|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 30 November 2005
doi: 10.1242/dev.02189
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Research Group Developmental Neurobiology, Max-Planck-Institute for Brain
Research, Deutschordenstrasse 46, 60528 Frankfurt/M, Germany.
2 Institute of Clinical Neuroanatomy, J.W.-Goethe University, Theodor-Stern-Kai
7, 60590 Frankfurt/M, Germany.
3 INSERM U564, CHU d'Angers, 4 rue Larrey, 49033 Angers Cedex, France.
4 Deptartment of Molecular Biology of the Cell I, German Cancer Research Center,
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Author for correspondence (e-mail:
rohrer{at}mpih-frankfurt.mpg.de)
Accepted 28 October 2005
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Cytokine, IL6/IL-6, Cholinergic, Sympathetic, VIP, VAChT
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Shah et al., 1996;
Varley and Maxwell, 1996;
Schneider et al., 1999). BMPs
induce a group of transcription factors, including MASH1 (ASCL1–Mouse
Genome Informatics), PHOX2A, PHOX2B, HAND2 and GATA2/GATA3, that, in turn,
control the expression of autonomic neuron-specific features (reviewed by
Goridis and Rohrer, 2002).
Subsequently, glial cell line-derived neurotrophic factor (GDNF) family
ligands (GFLs) produced by intermediate and final targets are essential for
sympathetic neuron migration, the development of axon projections and proper
target innervation (Honma et al.,
2002; Enomoto et al.,
2001; Hiltunen and Alraksinen,
2004). Neurotrophins control final stages of target organ
innervation, as well as target-dependent survival of sympathetic neurons
(Glebova and Ginty, 2004;
Francis and Landis, 1999).
Target-derived, retrogradely acting signals not only affect axon outgrowth
and neuron survival but also mediate the acquisition of neuronal traits, such
as the expression of distinct neurotransmitters. The best understood example
for target-dependent control of the neurotransmitter phenotype is the
sympathetic innervation of sweat glands in the footpads of rats and mice
(reviewed by Ernsberger and Rohrer,
1999; Francis and Landis,
1999). Sweat glands are innervated by noradrenergic sympathetic
axons shortly after birth. Because of signals derived from this target,
adrenergic traits such as catecholamine production are downregulated, whereas
the induction of cholinergic features, like choline acetyl transferase (ChAT),
vesicular acetylcholine transporter (VAChT) and the co-expressed neuropeptide
vasoactive intestinal peptide (VIP), leads to a functionally cholinergic sweat
gland innervation. The importance of the target tissue for this transmitter
phenotype switch to occur has been firmly established by sweat gland
transplantation, by replacement by parotid gland
(Schotzinger and Landis, 1990)
and by analysis of the tabby mouse mutant, which is devoid of sweat glands
(Guidry and Landis, 1995).
Cholinergic sympathetic neurons innervate, as additional target tissues, the
skeletal muscle vasculature and the periosteum, the connective tissue covering
the bone. For the periosteum of the sternum and ribs it has been demonstrated
that the initial innervation loses catecholaminergic markers and starts to
express cholinergic properties and VIP
(Asmus et al., 2000). The
sternum can induce cholinergic differentiation of sympathetic neurons both in
vitro and in vivo, upon transplantation to regions of hairy skin
(Asmus et al., 2001;
Asmus et al., 2000).
The interaction between sympathetic neurons and the sweat gland also
includes the induction and maintenance of the secretory responsiveness of
sweat glands (Francis and Landis,
1999; Landis,
1999). Secretory responsiveness, the ability of glands to produce
sweat after nerve stimulation or cholinergic agonist administration, does not
develop in the absence of sweat gland innervation
(Stevens and Landis, 1987).
Both catecholaminergic and cholinergic neurotransmission are required for the
induction of secretory responsiveness during development
(Tian et al., 2000;
Grant et al., 1995),
reflecting the developmental change in neurotransmitter phenotype. In addition
to eliciting sweat secretion in adult rodents, cholinergic transmission is
required for the maintenance of secretory responsiveness
(Grant et al., 1995). The
cholinergic differentiation factor(s) produced by the developing sweat gland
thus seems to trigger indirectly, by inducing cholinergic neurotransmission,
an essential step in the differentiation of this target tissue. However, the
molecular identity of the target-derived signal(s) has remained unclear.
