1 Departments of Pediatrics and Molecular Biology and Pharmacology, Washington
University School of Medicine, St Louis, MO 63110, USA
2 Departments of Pathology and Internal Medicine, Washington University School
of Medicine, St Louis, MO 63110, USA
3 Departments of Physiology and Medicine, Medical College of Virginia of
Virginia Commonwealth University, Richmond, VA 23298, USA
4 Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for
Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo
650-0047 Japan
* Author for correspondence (e-mail: heuckeroth{at}kids.wustl.edu)
Accepted 10 February 2003
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SUMMARY |
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Key words: GDNF, ENS, Apoptosis, Ret, Neurons, Mouse
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INTRODUCTION |
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In most parts of the central and peripheral nervous system up to twice as
many neurons are produced as will be needed in the mature organism
(Hutchins and Barger, 1998;
Macaya, 1996
;
Oppenheim, 1991
;
Roth and D'Sa, 2001
). Neurons
that extend their projections to the correct target receive target-derived
trophic factors that allow them to survive. Neurons that do not correctly
reach their targets or that do not receive an adequate amount of neurotrophic
factor are removed by programmed cell death (apoptosis). This allows the
number of innervating neurons to be matched to their target size. In addition,
the distance from the neuronal cell body to the innervation target of the
neuron dictates the length of neuronal fibers. For the ENS to function
properly, enteric neuron number and the extent of neuronal fibers must also be
closely controlled. Although neurotrophic factors essential for formation of
the ENS have been identified, the biological processes regulated by these
factors are not yet clear. Also, in contrast to the rest of the nervous
system, the environment at the cell body and the tip of the axon are
remarkably similar for many myenteric neurons. For this reason, it is more
difficult to understand how `target-derived' trophic factors could be used to
determine which enteric neurons survive or the extent of axonal projections
within the ENS.
To better understand the factors that regulate enteric neuron number and
the extent of neuronal fiber projections within the ENS, we have conducted a
detailed analysis of the enteric nervous system in mice with defects in the
Ret signaling system. Ret is a transmembrane tyrosine kinase that is essential
for formation of the ENS (Schuchardt et
al., 1996) and acts as a receptor for the glial cell line-derived
neurotrophic factor (GDNF) family of ligands (GFLs). There are four known GFLs
[GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN)]
(Baloh et al., 2000
) that
activate Ret. Ret activation also requires a glycosylphosphatidylinositol
(GPI)-linked co-receptor (GFR
1,GFR
2, GFR
3 or GFR
4)
that determines Ret ligand specificity. GFR
1 interacts preferentially
with GDNF (Jing et al., 1996
;
Treanor et al., 1996
),
GFR
2 with NRTN (Baloh et al.,
1997
; Jing et al.,
1997
; Sanicola et al.,
1997
; Suvanto et al.,
1997
), GFR
3 with ARTN
(Baloh et al., 1998a
;
Baloh et al., 1998b
;
Worby et al., 1998
) and
GFR
4 with PSPN (Enokido et al.,
1998
; Milbrandt et al.,
1998
). Both GDNF and NRTN are important for ENS morphogenesis and
function. However, despite the comparable ability of GDNF and NRTN to promote
ENS precursor proliferation and axonal extension in vitro
(Chalazonitis et al., 1998a
;
Hearn et al., 1998
;
Heuckeroth et al., 1998
;
Taraviras et al., 1999
), the
phenotype of Gdnf/ and
Nrtn/ mice is very different.
Gdnf/ mice have hypoganglionosis in the
stomach and aganglionosis of the small bowel and colon
(Moore et al., 1996
;
Pichel et al., 1996
;
Sanchez et al., 1996
).
Nrtn/ mice have a normal number of myenteric
neurons, but a reduced neuronal fiber density and abnormal intestinal
contractility (Heuckeroth et al.,
1999
).
To determine whether GDNF and NRTN play distinct or partially redundant
roles in controlling ENS morphogenesis, we have examined the ENS in mice
deficient in both GDNF and NRTN
(Gdnf//Nrtn/).
In addition, we have examined the ENS in mice heterozygous for GDNF
(Gdnf+/), as well as
Gdnf+//Nrtn/,
Ret+/ and Gfra1+/
animals. These studies demonstrate that GDNF and NRTN perform largely
non-redundant functions in the ENS. We have confirmed the recent observation
(Shen et al., 2002) that
Gdnf+/ mice have enteric hypoganglionosis. We have
also demonstrated that this hypoganglionosis occurs because GDNF availability
determines the rate of ENS precursor proliferation. Interestingly, we were
unable to find any significant role for programmed cell death in controlling
enteric neuron number. By contrast, NRTN availability determines
acetylcholinesterase-stained neuronal fiber density in the mature ENS, but
does not influence neuron number. In addition, we demonstrate that striking
changes in intestinal contractility can occur in the absence of dramatic
anatomic changes within the ENS.
