1 Department of Pathology and Immunology, Washington University School of
Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
2 Department of Urologic Surgery, Washington University School of Medicine, 660
South Euclid Avenue, St Louis, MO 63110, USA
3 Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
4 Department of Pediatrics, Washington University School of Medicine, 660 South
Euclid Avenue, St Louis, MO 63110, USA
* Author for correspondence (e-mail: jeff{at}pathbox.wustl.edu)
Accepted 25 August 2004
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SUMMARY |
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Key words: Ret, GDNF, Hirschsprung disease, Spermatogenesis
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Introduction |
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Numerous studies have demonstrated that Ret activation is essential for the
proper embryologic development of the kidneys, and the autonomic and enteric
nervous systems (ENS) (Enomoto et al.,
2001; Enomoto et al.,
2000
; Schuchardt et al.,
1994
). From the analyses of mice deficient in neurturin (NRTN) or
GFR
2, it has been suggested that Ret is also important for the
postnatal maintenance of the enteric and parasympathetic nervous systems,
while a role in spermatogenesis has been suggested following the analysis of
mice that overexpress GDNF in the testes
(Gianino et al., 2003
;
Heuckeroth et al., 1999
;
Meng et al., 2000
;
Rossi et al., 1999
).
Unfortunately, the perinatal lethality of Ret-null animals has made it
impossible to study the postnatal roles of Ret, and to develop animal models
that mimic Ret-related human diseases. This has hindered the molecular
understanding of these diseases, and the subsequent development and testing of
novel treatment strategies.
Ret signaling is mediated by the binding of the GFLs [GDNF, neurturin
(NRTN), artemin (ARTN) and persephin (PSPN)] with their cognate
glycophosphatidylinositol (GPI)-anchored GFR1-4 co-receptor, and their
subsequent interaction with the Ret extracellular domain
(Baloh et al., 2000
). These
interactions result in functional GFL-GFR
-Ret complexes and the
autophosphorylation of key Ret tyrosine residues that harbor consensus
sequences for adaptor proteins. Recruitment of these adaptor proteins to
phosphorylated Ret tyrosine residues activates multiple signaling pathways,
including the mitogen-activated protein kinase (MAPK) and the phosphoinositide
3-kinase (PI3K)-AKT pathways that regulate cell survival, proliferation,
migration and axonal outgrowth. The perturbation of these key processes leads
to the various abnormalities observed in Ret-null mice
(Airaksinen and Saarma, 2002
),
but the specific regions of Ret that regulate these cellular processes, and
are associated with the proper development and function of the nervous and
urinary systems, are unknown.
To better understand the roles of the key signaling domains of Ret in
mediating its diverse biological effects, we generated a mouse expressing a
Ret protein (RetDN) with mutations in the Ret cytoplasmic tail. Mice harboring
one copy of the RetDN allele (RetDN/+) exhibit
long-segment intestinal aganglionosis reminiscent of human patients with HSCR,
where aganglionosis due to RET mutations manifests in the
heterozygous state, and is typically restricted to the colon and/or distal
small intestine. The intestinal phenotype is distinct from previous Ret
loss-of-function murine models where the loss of one allele does not cause
significant morphologic abnormalities of the ENS
(Gianino et al., 2003),
whereas Ret-null homozygotes show a complete loss of ganglion cells in the
entire intestine (Schuchardt et al.,
1994
). These studies also highlight the differing requirements for
Ret signaling in the peripheral nervous system, as deficits are found in both
branches of the autonomic nervous system in Ret-null mice, but
RetDN/+ mice have defects only in the parasympathetic
nervous system. The postnatal survival of the RetDN/+ mice
allowed us to clearly demonstrate the postnatal importance of Ret signaling
for the maintenance of the ENS and parasympathetic nervous systems, and the
requirement for Ret during the first wave of spermatogenesis.
