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

Abstract

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.

A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

“…The Ret signaling pathway plays a critical role during ENS development. Appropriate levels of Ret activity are crucial for EPC proliferation, migration, and survival [ 8 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. In the ENS, migration, and proliferation are closely connected; decreased proliferation of EPCs leads to decreased EPC migration and vice versa [ 39 , 40 , 41 ].…”

“…At early stages of ENS development, enteric progenitor cells (EPCs) migrate to and along the gut [ 1 ]. During this stage, ret is expressed in migrating EPCs and Ret signaling promotes EPC proliferation, migration, and survival [ 8 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Ret continues to be expressed in neuronal EPCs and neurons into adulthood [ 15 , 20 , 22 , 23 ].…”

A New Transgenic Tool to Study the Ret Signaling Pathway in the Enteric Nervous System

“…The Ret signaling pathway plays a critical role during ENS development. Appropriate levels of Ret activity are crucial for EPC proliferation, migration, and survival [7,[13][14][15][16][17][18][19][20][21]. In the ENS, migration, and proliferation are closely connected; decreased proliferation of EPCs leads to decreased EPC migration and vice versa [35][36][37].…”

“…In mice and human, single-cell RNA-sequencing analysis found that Ret is expressed in most ENS neurons in juveniles and adults [22,[38][39][40]. Accordingly, a ret:EGFP transgenic mouse line showed EGFP expression in differentiating neurons [21]. Ret signaling activity is essential for neuronal fate in the ENS.…”

“…At early stages of ENS development, enteric progenitor cells (EPCs) migrate to and along the gut [12]. During this stage, ret is expressed in migrating EPCs and Ret signaling promotes EPC proliferation, migration, and survival [7,[13][14][15][16][17][18][19][20][21]. Ret continues to be expressed in neuronal EPCs and neurons into adulthood [15,20,22,23].…”