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MATERIALS AND METHODS |
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Whole mount preparation
Small bowel and colon were dissected from adult mice and mesentery was
removed. Tissues were incubated one hour in oxygenated Krebs-Ringer solution
(Sigma K4002) with nicardipine (2 mM) to relax muscles. Three (4 cm) pieces of
proximal small bowel and two (2 cm) pieces of distal colon were analyzed. The
most distal colon and two most proximal small bowel segments were
acetylcholinesterase stained (Enomoto et
al., 1998; Heuckeroth et al.,
1999
). Other segments were Cuprolinic Blue stained
(Karaosmanoglu et al.,
1996
).
PGP9.5/BrdU double labeling
E12 pregnant females were injected intraperitoneally with bromodeoxyuridine
(BrdU) (50 µg/g body weight). Embryos were harvested 3 hours later, fixed
(4% paraformaldehyde, 3 hours, room temperature) and paraffin wax embedded.
Sections (6 µm) were cut and processed as described
(Enomoto et al., 1998).
Sections were incubated with PGP9.5 rabbit antibody (Biogenesis, 1:200)
overnight, 4°C. A Cy3 tyramide signal amplification (TSA) kit
(Perkin-Elmer Life Sciences) was used to identify the PGP9.5-expressing cells.
Sections were then rinsed in TBS, denatured in 1 N HCl (45 minutes),
neutralized in 0.1 M sodium tetraborate (10 minutes) and blocked in 10% normal
donkey serum in TBST (1 hour) before incubation with biotinylated mouse
anti-BrdU antibody (Oncogene, undiluted) overnight. BrdU-positive cells were
visualized with a Fluorescein TSA kit. A total of 150 PGP9.5-expressing cells
from three wild-type and three Gdnf+/ E12 embryos
were evaluated for BrdU incorporation.
Single label fluorescent immunohistochemistry
Activated caspase 3 antibody (D175, Cell Signaling Technology, 1:100)
staining was performed on 6 µm paraffin wax-embedded specimens. GFR1
and GFR
2 (BAF560 and BAF429 respectively; R&D Systems, 1:40)
immunohistochemistry was performed on 12 µm fresh frozen sections. Primary
antibodies were placed on sections overnight at 4°C. Signals were detected
using either a Cy3 or Fluorescein TSA kit.
Myenteric plexus fiber counts
Myenteric fiber counts were performed on acetylcholinesterase-stained
wholemounts by counting fibers crossing a 0.12 mm2 grid
(Heuckeroth et al., 1999) with
five horizontal and six vertical lines. Twenty randomly selected fields per
mouse from three animals of each genotype were evaluated. Data was analyzed
using SigmaPlot and SigmaStat software (one way analysis of variants).
Cell counts
Cuprolinic Blue stained myenteric and acetylcholinesterase-stained
submucosal neurons were counted to determine the number of neurons within a
0.25 mm2 grid. Cells in 20 randomly selected fields were counted
from three animals of each genotype. `Total neuron numbers' were determined by
multiplying the neurons/unit area by the total area of the small bowel or
colon. Because colon and small bowel sizes were similar in all animals, data
in Figs 2 and
5 are similar to neuronal
density data.
|
|
Functional contractility studies
Contractility and transmitter release were measured in adult mouse small
bowel and colon as previously described
(Heuckeroth et al., 1999).
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RESULTS |
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As the severe loss of enteric neurons in Gdnf/ mice might make it difficult to appreciate a subtle role for NRTN in determining neuron number, we examined the ENS in adult Gdnf+/ and Gdnf+//Nrtn/ mice. Both Gdnf+/ and Gdnf+//Nrtn/ mice appeared healthy. We did not observe animals with intestinal distension or evidence of obstruction; however, the ENS appeared hypoganglionic in Gdnf+/ mice (Fig. 1). To establish more definitively whether Gdnf+/ mice have an abnormal number of enteric neurons, we performed quantitative analysis of neuron number in the myenteric and submucosal plexus of the adult gut (Fig. 2A). To determine whether the distal bowel was more significantly affected than the proximal bowel, as it is in Hirschsprung's disease, enteric neuron numbers were determined separately for proximal duodenum and distal colon. This analysis demonstrated that Gdnf heterozygotes have 43% fewer small bowel and 48% fewer colonic myenteric neurons than wild-type littermates (Fig. 2A, P<0.001). Gdnf+/ mice also have fewer submucosal neurons, with a 33% reduction in the small bowel (P=0.028) and a 32% reduction in the colon (P=0.08). Because of variability between animals, the reduced number of colon submucosal neurons does not reach statistical significance. Nonetheless, Gdnf haploinsufficiency clearly causes significant reductions in enteric neuron number.
