Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis
Summary
Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.
Abstract
Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.
Introduction
Development of multicellular organisms and the maintenance of homeostasis in their adult tissues reside in a fine-tuned regulation between self-renewal and differentiation of stem cells, which orchestrate in time and space normal tissue development and regeneration1. Subversion of this regulation causes developmental anomalies and cancers2. Thus, understanding the molecular and cellular mechanisms that orchestrate stem cell self-renewal and differentiation is of key interest in developmental and cancer biology.
Recent development of ex vivo organogenesis methods, in which tissue stem cells generate three-dimensional organoids have transformed our capabilities to study the dynamics of stem cells during mammalian organogenesis and maintenance of tissue homeostasis in a dish3. Organoids represent a good alternative to cumbersome genetically modified animal models to study these processes. Protocols for the development of organoids from tissue stem cells of many organs have now been developed3, including small intestine and colon, stomach, liver, pancreas, prostate, and mammary gland3. Additionally, the development of somatic genome-editing techniques in organoid-forming stem cells now enables to quickly interrogate the molecular and cellular mechanisms that control their biology4,5.
The primary cilium is a microtubule-based structure that is assembled at the surface of stem and/or differentiated cells of various tissues6. It is generally non-motile and is assembled as a single structure per cell7. Primary ciliogenesis is the dynamic process of assembling the primary cilium7. At the cell surface, the cilium acts as a cell signaling platform8. Thus, the primary cilium is thought to act as a key regulator of stem cell self-renewal and/or differentiation in many tissues, including the brain9,10, the mammary gland4,11, the adipose tissue12, and the olfactory epithelium13, among others. Primary ciliogenesis and/or ciliary signaling are dynamically regulated in distinct cell lineages and at different developmental stages4,13,14, but the underlying mechanisms remain to be largely determined.
Ex vivo organogenesis shows promise for the development of basic knowledge on the molecular and cellular mechanisms that control stem cell biology, including primary ciliogenesis and ciliary signaling. However, it relies on the ability to properly image whole mount organoids at the single cell level and at sub-cellular scales. We recently used a mouse mammary stem cell-derived organoid model to show that primary cilia positively control mouse mammary stem cell organoid-forming capacity4. Here we present a comprehensive protocol for the immunofluorescence staining of whole mount mouse mammary organoids (Figure 1A,B), which enables the analysis of primary cilia through light sheet microscopy during ex vivo organogenesis in three-dimension. Alternative methods were recently published for the immunofluorescence staining and imaging of organoids through confocal microscopy15,16. This protocol focuses instead on the preparation and imaging of organoids through light sheet microscopy.
Protocol
Representative Results
Discussion
The detailed protocol presented here enables the staining and imaging of mouse mammary organoids that grow in semi-solid medium. This protocol is presumably applicable to the staining of organoids mimicking the architecture of various tissues that grow in semi-solid and solid media. For organoids that grow in 100% Matrigel with medium on top, the recovery and fixation steps slightly differ. The culture medium must be removed from the culture well. After a quick PBS wash, the fixative solution (4% PFA) may be directly add…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Xavier Pinson for help in development of light sheet microscopy; the Biosit biotechnology center, including MRic, Arche, the Flow cytometry core facilities, and SFR Santé F. Bonamy, including the MicroPICell core facility, for technical support. This work was supported by Fondation ARC, Cancéropôle Grand Ouest, Université de Rennes 1, Fondation de France. M.D. was supported by a Graduate Fellowship from the University of Rennes. V.J.G. was supported by a Postdoctoral Fellowship from Fondation ARC.
Materials
| Anti-mouse IgG1 647 | Thermo-Fisher | A21240 | |
| Anti-mouse IgG2A 488 | Thermo-Fisher | A21131 | |
| Anti-rabbit 546 | Thermo-Fisher | A11035 | |
| Arl13b | NeuroMab | 73-287 | |
| EMS 16% Paraformaldehyde Aqueous Solution, EM Grade | Electron Microscopy Sciences | 15710 | |
| FBS | Thermo-Fisher | 10270106 | |
| gtubulin | Sigma-Aldrich | T5326 | |
| Hoechst 33342 | Thermo-Fisher | 62249 | |
| Integrin a6 | Biolegend | 313616 | |
| Light Sheet Capillary | Zeiss | 701908 | |
| Light Sheet plunger | Zeiss | 701998 | |
| Low binding Microcentrifuge tubes | BioScience | 27210 | |
| Normal Goat Serum Blocking Solution | Vector labs | S-1000 | |
| PBS | Sigma-Aldrich | p3587 | |
| Slug | Cell Signaling Technology | 9585 | |
| Triton-X100 | Sigma-Aldrich | T9284 | |
| Tween-20 | Euromedex | 9005-64-5 | |
| UltraPure Low Melting Point Agarose | Thermo-Fisher | 16520050 |
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