Volumetric Imaging and Analysis of Primary Cilia in Musculoskeletal Tissue using the ARL13B-CENTRIN-2 Mouse Model

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton and in coordinating cell responses to biochemical and mechanical cues from their surrounding environment. Due to its micrometer-scale size, the primary cilium is challenging to image in situ to characterize in tissues, in a high throughput manner and without optical biases. An understanding of its organization in its 3D environment in vivo will be key to understanding ciliary roles in physiology and disease. The ARL13B-CENTRIN-2 mouse line presents an mCherry tag on the ciliary axoneme protein ARL13B and a green fluorescent protein (GFP) tag on the centriole protein CENTRIN-2, localized to the base of the cilium. This mouse line allows for imaging of the primary cilium without the need for staining, which reduces the number of steps in image collection and circumnavigates issues with signal-to-noise. This publication describes a step-by-step protocol to image primary cilia in musculoskeletal tissues whilst preserving the fluorescent signal. It also describes a universally applicable image analysis pipeline, enabling unbiased quantification of cilia features, such as length and 3D orientation.