The first factor that was observed to induce cholinergic differentiation of
cultured sympathetic neurons was leukemia inhibitory factor (LIF)
(Yamamori et al., 1989). LIF
belongs to the IL6 family of cytokines
(Taga and Kishimoto, 1997),
which includes interleukin 6 (IL6), IL11, ciliary neurotrophic factor (CNTF),
oncostatin M (OSM), cardiotrophin 1 (CT1; CTF1–Mouse Genome
Informatics), cardiotrophin-like cytokine (CLC; CLCF1 – Mouse Genome
Informatics) (Elson et al.,
2000) [also known as novel neurotrophin 1/B-cell stimulating
factor 3 (Shi et al., 1999;
Senaldi et al., 1999)] and
neuropoietin/cardiotrophin 2 (NP; CTF2 – Mouse Genome Informatics)
(Derouet et al., 2004). CLC
interacts with the soluble receptor cytokine-like factor 1 (CLF; CRLF1 –
Mouse Genome Informatics), or with soluble CNTFR
, to form a functional
ligand for the CNTF receptor complex
(Elson et al., 1998;
Elson et al., 2000). All
family members analysed to date were shown to induce ChAT and VIP, and to
reduce noradrenergic gene expression in cultured sympathetic neurons
(Yamamori et al., 1989;
Saadat et al., 1989;
Geissen et al., 1998;
Rao et al., 1992a). This can
be explained by their common mechanism of action, activating receptor
complexes that share the signaling receptor subunit gp130 (IL6ST – Mouse
Genome Informatics), leading to the alternative term gp130 cytokines for IL6
cytokine family members (Taga and
Kishimoto, 1997; Heinrich et
al., 2003). The gp130 receptor family can be subdivided into
receptors that contain, as signaling subunits, either gp130 homodimers or
heterodimers, composed of gp130/LIFRß or gp130/OSMR. Additional
ligand-binding -receptor subunits can associate with the core signaling
receptors to form tripartite or even more complex (CLC/CLF) receptors
(reviewed by Heinrich et al.,
2003). In addition to IL6 cytokine family members, other signals
were also found to induce cholinergic sympathetic differentiation in vitro,
including the TGFß family member activin
(Fann and Patterson, 1995),
the GFL family member GDNF (Brodski et al.,
2002) and the neurotrophin NT3
(Brodski et al., 2000).
The role of IL6 cytokines in the cholinergic differentiation of sweat gland
innervation has been investigated by expression analysis and by
loss-of-function approaches (Rohrer,
1992; Rao and Landis,
1993; Habecker et al.,
1995a; Francis et al.,
1997). These studies excluded all known cytokines acting singly as
sweat gland-derived cholinergic differentiation factors. Neither was
cholinergic sweat gland innervation affected by the combined elimination of
LIF and CNTF (Francis et al.,
1997). IL6 cytokines are also implicated in the cholinergic
differentiation of periosteum innervating neurons, as antibodies against
LIFRß prevented ChAT induction in sympathetic neurons co-cultured with
periosteal cells (Asmus et al.,
2001). Although tissue homogenates from rat footpads and
supernatants of cultured sweat glands or sternum were shown to contain a
cholinergic differentiation activity with properties of a LIF-related cytokine
(Habecker et al., 1997), the
relevance of these findings for the in vivo situation is unclear, as
production and response to cytokines in neural cells is rapidly induced upon
in vivo lesioning or in tissue culture
(Freidin et al., 1992;
Rao and Landis, 1993;
Zigmond, 1996;
Yao et al., 1997).
To address the physiological importance of cytokine signaling for
target-dependent cholinergic sympathetic differentiation, we have selectively
eliminated gp130 in noradrenergic cells by crossing mice carrying a floxed
gp130 allele (Betz et al.,
1998; Hirota et al.,
1999) with a mouse line that expresses Cre recombinase under the
control of the dopamine ß-hydroxylase (DBH) promotor. The
observed complete lack of cholinergic fibers in mutant sweat glands, the
massive reduction in the number of cholinergic neurons in the stellate
ganglion and the maintenance of noradrenergic sweat gland innervation
demonstrates an essential function of Il6 cytokines for target-dependent
cholinergic differentiation. The co-expression of candidate cytokines
CNTF, CLC/CLF, CT1 and NP observed in sweat gland tissue
suggests that several factors may act together in this process, and explains
the lack of effects of single knockouts. As sweat glands show a normal
secretory response in mice displaying noradrenergic instead of cholinergic
innervation, cholinergic neurotransmission seems not to be required for the
acquisition and maintenance of secretory responsiveness.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Soriano,
1999). DBH-iCre mice were generated by introducing the
iCre sequence into a P1-derived bacterial artificial chromosome (PAC)
that harboured the gene for mouse DBH (R. Parlato et al.,
unpublished). Using homologous recombination in E. coli, the PAC was
modified to carry the improved coding sequence of Cre recombinase
(iCre) (Wintermantel et al.,
2002). Gp130-deficient mice were generated by backcrossing
gp130fl/fl animals and DBH-iCre animals with
C57Bl/6 mice to the fourth generation. Resulting animals were intercrossed and
bred to homozygosity.
