An integrated pipeline for the multidimensional analysis of branching morphogenesis.

Developmental branching morphogenesis establishes organ architecture, and it is driven by iterative interactions between epithelial and mesenchymal progenitor cell populations. We describe an approach for analyzing this interaction and how it contributes to organ development. After initial in vivo cell labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) and tissue-specific antibodies, optical projection tomography (OPT) and confocal microscopy are used to image the developing organ. These imaging data then inform a second analysis phase that quantifies (using Imaris and Tree Surveyor software), models and integrates these events at a cell and tissue level in 3D space and across developmental time. The protocol establishes a benchmark for assessing the impact of genetic change or fetal environment on organogenesis that does not rely on ex vivo organ culture or section-based reconstruction. By using this approach, examination of two developmental stages for an organ such as the kidney can be undertaken by a postdoctoral-level researcher in 6 weeks, with a full developmental analysis in mouse achievable in 5 months.