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Materials and methods |
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Histopathological analysis and immunohistochemistry
Mouse tissues were processed for histological analysis as previously
described (Enomoto et al.,
2000). Catecholaminergic neurons were visualized with
diaminobenzidine by means of tyrosine hydroxylase (TH) immunohistochemistry
(rabbit polyclonal antibody, Chemicon, 1:200) in whole-mount (for sympathetic
neurons) or in 60-µm unmounted floating sections (for midbrain dopaminergic
neurons) (Enomoto et al.,
2001
). The innervation of intraorbital harderian glands was
examined with PGP9.5 (rabbit polyclonal antibody, Biogenesis, 1:400) and
Alexa488-conjugated anti-rabbit immunoglobulin secondary antibody (1:250). The
small nociceptive DRG sensory neurons were visualized with P2X3
(guinea pig polyclonal antibody, Chemicon, 1:800) and Cy3-conjugated
antiguinea pig secondary antibody (1:400). Germ cells were identified with
germ-cell nuclear antigen (GCNA) immunohistochemistry (rabbit polyclonal
antibody, 1:500; gift from G. Enders, University of Kansas Medical Center,
Kansas, USA). In vivo 5-bromo-2-deoxyuridine (BrdU) labeling (200 mg/kg,
Sigma) and anti-BrdU immunohistochemistry (1:200) were used for proliferation
studies (Enomoto et al.,
2001
). Cell death was analyzed using TUNEL assay (Boehringer
Mannheim) (Enomoto et al.,
2000
). Quantitative analyses were performed with SigmaPlot
software (SPSS), and statistical significance was determined by Student's
t-test. For all studies, the sample size was three or more for each
genotype, unless otherwise stated.
Enteric neuron studies
Intestinal aganglionosis was evaluated by whole-mount acetylcholinesterase
(AChE) staining on dissected intestines of newborn to 3-week-old mice
(Enomoto et al., 1998).
Quantitative analysis of neuron number and myenteric plexus fibers was
performed as described previously (Gianino
et al., 2003
). The myenteric and submucosal neuron numbers were
determined using 20 randomly selected Cuprolinic Blue-stained intestine
regions and 10 randomly selected AChE-stained intestine regions, respectively
(0.25 mm2/counting grid areax10-20 areas/mousex3 mice
of each genotype). Myenteric plexus fiber density was determined from 10
randomly selected fields/mouse (30 regions per genotype), by counting the
number of fibers crossing the left and top edge of a 0.25-mm2
grid.
Parasympathetic neuron studies
The number of sphenopalatine ganglion neurons was determined from
thionin-stained, paraffin-embedded coronal head sections (6 µm) of newborn
mice. The entire ganglion was sectioned, and neurons with distinct nucleoli
were counted at 60-µm intervals (at least 3 animals, 6 ganglia, from each
genotype) (Enomoto et al.,
2000). Cell size analysis was performed on dissected postnatal
sphenopalatine ganglia (P21). Thirty neurons were used from each animal for
area determination with image analysis software
(http://www.chemie.unimarburg.de/~becker/image.html).
Spinal motor neurons cell counts
Spinal cords from 3-week-old animals were paraffin embedded, sectioned (12
µm), stained with thionin, and the number of motor neurons in the entire
lumbar enlargement, L1-L6 (counted at 120-µm intervals) was determined.
Cells with large cell bodies, granular cytoplasm and prominent nucleoli were
counted (Clarke and Oppenheim,
1995).
Analysis of the genitourinary system
To determine relative nephron number, we serially sectioned newborn kidneys
in their entirety and counted glomeruli every 120 µm
(Majumdar et al., 2003). For
spermatogenesis studies, testes were harvested at the indicated time points
for either histological or ploidy studies. For quantification, seminiferous
tubules appearing in cross section were used. Germ cell number (GCNA
immunohistochemistry), apoptosis (TUNEL-assay positive), or proliferation
(BrdU incorporation) in RetDN/+ and wild-type mice were
determined from 100 random seminiferous tubules of each animal.
Flow cytometry
For ploidy studies, testes were decapsulated and triturated into a single
cell suspension in Hank's balanced salt solution that contained propidium
iodide (50 µg/ml), citric acid (1 mg/ml), and Nonidet P40 (0.3%) for 30
minutes. Ten thousand cells were analyzed for their DNA content on a FACScan
(Becton Dickinson) with FlowJo software (Tree Star, Version 4.3).
In vitro experiments
The human Ret cDNAs used for these studies include Ret9,
Ret9(Y1062F), Ret9(K758M), Ret9-FLAG, RetDN-HA and Ret9(L985P, Y1062F), also
called RetDN, and either have been previously described
(Tsui-Pierchala et al., 2002)
or were generated in the pcDNA3.1 vector (Invitrogen) using standard methods.
Lentiviruses that harbor the different Ret cDNAs in pFCIV-1
(Crowder et al., 2004
) were
prepared by standard methods (Lois et al.,
2002
; Naldini et al.,
1996
). Cell populations expressing equivalent levels of each of
the Ret proteins in the infected CHP126, 293T or Neuro2A
1 cell lines
were obtained as described (Crowder et al.,
2004
). For ligand stimulation, cells were grown in low serum
(0.5%) for 4 hours and then exposed to GDNF (25 ng/ml) for 10 minutes before
harvest.
