochondrial fission and fragmentation. Heterozygous mutants show a slow onset of degeneration in the optic nerve electron microscopy. Furthermore, they demonstrate a functional reduction in visual function on testing with the optokinetic drum and the circadian running wheel. These findings indicate that the opa1 GTPase contains crucial information required for the survival of RGCs and that Opa1 is essential for early embryonic survival. The Opa1 +/- mice described here provide a means to directly investigate the cellular pathophysiology of OPA1 ADOA.

Toxicogenomics of drug delivery systems: Exploiting delivery system-induced changes in target gene expression to enhance siRNA activity.

Synthetic siRNAs are typically formulated with drug delivery systems (DDS) that improve cellular uptake for optimal gene silencing activity. Here, we show that two PAMAM dendrimer DDS, differing only in their structural architecture, elicit many different gene expression changes in human cells including opposing effects on the expression of epidermal growth factor receptor (EGFR), a gene targeted for silencing by siRNA. Despite providing similar improvements in siRNA uptake, these two formulations led to a approximately 10-fold variation in anti-EGFR siRNA activity. These data show that gene expression changes induced by DDS, separate from their ability to enhance cell uptake, determine 'apparent' siRNA potency and thus offer the possibility of tailoring delivery system-siRNA combinations for additive or synergistic effects on gene silencing.

Delivery strategies for siRNA-mediated gene silencing.

RNA interference (RNAi) represents a promising new gene silencing technology for functional genomics and a potential therapeutic strategy for a variety of genetic diseases. RNAi involves the targeted post-transcriptional degradation of messenger RNA thereby inhibiting the synthesis of the desired protein. This effectively leads to silencing of gene expression. The effectors of this process are short interfering RNA (siRNA) duplexes (approximately 21-23nt) that are key intermediaries in the specific degradation of target mRNA following incorporation into the RNA-induced silencing complex (RISC) in the cytosol. However, due to the large molecular weight and negative charge of siRNA duplexes the effective cellular uptake and intracellular delivery appear to represent a major challenge for the widespread use of RNAi in vivo. This review summarises some of the main delivery strategies that have been attempted for the transfection of siRNA to cells in vitro and in vivo.

Diabetes-induced renal vascular dysfunction is normalized by inhibition of epidermal growth factor receptor tyrosine kinase.

Contribution of receptor tyrosine kinase activation to development of diabetes-induced renal artery dysfunction is not known. We investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of tyrosine kinases (TKs), and AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) TK activity, to modulate the altered vasoreactivity of isolated renal artery ring segments to common vasoconstrictors in streptozotocin-induced diabetes. In diabetic renal artery, the vasoconstrictor responses induced by norepinephrine, endothelin-1 and angiotensin II were significantly increased. Inhibition of TKs or the EGFR pathway did not affect the agonist-induced vasoconstrictor responses in the non-diabetic control animals. However, inhibition of TKs by genistein or EGFR TK by AG1478 treatment produced a significant normalization of the altered agonist-induced vasoconstrictor responses without affecting blood glucose levels. Treatment with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor responses in the diabetic animals. Western blotting showed that phosphorylated EGFR protein levels were increased in vehicle-treated diabetic animals. In renal arteries from AG1478-treated diabetic animals, EGFR protein levels were similar to non-diabetic control animals. These data suggest that activation of TK-mediated pathways, including the EGFR TK signalling pathway, are involved in the development of diabetic vascular dysfunction in the renal artery.

The design and exogenous delivery of siRNA for post-transcriptional gene silencing.

RNA interference (RNAi) is a natural cellular process that effects post-transcriptional gene silencing in eukaryotic systems. Small interfering RNA (siRNA) molecules are the key intermediaries in this process which when exogenously administered can inhibit or "silence" the expression of any given target gene. Thus, siRNA molecules hold great promise as biological tools and as potential therapeutic agents for targeted inhibition of disease-causing genes. However, key challenges to the effective and widespread use of these polyanionic, macromolecular duplexes of RNA are their appropriate design and efficient delivery to cells in vitro and in vivo. This review highlights the current strategies used in the design of effective siRNA molecules and also summarises the main strategies being considered for the exogenous delivery of siRNA for both in vitro and in vivo applications.

A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides.

The optimal design of hybridisation-competent antisense oligonucleotides (ODNs) coupled with an efficient delivery system appear to be important prerequisites for the successful use of antisense reagents for gene silencing. We selected an antisense ODN complementary to an accessible region of the epidermal growth factor receptor (EGFR) mRNA with the aid of an antisense oligonucleotide scanning array. The scanning array comprised 2684 antisense ODN sequences targeting the first 120 nts in the coding region of EGFR mRNA. The array-designed antisense ODN was covalently conjugated to a novel anionic dendrimer using a pentaerythritol-based phosphoroamidite synthon via automated DNA synthesis and the ability of this conjugate to effectively deliver and down-regulate EGFR expression in cancer cells was evaluated. Each dendrimeric structure had nine ODN molecules covalently linked to a common centre at their 3' termini. This dendrimer conjugate was markedly more stable to serum nucleases compared to the free ODNs and the cellular uptake of ODN-dendrimer conjugates was up to 100-fold greater as compared to mannitol, a marker for fluid phase endocytosis, and up to 4-fold greater than naked ODN in cancer cells. ODN-dendrimer uptake was energy-dependent and mediated, at least in part, via binding to cell surface proteins; a process that was inhibited by self-competition and by competition with free ODN, salmon sperm DNA, heparin and dextran sulphate. Fluorescent microscopy studies showed a combination of punctate and more diffuse cytosolic distribution pattern for fluorescently labelled ODN-dendrimer conjugate in A431 cells implying internalization by endocytosis followed by release and sequestration of the conjugate into the cytosol. Little or no conjugate appeared to be present in the nuclei of A431 cells. In vitro RNase H-mediated cleavage assays confirmed that covalently conjugated antisense ODNs in the dendrimer conjugate were able to hybridize and cleave the array-defined hybridisation target site within the EGFR mRNA without the need for ODN dissociation from the conjugate. In cell culture, ODN-dendrimer conjugates were effective in inhibiting cancer cell growth that correlated with a marked knockdown in EGFR protein expression. These data highlight a novel anionic dendrimer delivery system for gene silencing oligonucleotides that improved their biological stability, cellular delivery and antisense activity in cultured cancer cells.

