No getting around RET

Researchers find no role for RET-independent GFR-alpha in development or regeneration -
Neurons depend on external molecular signals for their very survival. These molecules, collectively referred to as neurotrophic factors, include a family of
four GDNF Family Ligands (GFLs) that bind to specific receptor sites on the surfaces of neural cells. These sites allow GFLs to signal through a receptor
complex composed of the RET tyrosine kinase and a GFRб-family receptor. Tyrosine kinases, such as RET, are well-known for their function in
phosphorylation cascades that span the cell membrane. The role of the GFRб co-receptors in these complexes was long thought to be limited to as a co
-receptor for RET, but GFRs have recently been suggested to play other roles as well.


The individual functions of the RET and GFRб subunits in these receptor complexes, which are important in developmental milieux from peripheral
neurogenesis to the developing kidney, remains a thorny question complicated by the fact that GFRб is much more widely expressed in the body than is
RET and that, in vitro, cells expressing GFRб1 without RET have been shown to respond to GDNF signals. A report by Hideki Enomoto (Team Leader,
Laboratory for Neuronal Differentiation and Regeneration) and colleagues at the RIKEN Center for Developmental Biology and the Washington University
School of Medicine published in the November 18 issue of Neuron now challenges the view that RET-independent GFRб1 signaling plays a significant
physiological role in either development or regeneration.


Enomoto first devised an elegant experimental system to make it possible to generate mice specifically lacking RET-independent GFRб1. The study of
GFRб deficiencies in vivo is dogged by the lethality of the phenotype, in which the absence of enteric neurons and functioning kidneys results in death
soon after birth. In vitro studies and the proximity of RET-independent GFRб and RET-expressing cells in some developmental regions, however, have
prompted strong speculation that GFRб might be able to operate even in the absence of RET indigenous to the cell. It has been suggested that this might
take the form of either trans signaling, in which the GFRб receptor captures diffusible GFLs and presents them to a neighboring RET-expressing cell, or
through a separate signaling mechanism mediated by GFL-activated neural cell adhesion molecules (NCAMs).


Given this body of work showing the likelihood of a physiological role for RET-independent GFRб1 activity, Enomoto et al. decided to test whether the in
vitro evidence would be borne out in living mice. The team first showed that mice homozygous for a transgene deleting an important segment of the GFR
б1 gene died in the perinatal period, while heterozygotes (which carried only a single copy of the transgene) were healthy and fertile. On comparing
specific embryonic regions in hetero- and homozygous mice, they found associations between RET-expressing and RET-independent GFRб1 cells in
kidney, enteric and motor neurons, as well as the expected disturbances in development. However, when they next generated mice that were only capable
of expressing GFRб1 only in the RET-expressing cells (by cloning GFRб1 cDNA into a region under the control of the Ret promoter and crossbreeding
the resulting animals with GFRб1 heterozygotes), they were surprised to discover the mice were born healthy and free of any evident developmental
defects in the kidney or nervous system. They found no trace of GFRб1 mRNA in non-Ret-expressing cells in these mice (which they named Cis-only
mice, for their lack of trans signaling), while GFRб1 transcripts were detected as expected in RET-positive cells, proving that the conditional expression
scheme had worked.















Analysis of individual regions known to be susceptible developmental failure on loss of GFRб1 function, such as the kidneys, motor and enteric neurons
and certain parts of the central nervous system during development and following injury, showed that Cis-only mice develop and regenerate structures
that are both morphologically normal and fully functional.


Investigating the second question of a possible alternate RET-independent GDNF receptor complex thought to involve neural cell adhesion molecules,
they next examined Cis-only mouse olfactory bulbs. These bulbs are reduced in size in NCAM-deficient mice as the result of impaired migration of neural
precursors through a zone called the rostral migratory stream and swell with cells that have failed to reach their normal destination; this phenotype is seen
in mice only weakly lacking GFRб1 (which is thought by some to regulate NCAM-mediated cell adhesion), but not in mice lacking RET. Again, the Cis-only
mice showed no discernible differences from wild type.


This comprehensive series of experiments makes a convincing case against any essential physiological role for RET-independent GFRб1, but leaves the
question of why GFRб1 would be more widely expressed if it indeed plays no role without RET. It may be the case that GFRб receptors associate with
other partners that have yet to be identified. Whatever the answer, by laying to rest a theory that had been strongly supported by in vitro evidence, the
Enomoto report serves to underscore the importance of differences between the behavior of cells in the body and cells in a dish.


Contact: Doug Sipp

sippcdb.riken.jp

RIKEN Center for Developmental Biology