Western blot and immunoprecipitation of cell extracts from cell lines or
primary superior cervical ganglion (SCG) cultures were performed as described
previously (Tsui-Pierchala et al.,
2002). The primary antibodies (1:1000) were as follows: rabbit
anti-panRet and anti-pY1062
(Tsui-Pierchala et al., 2002
),
rabbit anti-AKT and anti-pAKT (Cell Signaling Technologies), rabbit anti-pMAPK
(New England Biolabs), rabbit anti-pY20 (BD Biosciences), and rabbit anti-FLAG
and rabbit anti-Actin (Sigma, St Louis). The secondary peroxidase-conjugated
anti-rabbit antibodies (Jackson Immuno) were used at 1:10,000. Signals were
detected with chemiluminescence with the Super Signal West Dura kit (Pierce)
on an EPI CHEM II Darkroom instrument (UVP). The normalization and analysis
for ligand-dependent phosphorylation of AKT was performed using LabWorks Image
Acquisition and Analysis software (UVP). Phospho-AKT levels were normalized to
the amount of total AKT in the cells.
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Results |
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The mutant RetDN allele was inactive when the neomycin resistance
cassette was present, but was activated when this cassette was excised by
mating with ß-actin Cre animals. Interestingly, all of the F1 hemizygous
RetDN mice produced in this manner displayed growth retardation and
died by 4 weeks of age (Fig.
1D,E). This was surprising as one wild-type mouse Ret
allele (Ret-/+ or Ret TGM/+) is
sufficient for viability, fertility, and proper development of all known
Ret-dependent tissues (Enomoto et al.,
2001; Gianino et al.,
2003
; Schuchardt et al.,
1994
). Furthermore, hemizygous mice expressing wild-type human
RET9 (Ret9/+) were viable and fertile with no
developmental defects (Fig.
1B,C; data not shown), confirming that expression of the mutant
RetDN allele is responsible for the observed phenotype. Because of
the dominant effect of this Ret mutant, we refer to this protein as RetDN and
to mice harboring this mutant allele as RetDN/+. We
detected mRNAs specific to the recombined Ret9 or RetDN
alleles by RT-PCR at several sites of Ret expression (e.g. brain, spinal cord
and eyes; Fig. 1F; data not
shown). The early lethality of RetDN/+ mice made the
propagation of this line difficult. However, we were able to maintain this
line by taking advantage of the diminished expression of the mutant
RetDN allele caused by retention of the neomycin resistance cassette
(RetDNneo/+) (Fig.
1B,C,F) (Barrow and Capecchi,
1996
; Rijli et al.,
1994
; Schuchardt et al.,
1994
). The RetDNneo/+ mice had no overt
abnormalities (see below, and data not shown).
Long-segment distal intestinal aganglionosis in RetDN/+ mice
RetDN/+ mice developed increasing abdominal distension
with age. Because Ret null mice have extensive intestinal
aganglionosis (i.e. no enteric neurons in the small bowel or colon) and
patients with heterozygous-inactivating RET mutations have HSCR, we
postulated that failure to thrive in RetDN/+ mice could
result from defective ENS development. Indeed, gross examination of the
gastrointestinal system showed narrowing of the distal bowel, preceded by
dilation of the proximal colon or ileum typical of gut morphology in patients
with HSCR (Fig. 2A).
|
To determine whether the ENS anomalies observed in
RetDN/+ mice were restricted to the distal bowel, we also
performed quantitative analysis of the ENS on whole-mount preparations of the
proximal duodenum in wild-type and mutant mice. Although the proximal bowel
has generally been assumed (with little data) to be `normal' in patients with
HSCR, RetDN/+ mice had a remarkable reduction in both
neuron number and neuronal fiber density in the proximal small bowel
(Fig. 2B,D). These reductions
are reminiscent of ENS deficits that appear in GDNF (decreased neuron number)
or NRTN (decreased fiber density) deficient mice, indicating that
RetDN/+ mice have a combination of deficits resulting from
an overall reduction in GFL-mediated Ret activation
(Gianino et al., 2003;
Heuckeroth et al., 1999
;
Rossi et al., 1999
).
Defects in the parasympathetic, but not sympathetic, nervous system in RetDN/+ mice
Among the parasympathetic cranial ganglia, the sphenopalatine ganglia (SPG)
are the most severely affected in GDNF- and Ret-null mice.