Tissue preparation
Animals were killed and immediately dissected to collect front feet,
stellate and superior cervical ganglia. Tissues were deep frozen on blocks of
dry ice and stored at –20°C. Prior to immunohistochemistry and in
situ hybridization, tissues were cryosected and 14 µm sections were
collected on glass slides.
ß-galactosidase staining
Embryos or tissue sections were fixed in 0.4% glutaraldehyde for 2-4 hours
or 15 minutes, respectively. Staining was carried out overnight in a solution
containing 0.1% sodium deoxycholate, 0.2% Nonidet NP-40, 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide and 1 mg/ml X-Gal.
Immunohistochemistry and in situ hybridization
For in situ hybridization and immunohistochemistry, slices were postfixed
for 15 minutes in 0.1 M NaH2PO4 with 4% paraformaldehyde
and rinsed in PBS. For antibody staining on sections, slices were
pre-incubated for 1 hour in PBS with 5% fetal calf serum and 0.2% Triton X-100
(antibody buffer). Primary antibodies in antibody buffer were incubated
overnight at room temperature and slices were washed in PBS with 0.2% Triton
X-100 (PBT). Secondary antibodies in PBT with 0.1% DAPI were incubated for two
hours and washed off.
The following antibodies were used: mouse monoclonal anti-neuronal class III ß-tubulin (Tuj1; Hiss Diagnostics, Freiburg, Germany), rabbit polyclonal anti-porcine VIP (PROGEN, Heidelberg, Germany), rabbit polyclonal anti-VAChT serum (Phoenix Pharmaceuticals, Belmont, CA, USA), rabbit polyclonal anti-ChT1 (kindly provided by T. Okuda, Tokyo, Japan), rabbit polyclonal anti-TH (BioTrend, Cologne, Germany).
In situ hybridization was performed according to established protocols
(Ernsberger and Rohrer, 1997).
Slices were incubated with Digoxigenin (DIG)-labeled antisense RNA probes at
68°C overnight in hybridization buffer with 1xSSC and 50% formamide.
Alkaline phosphatase-coupled anti-DIG antibody was applied overnight and the
staining reaction was carried out using NBT/BCIP as substrate. Antibody
staining following hybridization was performed following the standard protocol
given above.
Antisense probes for CLC, CLF and CT1 were generated using IMAGE Consortium cDNA clones CloneID 3411865, CloneID 3710164 and CloneID 315363, respectively (RZPD, Berlin, Germany). The antisense probe for NP was generated using a cDNA encompassing the coding sequence.
Morphometric analysis
Pictures of immunostained sweat glands were taken at a Zeiss Axioplan
microscope stand using a Visitron SPOT CCD camera and 20x and 40x
objective magnification. The pictures of sweat glands were imported into the
NIH-imaging software ImageJ to determine the number of pixels covering the
area of the sweat glands and the percentage of pixels covering immunopositive
fibers within that area.
For stellate ganglia sections, pictures were taken likewise with 10x objective magnification. The number of pixels covering the area of the cross sections was measured with ImageJ and calibrated to metric values. Immunoreactive cells containing DAPI-stained nuclear profiles in the sections plane were counted manually using 40x objective magnification. Student's t-test was performed to test for statistical significance of differences.
Functional sweat gland test
Mice were collected at postnatal days 52 to 58. The animals were
anesthetized by intraperitoneal (ip) injections of a mixture of ketamine (0.2
mg per animal) and xylazine (0.08 mg per animal). To induce sweat response,
animals received 3 µg pilocarpine per gram body weight (ip). As soon as the
animals showed robust salivation, both hind feet were wiped with ethanol and
coated with Coltexfine dental paste (Coltene/Whaledent AG Altstaetten,
Switzerland). After polymerization of the material, the moulds were removed
and the process was repeated once. At the end of the experiment all animals
were killed by decapitation and tail tissue was collected for genotyping.
