Live visualization of a functional RET‐EGFP chimeric receptor in homozygous knock‐in mice

• Published in: Development, growth & differentiation Vol. 63; no. 6; pp. 285 - 294
• Main Authors: Sunardi, Mukhamad, Ito, Keisuke, Enomoto, Hideki
• Format: Journal Article
• Language: English
• Published: Japan Wiley Subscription Services, Inc 01.08.2021
• Subjects: Active transport; Animals; Axon guidance; Cell membranes; Cell migration; Cell Movement; Cytoplasm; Embryos; Endosomes; Enteric Nervous System; GDNF; Glial cell line-derived neurotrophic factor; Glial Cell Line-Derived Neurotrophic Factor - genetics; Kidneys; live imaging; Localization; Mice; Neural stem cells; Neurons; neurotrophic factors; Organogenesis; Polarity; Progenitor cells; Protein turnover; Protein-tyrosine kinase receptors; Proteins; receptor trafficking; RET; Ret protein; Signal Transduction; Visualization; RET; live imaging; GDNF; neurotrophic factors; receptor trafficking
• ISSN: 0012-1592; 1440-169X
• DOI: 10.1111/dgd.12740

The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET‐dependent developmental processes involve long‐distance cell‐cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs. Little is known, however, about the dynamics of RET protein in cells. Addressing this issue requires development of a system that allows visualization of RET in living cells. In this study, we report generation of a novel knock‐in mouse line in which the RET‐EGFP chimeric receptor is expressed under the Ret promoter. Unlike Ret‐deficient mice that die after birth due to the absence of the enteric nervous system (ENS) and kidneys, RetRET‐EGFP/RET‐EGFP mice were viable and grew to adulthood with no overt abnormality, which indicated that RET‐EGFP exerts function comparable to RET. In neurons and ENS progenitors, RET‐EGFP signals were detected both on the cell membrane and in the cytoplasm, the latter of which appeared as a punctate pattern. Time‐lapse imaging of cultured neural cells and embryos revealed active transport of RET‐EGFP puncta in neuronal axons and cell bodies. Immunohistochemical analyses detected RET‐EGFP signals in early and recycling endosomes, indicating that RET‐EGFP is trafficked via the endocytic pathway. RetRET‐EGFP/RET‐EGFP mice enable visualization of functional RET protein in vivo for the first time and provide a unique platform to examine the dynamics and physiology of RET trafficking. We report generation of a novel knock‐in mouse line in which the RET‐EGFP chimeric receptor is expressed under the Ret promoter. Time‐lapse imaging of cultured neural cells and embryos of this mouse line revealed active transport of RET‐EGFP puncta in neuronal axons and cell bodies. RET‐EGFP knockin mice allow visualization of functional RET protein in vivo and provide a unique platform to examine the dynamics and physiology of RET trafficking.