In these mutant mice, the SPG have a marked reduction or absence of neurons
that can be primarily attributed to a reduction in neuronal precursor
proliferation (Enomoto et al.,
2000). We observed a 50% decrease in neuron number in newborn
RetDN/+ mice, when compared with controls (wild type,
Ret9/+, RetTGM/+)
(Fig. 3A). While GDNF-mediated
Ret activation is crucial for formation of SPG neurons, NRTN and GFR
2
are necessary for SPG trophic maintenance and innervation of the intraorbital
harderian glands (Heuckeroth et al.,
1999
; Rossi et al.,
1999
). The postnatal survival of RetDN/+ mice
allowed us to determine whether the SPG neurons that were generated are
affected by the decreased Ret activity. We found that
RetDN/+ SPG neurons are smaller in size than those of
wild-type littermates at 3 weeks of age
(Fig. 3A). Additionally, PGP9.5
immunostaining revealed abnormal innervation of the harderian glands by
RetDN/+ SPG neurons
(Fig. 3A). Thus,
RetDN/+ mice have deficits in SPG neuron number, size and
target innervation that are a combination of the defects observed in
GDNF- and NRTN-null mice, supporting the hypothesis that
persistent Ret signaling is required for both the formation and proper
function of SPG neurons.
|
Normal development of DRG sensory, spinal motor and midbrain dopaminergic neurons in RetDN/+ mice
GFL signaling has been implicated in the survival of midbrain dopaminergic,
spinal motor and DRG sensory neurons in cell-culture and injury models
(Beck et al., 1995;
Bennett et al., 1998
;
Bowenkamp et al., 1995
;
Cacalano et al., 1998
;
Henderson et al., 1994
;
Lin et al., 1993
;
Molliver et al., 1997
;
Moore et al., 1996
;
Oppenheim et al., 1995
;
Oppenheim et al., 2000
;
Sanchez et al., 1996
). Because
these neurons continue to mature postnatally, and Ret-null mice die at birth,
the importance of Ret activity for their proper postnatal function is unknown.
We examined these neuronal populations in 3-week-old
RetDN/+ mice, and found that they were morphologically
normal. Thus, these neurons, unlike enteric and parasympathetic neurons, do
not appear to be overtly sensitive to the diminished Ret activity caused by
the RetDN allele (see Fig. S1 in supplementary material).
Abnormal renal development in RetDN/+ mice
Mice with altered GDNF-GFR1-Ret signaling have renal deficits with
variable expressivity that range from cortical cysts to renal agenesis
(de Graaff et al., 2001
;
Enomoto et al., 1998
;
Enomoto et al., 2001
;
Pichel et al., 1996
;
Sanchez et al., 1996
;
Schuchardt et al., 1996
).
These observations suggest that, as in other GDNF-dependent systems, renal
development is sensitive to the level of effective GDNF signaling. The
RetDN/+ kidneys were also variably affected, as animals
were born either with both kidneys (57%), or with bilateral (18%) or
unilateral renal agenesis (25%) (Fig.
4A). All kidneys in RetDN/+ mice were
hypoplastic, with an approximate 50% decrease in glomeruli compared with that
of their wild-type littermates (Fig.
4A). Unlike the distorted architecture of kidney rudiments
observed in the few Ret-null mice that do not have complete renal agenesis,
the hypoplastic RetDN/+ kidneys had histologically normal
organization of the cortex and medulla at birth (n>9, data not
shown). Among the animals surviving perinatal lethality, 70% (29/43) had both
kidneys and 30% (14/43) had unilateral renal agenesis. Interestingly, 50% of
RetDN/+ mice (6/12) at 3 to 4 weeks of age had renal
tubular cysts and proteinaceous casts (Fig.
4A). The severity of the renal cystic anomalies ranged from a few
cystic tubules to extensive parenchymal involvement. No renal abnormalities
were noted in control mice (RetDNneo/+, n=5;
RetTGM/+, n=10; Ret9/+, n=15;
Ret+/+, n=20).
|
To further characterize the spermatogenesis defects in
RetDN/+ mice, we first used TUNEL staining to examine
apoptosis. Compared with wild-type mice at 4 weeks of age, the
RetDN/+ seminiferous tubules contained large numbers of
apoptotic cells that were primarily spermatocytes. The increased TUNEL
staining was detected as early as postnatal day (P) 17 in
RetDN/+ mice (Fig.