|
Neurturin availability determines cell size and neuronal fiber
density for mature enteric neurons
Our previous analysis demonstrated that
Nrtn/ mice have smaller myenteric neurons
than wild-type animals and a reduced neuronal fiber density in the small bowel
(Heuckeroth et al., 1999). To
establish whether NRTN provides equivalent trophic support to small bowel and
colonic myenteric neurons, ENS neuronal cell size was determined in both
regions of the bowel (Fig. 2B).
Myenteric neuron size was reduced by 29% in the small bowel and 33% in the
colon of Nrtn/ mice (P<0.001).
In addition, the density of acetylcholinesterase-stained neuronal fibers in
the myenteric plexus is reduced by 32% in the small bowel
(P<0.001) and 22% in the colon (P<0.001) of
Nrtn/ mice
(Fig. 2C). Thus, NRTN provides
equivalent trophic support to small bowel and colonic myenteric neurons. The
effect of NRTN on submucosal neuron size is more subtle
(Fig. 2B). Although cell size
is normal in both Nrtn/ and
Gdnf+/ mice,
Gdnf+//Nrtn/
animals have smaller than normal submucosal neurons in both the small bowel
(14%, P<0.001) and in the colon (17%, P<0.001). This
suggests that both GDNF and NRTN may provide trophic support for mature
submucosal neurons.
To establish more definitively whether GDNF provides trophic support to mature myenteric neurons, we determined acetylcholinesterase-stained myenteric plexus fiber counts in the small bowel and colon of Gdnf+/ and Nrtn//Gdnf+/ mice. If GDNF were important for determining neuronal fiber outgrowth from myenteric neurons, then Gdnf+/ mice would be expected to have fewer neuronal fibers than wild-type littermates. Surprisingly, acetylcholinesterase-stained myenteric plexus fiber counts in both the colon and small bowel of Gdnf+/ mice are normal, despite the 43-48% loss of myenteric neurons (Fig. 2C). The limited role for GDNF in determining the density of acetylcholinesterase-stained fibers within the myenteric plexus is also supported by the observation that neuronal fiber density is comparably reduced in Nrtn/ and Gdnf+//Nrtn/ mice (Fig. 2C). Although we cannot exclude the possibility that the normal density of acetylcholinesterase-stained fibers in the ENS of Gdnf+/ mice results in part from increased extrinsic innervation, these studies demonstrate that the acetylcholinesterase-stained fiber density in the ENS is critically dependent on the availability of NRTN and not GDNF.
Heterozygosity for GFR1 or Ret causes only mild changes in ENS
anatomy
Because Hirschsprung's disease in humans is frequently caused by
haploinsufficiency for Ret (Edery et al.,
1994; Romeo et al.,
1994
), we examined the ENS in Ret+/ and
Gfra1+/ mice. Remarkably, even with detailed
quantitative analysis, myenteric (P>0.8) and submucosal
(P>0.29) neuron numbers in the small bowel and colon of
Gfra1+/ and Ret+/ mice
(Fig. 2A) were identical to
wild-type animals. Thus, in contrast to the results with GDNF,
haploinsufficiency for Ret or GFR
1 does not appear to affect ENS neuron
number in mice.
To establish whether Ret or GFR1 heterozygosity influences mature
enteric neurons, both cell size and fiber densities were determined for
Ret+/ and Gfra1+/ mice
(Fig. 2B,C). In the myenteric
plexus there were significant reductions in cell size in the small bowel (35%;
P<0.001), and colon (34%; P<0.001) of
Ret+/ mice. Ret+/ mice
also had 16% smaller colon submucosal neurons (P<0.001), but cell
size was normal for small bowel submucosal neurons. These changes in neuronal
cell size were similar to the reductions seen in
Nrtn/ mice. Myenteric neuron
acetylcholinesterase-stained fiber counts were also reduced (11-13%,
P<0.001) in Ret+/ mice, but not to the
extent seen in Nrtn/ animals
(Fig. 2C).