The moulds were analyzed under stereoscopic magnification. Imprints of sweat droplets were counted for each of the two interdigital foot pads. The mean number of interdigital sweat glands was calculated from all counts derived from a single animal. The overall mean and its standard error were calculated for the groups of wild-type and mutant animals. Statistical significance of differences was tested for by Student's t-test.
Laser dissection of sweat glands and total RNA isolation
Freshly dissected tissue was frozen and embedded in tissue-Tec (Sakura, The
Netherlands). Serial cryostate sections (10 µm) were cut and mounted on
autoclaved polytarthalene (PEP) foil stretched on a metal frame (Leica).
Sections were then fixed in ice-cold acetone (2 minutes), dried on a heater
(40°C, 10 minutes) and stained with 1% Toluidine Blue (Merk, Darmstadt,
Germany). After differentiation in 75% ethanol (3 minutes), the sections were
dried (40°C, 10 minutes) and subjected to laser microdissection (LMD)
(Burbach et al., 2003) using
the AS LMD system from Leica Microsystems. Total RNA from LMD-isolated sweat
gland coils was obtained by using the RNeasy Micro Kit (Qiagen, Hilden,
Germany).
RT-PCR on sweat gland RNA
cDNA synthesis was performed using the Thermoscript RT-PCR System and
oligo-dT primers (Invitrogen, Karlsruhe, Germany). For the detection of
neuropoietic cytokines, the following primer combinations and the Hot Star Taq
Master Mix Kit (Qiagen, Hilden, Germany) were used:
For all reactions, a standardized PCR protocol was used: 40 cycles of 30 seconds at 94°C, 30 seconds at 65°C and 30 seconds at 72°C.
Detection of recombined gp130 allele
Primers used for detection of the recombined gp130 allele
(Betz et al., 1998) were
5'-TTTCAAGTACCCTGGGGATGG-3' (forward) and
5'-TGAGGCAGAAACACACTCATGC-3' (reverse), and are expected to
produce a PCR product of >4 kb for gp130fl/fl and about
800 bp for the recombined gp130 allele in noradrenergic cells of
gp130DBHcre mice. The PCR protocol used was 42 cycles of 1
minute at 94°C, 30 seconds at 58°C and 2 minutes at 68°C.
Sympathetic neuron culture
Cultures of embryonic day (E) 7 chick sympathetic neurons were prepared and
maintained as described previously
(Ernsberger et al., 1989).
Cytokines were added immediately to induce the expression of VIP (GPA, 2
ng/ml; CNTF, 1.5 ng/ml; CLC/CLF, 100 ng/ml; NP, 500 ng/ml). After 4 days, the
cells were stained for VIP, as described previously
(Ernsberger et al., 1989).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). This
floxed gp130 allele contains two loxP sites flanking exon 16, which
encodes the transmembrane domain of gp130. Recombination leads to the loss of
gp130 from the plasma membrane, resulting in a complete lack of responsiveness
to IL6 cytokine family members (Betz et
al., 1998; Hirota et al.,
1999). To mutate this allele in sympathetic neurons, we have used
a transgenic mouse line carrying the Cre recombinase under the control of the
DBH gene, expressed selectively in noradrenergic neurons (R.P., C.
Otto and G. Schütz, unpublished). As previous plasmid-based transgenesis
using a 5.8 kb DBH promotor did not fully reproduce the normal DBH
expression pattern (Hoyle et al.,
1994; Mercer et al.,
1991), Cre recombinase was expressed under the control of the
DBH gene, embedded in a 150 kB P1-derived bacterial artificial
chromosome.
The transgenic mouse line carrying the DBH-Cre PAC (DBH-Cre) shows
Cre expression exclusively in noradrenergic neurons of the peripheral (PNS)
and central (CNS) nervous system, as revealed by immunohistological analysis
of Cre expression (data not shown) and by analysing Cre activity using the
ROSA26 reporter mouse line (Soriano,
1999). In the PNS, Cre-mediated recombination leading to
lacZ expression was restricted to DBH-expressing cells,
including paravertebral sympathetic ganglia
(Fig. 1A), and was observed as
early as at E10.5 (data not shown). On cross sections of E16.5 sympathetic
ganglia, virtually all ganglion neurons seemed to be lacZ-positive
(Fig. 1B). Peripheral targets
of cholinergic sympathetic neurons, i.e. sweat glands in the fore- and
hindlimb footpads, did not show any recombination
(Fig. 1C), as expected from the
endogenous DBH expression pattern.