5B). Because abnormal spermatogenesis, by default, leads to
apoptosis, we examined earlier time points for spermatogenesis defects. We
first used a marker of early germ cells, germ cell nuclear antigen (GCNA), to
determine whether the number of germ cells was decreased in
RetDN/+ mice (Enders
and May, 1994). We found that, in newborn mice, the number of
GCNA-stained germ cells was similar between wild-type and
RetDN/+ testes (Fig.
5C). However, at P10, an age when spermatogonia (2n) typically
differentiate into spermatocytes (4n) and then enter meiosis, the
RetDN/+ testis had a marked decrease in total and
proliferating germ cells per tubule, as assessed by GCNA and BrdU
immunohistochemistry, respectively (Fig.
5C). We also tested whether there were alterations in the
proportion of 2n and 4n germ cells in RetDN/+ testes, as
this would reflect aberrant spermatogonial maturation. Using cell ploidy
analysis, we found that the proportion of 4n cells was always lower in
RetDN/+ testes, resulting in an increased 2n to 4n ratio
at all time points (P10, P12 and P17) examined
(Fig. 5D; data not shown). The
above results suggest that a reduced number of precursors was available for
meiosis, and explains the reduction in meiotic products (round and elongated
spermatids) observed in P28 RetDN/+ mice
(Fig. 4D). Hence, we conclude
that Ret signaling is required in early spermatogenesis (between P0 and P10)
to establish normal germ cell number and spermatogonial maturation.
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Discussion |
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Second, it is puzzling why Ret mutations act dominantly to give rise to
HSCR in humans, but Ret heterozygote mice have normal ENS development. The ENS
defects in RetDN/+ mice suggest that intestinal
aganglionosis is determined by the total amount of Ret signaling. For example,
although an absence of Ret signaling results in complete intestinal
aganglionosis in both humans and mice
(Enomoto et al., 2001;
Schuchardt et al., 1994
;
Shimotake et al., 2001
;
Solari et al., 2003
), reduced
Ret activity appears to be tolerated until a certain threshold is reached.
Further decreases in activity then impact ENS precursor survival,
proliferation and migration, and result in an aganglionic distal bowel. The
aganglionosis in RetDN/+ mice is remarkably similar to
long-segment HSCR caused by RET mutations in human populations, and
makes these animals a useful model for studying HSCR pathobiology. For
instance, we found profound hypoganglionosis in the proximal small bowel of
RetDN/+ mice. Although it is not known whether small
intestine hypoganglionosis occurs in children with HSCR because this bowel
region is generally not evaluated, this finding provides a potential
explanation for the persistent intestinal dysmotility observed in a large
proportion of these patients even after resection of the aganglionic bowel
(Tsuji et al., 1999
).
Third, it is unknown whether the organs affected by Ret deficiency are
differentially susceptible to levels of Ret activity. The surprisingly normal
sympathetic nervous system but abnormally developed enteric and
parasympathetic nervous systems and kidneys in the RetDN/+
mice suggest that the threshold of Ret signaling required for normal
sympathetic nervous system development is different from that of other
Ret-affected tissues. This `sparing' effect may result from the preponderance
of the Ret51 isoform in sympathetic neurons. As the RetDN allele
produces a mutant Ret9 isoform that may not interact with or inhibit Ret51
(Tsui-Pierchala et al., 2002),
residual Ret51 activity in RetDN/+ SCG neurons may be
sufficient for normal sympathetic nervous system development. Alternatively,
the DN mutant could affect both Ret isoforms to some extent, and the remaining
endogenous Ret activity (from both Ret9 and Ret51 isoforms) could be
sufficient to support development of the sympathetic, but not the
parasympathetic, nervous system.
Fourth, the role of Ret in kidney development after ureteric bud induction
and early branching is unknown because Ret-null mice have renal agenesis and
die at birth. The fact that RetDN/+ kidneys are
hypoplastic but show normal renal architecture at birth only to degenerate
over the next few weeks, suggests that Ret activity is required throughout
nephrogenesis to maintain normal renal function. Hypoplastic kidneys due to
decreased Ret signaling are also observed in transgenic mice that express Ret
under the Hox7b promoter, and in Ret51 monoisomorphic mice that do
not express the Ret9 isoform (Davies and
Fisher, 2002; de Graaff et
al., 2001
; Lechner and
Dressler, 1997
; Srinivas et
al., 1999
). Because Ret9/Ret51 heterodimers are rare, and Ret9 is
important for embryological development, the RetDN/+
kidney defects at birth are likely to be due to inhibition of the Ret9 isoform
by the mutant RetDN allele (de
Graaff et al., 2001
;
Tsui-Pierchala et al., 2002
).