Because GFR1 is the preferred GDNF co-receptor and GDNF is not
important for determining ENS neuronal cell size or
acetylcholinesterase-stained fiber density, we expected these parameters to be
normal in Gfra1+/ mice. However, neuronal cell size
was reduced in colon submucosal neurons (19%; P<0.001), and in
colonic (20% decrease; P<0.001) and small bowel (31% decrease;
P<0.001) myenteric neurons
(Fig. 2B) compared with
wild-type littermates. Gfra1+/ mice also had 11%
fewer small bowel (P=0.002) and a 12% fewer colonic
(P<0.001) acetylcholinesterase-stained myenteric plexus fibers
(Fig. 2C). These findings raise
the possibility that GFR
1 and Ret heterozygotes may have abnormal
intestinal motility, despite normal enteric neuron numbers, and imply that
GFR
1/Ret signaling is important for the function of some mature enteric
neurons.
GFR1 and GFR
2 expression patterns in the ENS reflect
the function of these receptors
During development, ENS precursors actively divide within the intestine to
produce enough neurons to populate the gut. Although GDNF and NRTN have
similar effects on ENS precursors in culture
(Heuckeroth et al., 1998),
Gdnf/ and
Nrtn/ mice have strikingly different ENS
phenotypes. These differences would be best explained if GFR
1 was
expressed at high levels early in development, while ENS precursors are
proliferating and GFR
2 expressed at high levels in the mature gut. To
test this hypothesis, immunohistochemical staining for GFR
1 and
GFR
2 was performed on the gut of wild-type E14, P0, P7 and adult mice.
At E14, GFR
1 is easily seen within the gut wall in a region broader
than that occupied by the neural crest
(Fig. 3A). This suggests that
GFR
1 protein is present on both neural crest and mesenchymal components
of the gut wall. The location and number of the neural crest cells within the
gut is indicated by PGP9.5 staining (Fig.
3B). By contrast, GFR
2 expression was not detected at E14
(FITC staining, Fig. 3B). At
P0, GFR
1 and GFR
2 are both visible within the ENS
(Fig. 3C,D), but GFR
1
staining is less intense than at E14. GFR
1 is also more tightly
associated with the developing ENS than at E14. Staining at P7 is similar to
P0. In the adult mouse, GFR
2 continues to be strongly expressed
(Fig. 3F) in the ENS.
GFR
1 is also seen within myenteric and submucosal ganglia, but cells
are less intensely stained than at E14
(Fig. 3E).
|
|
GDNF heterozygotes have reduced ENS precursor proliferation in
vivo
Because we could not find evidence of apoptosis in the ENS, we performed
BrdU/PGP9.5 double label immunohistochemistry to determine whether reduced ENS
precursor proliferation could be responsible for hypoganglionosis in
Gdnf+/ mice. PGP9.5 is expressed in the developing
and mature enteric neural crest
(Sidebotham et al., 2001), and
BrdU is incorporated into proliferating cells. In wild-type embryos, 14% of
PGP9.5-expressing cells were BrdU labeled
(Fig. 6).
Gdnf+/ embryos had 43% fewer
BrdU+/PGP9.5+ cells (P=0.01). These findings
suggest that the amount of GDNF available to proliferating ENS precursors
critically determines the proliferative capacity of these cells and that the
extent of ENS precursor proliferation ultimately determines the number of
enteric neurons in the adult gut. Thus, in contrast to the rest of the nervous
system, cell number in the adult ENS appears to be largely determined by cell
proliferation rather than cell death.
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DISCUSSION |
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Programmed cell death and the ENS
In most parts of the central and peripheral nervous system, neurons are
created by active proliferation of neuronal precursors. As in the ENS, these
precursors often migrate during development to reach their final destination.
The neurons then differentiate, stop migrating and extend neuronal processes
toward their targets. The innervation targets secrete trophic factors that
innervating neurons depend on for survival. Many neurons are particularly
sensitive to trophic factor deprivation at the time of target innervation
(Bennet et al., 2002;
Ernfors, 2001
;
Giehl, 2001
;
Korsching, 1993
). Neurons that
fail to extend processes to their targets, or to compete for an adequate
amount of trophic factor are eliminated by programmed cell death. This
paradigm for developmentally regulated apoptosis is widespread in the
peripheral and central nervous system. In fact, in most parts of the nervous
system, 20-80% of all neurons produced during embryogenesis die before
adulthood (Oppenheim, 1991
).
