In Situ Hybridization Analysis of the Expression of the Type II Collagen Gene in the Developing Chicken Limb Bud

Abstract

In situ hybridization with ³²P- or ³¹S-labeled double-stranded DNA or single-strandedRNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo. When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18–25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues. In contrast, sections hybridized with a probe complementary to mRNA for the α1 chain of type I collagen exhibited an intense hybridization signal over the perichondrium and little or no signal over the cartilage primordia. At all stages of development examined, ³²P-labeled double-stranded DNA probes or single-stranded RNA probes labeled with either ³²P or ³⁵S provided adequate hybridization signals. Several experimental protocols were employed to control for the potential cross-hybridization and non-specific hybridization of the type II collagen probes. These included the utilization of labeled noncomplementary “sensestrand” type II collagen RNA as a control probe for nonspecific background, and prehybridization with a large excess of appropriate unlabeled RNA to block sequences in heterologous collagen RNAs that might cross-hybridize to the specific labeled probe.