Expression studies suggest that the Ret51 isoform may be important in
postnatal renal development and maintenance
(Lee et al., 2002
). Thus, the
postnatal cystic abnormalities in RetDN/+ mice may be due
to decreased Ret9 activity along with Ret51 haploinsufficiency. Interestingly,
40% of patients with renal agenesis harbor dominant RET mutations
predominantly in the tyrosine kinase domain (M. Skinner, personal
communication), thus making the RetDN/+ mice a useful
model of congenital human kidney disease.
Finally, while Gdnf+/ adult mice have abnormal
spermatogenesis (Meng et al.,
2000), the role of Ret activation in testes biology is uncertain
because of the fact that spermatogenesis initiates well after birth. The
survival of RetDN/+ mice beyond P0 allowed us to obtain
definitive evidence that Ret signaling is required for the first wave of
spermatogenesis. The RetDN/+ testes phenotype is much more
severe than that of Gdnf+/ mice, because the first
wave of spermatogenesis is distinctly abnormal, and this process occurs
normally in Gdnf+/ mice. The defect in the first
wave of spermatogenesis is apparent as early as P10, even before the onset of
meiosis, and is consistent with the prepubertal expression of Ret in
spermatogonia (Meng et al.,
2000
). These results suggest that Ret activity in
RetDN/+ testes is even less than that of
Gdnf+/ mice, and correlates well with abnormalities
in the RetDN/+ ENS, the parasympathetic nervous system and
the kidneys, which are also more severe than those observed in
Gdnf+/ mice. Furthermore, it appears that certain
events in spermatogenesis require different levels of Ret activity. For
instance, small reductions in Ret activity in
Gdnf+/ mice cause reduced spermatogonial cell
renewal (Meng et al., 2000
),
whereas more severe reductions in Ret signaling in RetDN/+
animals prevent spermatogonial differentiation and cause apoptosis of
spermatid precursors.
The phenotypic deficits observed in RetDN/+ mice are
mediated by mutations L985P (kinase domain) and Y1062F (Shc docking site) of
the RetDN allele. Mutations in these Ret regions have been described
in patients with HSCR (Geneste et al.,
1999; Iwashita et al.,
2001
) and congenital kidney disease (M. Skinner, personal
communication), and affect MAPK and/or AKT activation. Biochemical analysis
from experiments in cell lines and primary SCG neurons expressing RetDN also
show a reduction in AKT and MAPK activity. The preferential inhibition of AKT
in vitro suggests that the developmental abnormalities observed in
RetDN/+ mice are partly due to decreased signaling via the
Ret(Y1062)-AKT pathway. This is consistent with several studies demonstrating
that GFL-mediated activation of the PI3K-AKT pathway preferentially affects
ENS and kidney development, and is crucial for neural crest precursor
proliferation, survival and migration
(Natarajan et al., 2002
;
Tang et al., 2002
) (R.H.,
unpublished). However, it is possible that other Ret-stimulated signal
transduction pathways, such as PLC
, could also contribute to the
phenotype observed in RetDN/+ mice. In this regard, it has
been shown that mutations near the Ret(L985) in HSCR patients result in
diminished PLC
activity in vitro
(Iwashita et al., 2001
). We
have not directly determined whether the abnormalities in
RetDN/+ mice are due to mutations in the residues L985P or
Y1062F, or both. However, the dominant-negative activity of this Ret mutant is
likely to result from the RetL985P mutation, as the Ret9(Y1062F) mutant only
affects phosphorylation of Ret at Y1062
(Encinas et al., 2004
),
Ret(L985P) alone is kinase deficient (data not shown), and recently generated
mice that are heterozygous for a Ret9(Y1062F) allele
(RetY1062F/+) are viable and fertile (S.J.,
unpublished).
The analysis of RetDN/+ mice has enhanced our understanding of Ret function in postnatal development, provided novel insights into the function of GFL-Ret signaling complexes in vivo, and provided a valuable model to study how deficiencies in Ret signaling result in human diseases such as HSCR, and congenital kidney abnormalities. These mice will also be useful for investigating the importance of GFL-mediated Ret activation in adult animals. In particular, the ability to conditionally activate the RetDN inhibitory allele through Cre recombinase provides a method of assessing its role in maintaining specific neuronal populations, such as those affected in neurodegenerative diseases.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/21/5503/DC1
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