This process is thought to provide an efficient means for matching neuronal
populations to the size of the innervation targets and for ensuring that
neuronal processes are correctly targeted.
Although apoptosis in the nervous system is a common way of determining the
final number of neurons, there are some neuronal populations that do not
appear to undergo naturally occurring cell loss. This includes neurons in the
pontine nuclei (Armstrong and Clarke,
1979), red nucleus, locus ceruleus
(Oppenheim, 1981
) and chick
spinal cord interneurons (McKay and
Oppenheim, 1991
). Similarly, we have been unable to find evidence
of naturally occurring cell death in the ENS using activated caspase 3
staining, a method specific for detecting cells undergoing apoptosis
(Srinivasan et al., 1998
).
To look for additional evidence that apoptosis is important for determining
enteric neuron number, we analyzed the ENS in Bax- and Bid-deficient mice. For
most parts of the nervous system, programmed cell death depends on the
presence Bax (White et al.,
1998). Bax deficiency in mice causes increased neuronal cell
numbers and virtually eliminates apoptosis in spinal cord motor neurons,
trigeminal ganglia, trigeminal brain stem nuclear complex, facial nucleus,
DRG, sympathetic ganglia, cochleovestibular ganglia, cerebellum, post-natal
retinal ganglion cells and in the hippocampus. The failure of Bax deficiency
to alter enteric neuron number, even in the setting of GDNF
haploinsufficiency, is therefore consistent with the idea that cell death is
not a major determinant of neuronal cell number in the ENS. In some regions of
the nervous system, however, cell death is Bax independent (e.g. retina from
E11.5 to P1) (White et al.,
1998
), and it remains possible that other Bcl2 family members
regulate cell death within the ENS. One potential candidate for regulating
neuronal cell death in the ENS was Bid. Although Bid deficiency is not
important for programmed cell death in most parts of the nervous system
(Leonard et al., 2001
), Bid
has been implicated in neuronal apoptosis in the setting of nerve injury
(Henshall et al., 2001
;
Plesnila et al., 2001
).
However, Bid deficiency also does not alter enteric neuron number.
Trophic factors and the ENS
These findings do not imply that enteric neurons or ENS precursors are
trophic factor independent. In fact, the hypoganglionosis in
Gdnf+/ mice and the reduction in neuronal cell size
and acetylcholinesterase-stained myenteric fiber density in
Nrtn/ mice provides strong evidence that
mature enteric neurons and their precursors are trophic factor dependent. The
BrdU labeling studies, however, are consistent with the idea that enteric
neuron number is determined by controlled cell proliferation rather than by
apoptosis in wild-type mice. We do not propose that ENS precursors are
resistant to apoptosis, but rather that apoptosis is not important for
determining neuron number during normal ENS development. Indeed, cell death of
ENS precursors is seen in the esophagus of
Ret/
(Taraviras et al., 1999) and
Phox2b/ mice
(Pattyn et al., 1999
), and in
the mesenchyme surrounding the neural tube in
Sox10Dom/Sox10Dom mice
(Kapur, 1999
;
Southard-Smith et al., 1998
).
In addition, adult guinea pig enteric neurons undergo apoptosis in response to
glutamate (Kirchgessner et al.,
1997
), and trkC-expressing rat ENS precursors undergo apoptosis in
response to NT3 withdrawal (Chalazonitis
et al., 2001
). Thus, despite demonstrable trophic factor
dependence, the absence of identifiable cell death in wild-type mice suggests
that during normal development ENS precursors receive an adequate amount of
trophic factor for survival.
Trophic factor dependence changes during ENS development
Like many other neuronal populations, enteric neurons and their precursors
appear to switch trophic factor dependence during development. In the early
stages of cell migration and proliferation, GDNF activation of Ret via
GFR1 is absolutely required for both the survival and proliferation of
all ENS precursors in the small bowel and colon. As development proceeds, many
enteric neurons become dependent on neurturin and GFR
2 for trophic
support. This is demonstrated by the reduced size of myenteric neurons within
the ENS of adult Nrtn/ mice and by the loss
of acetylcholinesterase-stained fibers in these animals. The switch in ENS
trophic factor dependence from GDNF to NRTN is similar to the change in
trophic factor dependence that occurs in the parasympathetic nervous system
(Enomoto et al., 2000
) where
GDNF is required early for proliferation and migration of neuronal precursors,
but NRTN is essential for maintenance of neuronal projections in the mature
animal. Because of the complexity of the ENS, however, it seems likely that
subpopulations of enteric neurons will be supported by distinct neurotrophic
factors and neuropoeitic cytokines. This hypothesis is supported by the
observation that Gfra2/ mice have normal
appearing NADPH diaphorase-stained neuronal fibers
(Rossi et al., 1998
) and, at
least in the rat, NADPH diaphorase and acetylcholinesterase stain largely
non-overlapping populations of neurons
(Aimi et al., 1993
). The
dependence of enteric neuron subsets on other trophic factors is also
supported by the observation that both Nt3/
and Ntrk3/ (trkC) mice have a reduced number
of enteric neurons and that only a subset of enteric neurons retrogradely
transport NT3 (Chalazonitis et al.,
2001
). Similarly, CNTF receptor
and LIF receptor ß
are expressed on enteric neuron subsets, and activation of these receptors
promotes the development of NADPH diaphorase-expressing neurons
(Chalazonitis et al., 1998b
).