Evaluation of generation 2 and 3 poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides targeting the epidermal growth factor receptor.

PURPOSE: To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. METHODS: Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. RESULTS: G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. CONCLUSIONS: G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.

Toxicogenomics of non-viral vectors for gene therapy: a microarray study of lipofectin- and oligofectamine-induced gene expression changes in human epithelial cells.

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

Targeting caveolae for vesicular drug transport.

Caveolae are morphologically evident as omega-shaped invaginations of the plasma membrane with a diameter of 50-100 nm. They may also exist in a variety of other forms including flattened domains indistinguishable from the plasma membrane itself. At least in some cell types caveolae undertake transport functions including that of the endocytic and transcytotic movement of macromolecules, and indeed microbes and microbial toxins. Opportunities exist for basic and applied investigators working within the pharmaceutical sciences to exploit caveolae membrane interactions with the aim to develop of novel cellular or transcellular drug delivery strategies. This overview article will provide: pertinent information on the biology of the caveolae membrane system; review the various caveolae isolation methods; highlight some of the literature evidence showing that caveolae are functional with regard to macromolecule transport; discuss the role that caveolae could fulfill in the pulmonary absorption of therapeutic proteins from alveolar airspace to capillary blood following inhalational drug delivery, and finally review some very recent work showing proof-of-principle that caveolae domains can be targeted in a tissue-specific manner with highly selective ligands.

Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system.

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.

Constitutive expression of p-glycoprotein in normal lung alveolar epithelium and functionality in primary alveolar epithelial cultures.

The multidrug resistant (MDR) transporter P-glycoprotein (P-gp) is constitutively expressed in normal tissues, where its spatial distribution defines it as an important element reducing the systemic exposure and tissue access of potentially harmful xenobiotics. We sought to determine whether P-gp is functionally expressed within alveolar epithelium of lung, in particular within the predominant cell type of this barrier, the alveolar epithelial (AE) type I cell. By immunohistochemistry, MDR-1/mdr-1 P-gp was localized to luminal membranes of AE type I epithelium within normal human and rat lung tissue. Using a primary rat cell culture model affording study of AE type II to AE type I differentiation, we observed increased expression (reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunoflow cytometry techniques) of mdr-1a and mdr-1b P-gp in the cultures as they adopted an AE type I phenotype; freshly isolated AE type II cells were negative for mdr-1/P-gp. The functionality of P-gp within the AE cultures was demonstrated by a flow cytometric accumulation-retention assay using rhodamine-123 as substrate, and also by the polarized transport of vinblastine across confluent AE type I monolayers (basal-to-apical permeability was 3-fold that of apical-to-basal permeability), which was found to be comparable with the P-gp transport barrier presented by Caco-2 cell monolayers. The implications of localizing P-gp within alveolar epithelium is of significance to studies of fundamental respiratory cell biology as well as to further clarifying the nature of the barrier to xenobiotic transfer from alveolar airspace to pulmonary interstitium and capillary blood.

Caveolin expression during chondrogenesis in the avian limb.

Caveolin is the principal component and critical structural and functional element of caveolae, omega-shaped plasmalemmal invaginations, which have been implicated in a wide range of cellular processes in several different tissues. In the present study, we have investigated both the spatial and temporal expression of caveolin proteins during chondrogenesis in the avian tibiotarsus at days 10-20 of embryonic development. By using semiquantitative Western blotting, we found that caveolin-1 was clearly expressed in developing avian cartilage. The positive expression of caveolin-1 in cartilage showed an upward trend of accumulation temporally, with the highest levels of expression at 20 days of development. By using immunocytochemistry, we detected all three caveolin proteins in the cells of the outer fibrous articular surface, although caveolin-1 demonstrated the strongest and most consistent reactivity. In all cases, however, immunoreactivity appeared to be concentrated in cells facing the articular cavity. In the epiphyseal cartilage, immunocytochemistry revealed that caveolin-1 was present in the majority of chondrocytes within all layers of the cartilage and at all stages examined. A discrete, intense band of caveolin-1 immunoreactivity was apparent within the layer of flattened cells immediately underlying the proliferating rounded chondrocytes and suggests that caveolin-1 might be involved in regulating the progression of cells through these gradually maturing cell layers. In contrast to the results for caveolin-1, in the case of caveolin-2 and -3, chondrocytes were devoid of immunoreactivity in all regions of the epiphyseal cartilage. Overall, this study demonstrates that caveolin-1, -2, and -3 are expressed during chondrogenesis in the developing avian limb, although the patterns of expression are restricted both spatially and temporally throughout the differentiating cell layers of the cartilage. The results suggest that caveolin proteins might play a differentiation-dependent role during avian chondrogenesis.