Gene Expression Detection in Developing Mouse Tissue Using In Situ Hybridization and microCT Imaging

Abstract

High resolution and noninvasiveness have made soft tissue X-ray microtomography ({micro}CT) a widely applicable three-dimensional (3D) imaging method in studies of morphology and development. However, scarcity of molecular probes to visualize gene activity with {micro}CT has remained a challenge. Here we apply horseradish peroxidase -assisted reduction of silver and catalytic gold enhancement of the silver deposit to in situ hybridization in order to detect gene expression in developing tissues with {micro}CT (here called GECT, Gene Expression CT). We show that GECT detects expression patterns of collagen type II alpha 1 (Col2a1) and sonic hedgehog (Shh) in developing mouse tissues comparably with an alkaline phosphatase-based detection method. After detection, expression patterns are visualized with laboratory {micro}CT, demonstrating that GECT is compatible with varying levels of gene expression and varying sizes of expression regions. Additionally, we show that the method is compatible with prior phosphotungstic acid (PTA) staining, a conventional contrast staining approach in {micro}CT imaging of soft tissues. Overall, GECT is a method that can be integrated with existing laboratory routines to obtain spatially accurate 3D detection of gene expression.