It has been suggested that the matrix proteoglycan, versican, may perform a functional role during early events of limb skeletogenesis largely by virtue of its spatiotemporal expression pattern in precartilage mesenchymal aggregations. The versican-deficient hdf transgenic mouse has provided the first model to explore the implications of a null mature versican on limb chondrogenesis. Due to lethality of hdf homozygous embryos prior to limb cartilage differentiation, high-density micromass cultures were employed to compare the chondrogenic capacity of hdf mutant limb mesenchyme to that of wild-type. In homozygous hdf mesenchyme, aggregation was severely compromised and neither cartilage-characteristic Type 11 collagen nor alcian blue positive foci were detected during a 6-day period of culture. Three-dimensional culture of hdf mutant mesenchyme, however, showed that in a permissive environment mutant cells also expressed Type II collagen. Results strongly suggest that mature versican proteoglycan is essential for precartilage aggregation and subsequent cartilage differentiation. (c) 2005 Elsevier Inc. All rights reserved.