99 - Cartilage Nodule Interfaces in Micromass Cultures: Developing an in Vitro Model for Early Joint Development

Poster Board Number: 99
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Abstract Body : Studying joint development can be a difficult and slow-paced process because most studies must be done in vivo to maintain location-specific gene expression and patterning information. Micromass cultures have been used to study cartilage and skeletal development but has not been explored as a model for joint development. Cartilage nodules in micromass cultures have cell arrangements similar to the articular cartilage of joints. Hence, we hypothesized that micromass cultures will generate joint-associated cell types, gene expression, and transcriptional regulation in a pattern similar to a developing joint.

To test this hypothesis, mesoderm from chicken limb buds (with or without somites) were densely plated and grown for seven days to develop micromass cultures. The micromass cultures were then evaluated for the presence of joint-associated tissues, transcription factors, and enhancers using in situ hybridization and fluorescence microscopy. Cartilage nodules were stained with alcian blue.

Using markers, we identified various joint-associated cell types such as cartilage (COL2A1, COL9, ACAN), GDF5-positive interzone cells, connective tissues (COL1A2, SCX), and myogenic precursors (MYOD1) present within the micromass cultures. The flattened arrangement of cells surrounding cartilage nodules in the micromass cultures was similar to the arrangement found in abutting cartilage anlagen of a forming joint. Importantly, a GDF5-enhancer displayed activity that overlapped GDF5 expression similar to developing joints. Joint-associated transcription factors such as FOX and SOX were also expressed in the micromass cultures.

Our findings suggest that micromass cultures can provide a model suitable for evaluating early joint-associated tissue differentiation. Further studies will help to determine which joint-associated tissues, genes, and pathways adequately recapitulate the early in vivo process of joint formation and thus can be appropriately exploited in vitro.