How to Wire the Diaphragm: Wholemount Staining Methods to Analyze Mammalian Respiratory Innervation

Direct or indirect impairment of breathing in humans by diseases or environmental factors can either cause long-term disability and pain, or can ultimately result in death. Automatic respiratory centers in the brainstem control the highly structured process of breathing and signal to a specialized group of motor neurons in the cervical spinal cord that constitute the phrenic nerves. In mammals, the thoracic diaphragm separates the thorax from the abdomen and adopts the function of the primary respiratory musculature. Faithful innervation by the phrenic nerves is a prerequisite for correct functionality of this highly specialized musculature and thus, ultimately, the viability of the entire organism.To analyze the effects of diseases and genetic defects responsible for deleterious or lethal respiratory phenotypes, accurate imaging of respiratory innervation during embryonic development, e.g., in genetically modified mouse models enables the characterization of specific marker genes and pathways that underlie appropriate wiring of the diaphragm. Among the different available immunostaining techniques, wholemount staining methods provide the advantage of clear and faithful three-dimensional information about the location of the antigens of interest. In comparison to routine histological techniques, however, the researcher has to deal with technical challenges, such as antibody penetration, the stability and availability of the antigen, and clearing of the relevant tissue, and the need to be equipped with state-of-the-art microscope equipment.In this methodological chapter, we explain and share our expertise concerning wholemount processing of mouse embryos and thoracic diaphragms for the analysis of mammalian respiratory innervation.