Thus, as in the DRG (Ernfors,
2001
; Rifkin et al.,
2000
; Snider and
Silos-Santiago, 1996
), different functional classes of enteric
neurons are likely to be supported by different trophic factors.
Heterozygosity for Ret signaling components results in a range of
abnormalities in the ENS structure and function
Gene dosage effects, such as the dramatic reduction in enteric neuron
number seen in Gdnf+/ mice, suggest that GDNF is
produced in limiting quantities during murine ENS development. This is
consistent with the idea that the quantity of neurotrophic factor available
often limits neuronal survival or proliferation. The normal cell numbers in
Ret+/ and Gfra1+/ mice
implies that while ENS precursors are proliferating, wild-type mice have a
significant excess of Ret and GFR1 and thus ENS precursors
proliferation is not affected by reduced Ret or GFR
1 gene dosages. The
reductions in neuron size and acetylcholinesterase-stained fiber counts
observed in adult Ret+/ and
Gfra1+/ mice imply more limited production of these
signaling components in the mature mouse ENS. This is consistent with the
reduced intensity of GFR
1 immunohistochemical staining in the adult
mouse ENS compared with the level seen at E14. Interestingly, Ret mutations
that cause Hirschsprung's disease (distal intestinal aganglionosis) in humans
are typically inactivating mutations that are penetrant in the heterozygous
state. This implies that human ENS precursors have more limiting Ret
expression than the corresponding murine cells. Recent data also suggest that
in human populations the penetrance of Ret mutations may depend on the
presence of second site modifiers that could influence the level of Ret
expression (Gabriel et al.,
2002
).
Enteric neuron number is controlled by regulating proliferation
instead of cell death
Mechanisms that control many aspects of enteric neural crest patterning are
similar to those used in other parts of the peripheral nervous system. The
reliance on controlled cell proliferation in the ENS rather than programmed
cell death to determine neuron number, however, may derive from anatomic
differences between the ENS and other parts of the nervous system. For
example, the distance from the DRG cell body to its innervation target
determines neuronal fiber length for DRG neurons. Cells with inadequate
dendritic extension to reach their targets are eliminated by apoptosis.
Similarly, neurons whose processes extend to `incorrect' targets do not
receive the right type of neurotrophic factor and are also eliminated by
apoptosis. For many neurons within the ENS, these mechanisms cannot apply.
Several types of myenteric neuron, for example, extend their processes within
the muscular layer of the gut (Costa and
Brookes, 1994; Furness,
2000
). For these neurons, the environment at the tip of the axon
is similar to the environment near the cell body. If the only requirement for
these axons is that the fibers reach a source of neurturin, this could be met
within the tissues directly adjacent to the neuronal cell body eliminating the
need for the axon to extend any significant distance. As an alternative
strategy, it appears that the amount of neurturin available to each neuron
determines the extent of acetylcholinesterase-stained fibers. This would
explain why Nrtn/ mice have a reduction in
acetylcholinesterase-stained fibers while Gdnf+/
mice have an increased ratio of neuronal fibers to neuronal cell number, but a
normal total number of fibers. Using apoptosis to eliminate neurons within the
ENS with `incorrectly' targeted axons would also be difficult for many
myenteric neurons because within the myenteric plexus, the environment is
similar in all directions. Instead, it appears that ENS cell number is largely
determined by the ability of GDNF to control cell proliferation. Thus,
although GDNF and neurturin availability, respectively, control neuronal cell
number and neuronal fiber density within the ENS, mechanisms that control axon
targeting within the ENS will need further investigation.
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ACKNOWLEDGMENTS |
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