Crossing of DBH-Cre with gp130fl/fl mice produced DBHCre;gp130fl/fl (abbreviated to gp130DBHCre) offspring, born in normal Mendelian ratio, that were viable, with normal weight (25.5±0.5 g in fl/fl versus 23.8±0.4 g in gp130 DBHcre) and without any obvious impairments. The gp130DBHcre mice breed and can be maintained as homozygous line. The very high recombination efficiency in sympathetic ganglia observed in the ROSA26 mouse line implies that functional gp130 is eliminated in sympathetic neurons of gp130DBHcre mice. We were unable to analyse gp130 protein expression in wild-type and mutant sympathetic ganglia, most likely due to low expression levels and the small amount of tissue available (data not shown). However, Cre-mediated elimination of exon 16 in DBH-expressing sympathetic ganglia and adrenal glands of gp130DBHcre mutant mice could be demonstrated at genomic level by PCR (Fig. 1D).
|
Whereas VAChT, ChT1 and VIP were expressed by the sweat gland innervation
in wild-type and control gp130fl/fl mice
(Fig. 2C,I,P), cholinergic
markers were virtually absent in the sweat gland innervation of
gp130DBHcre mice (Fig.
2F,M,S). The extent of cholinergic innervation was quantified
morphometrically, revealing a nearly complete loss of cholinergic features
(VAChT, ChT1) and VIP, when compared with control mice. This effect was not
due to a decreased sweat gland innervation or reduced terminal sprouting
(Hiltunen and Alraksinen,
2004), as the expression of the general neuronal marker TUJ1 was
not reduced, as shown in the double immunostainings
(Fig. 2D,E,K,L,Q,R) and by the
quantitative analysis of TUJ1 immunostaining
(Fig. 3A-C). The expression of
neurofilament immunoreactivity was investigated as an additional neuronal
marker, revealing no significant change following the elimination of gp130
signaling (128±21%; mean±s.e.m., n=3-4) in
gp130DBHcre when compared with controls. In
addition, the expression of the adrenergic marker TH was not significantly
different between gp130DBHcre mice and
gp130fl/fl controls
(Fig. 3D-F). Taken together,
these results strongly suggest that in the absence of gp130 signaling
noradrenergic properties are maintained, as in the wild type, and cholinergic
characteristics are not acquired.
Elimination of gp130 results in a reduced number of VAChT- and VIP-positive neurons in the sympathetic stellate ganglion
The lack of VAChT and VIP immunoreactivity in the sweat gland innervation
of gp130DBHcre mice could be due to a general switch of
the neurotransmitter phenotype or to a restricted effect on the distal
processes. Selective effects on the cholinergic properties of sweat gland
innervation have been observed in Gfra2-deficient mice
(Hiltunen and Alraksinen,
2004). It has also been suggested that the number of cholinergic
neurons in the stellate ganglion would be constant from the latter half of
gestation into adulthood, with increasing expression levels of cholinergic
markers in the sweat gland innervation
(Schäfer et al., 1997).
To address these issues, the number of cholinergic neurons was analysed in the
stellate ganglion of wild-type and gp130DBHcre mice at
postnatal day (P) 60 and P2. The stellate ganglion is the source of forelimb
sweat gland innervation (Morales et al.,
1995; Schäfer et al.,
1998) and may also contribute to the cholinergic sympathetic
innervation of the periosteum of sternum and ribs
(Asmus et al., 2000).
We observed in stellate ganglia of P60 gp130DBHcre mice a strong reduction in the density of VIP-positive neuronal cell bodies (from 35.4±4.5 to 7.8±2.7 VIP+ cells/mm2; mean±sem, n=3-5; P=0.013; Fig. 4A-C). As the mean ganglion area was not significantly altered (90±10.6% in gp130DBHcre) when compared with controls (P>0.5), we conclude that the reduced density of VIP-immunoreactive (VIP-IR) cells reflects a lower number of VIP-positive cells/ganglion. Also, the number of VAChT-positive cell bodies was reduced by 70% (from 25±2 to 7.5±1.5 VAChT+ cell bodies/mm2; mean±s.e.m., n=3-5, P<0.01). The observed lower density of VAChT-IR cells, when compared with VIP-IR cells can be explained by the lower intensity of VAChT-IR. The strong reduction in the number of cholinergic neurons may either reflect the fact that the majority of stellate cholinergic neurons innervate the forelimb sweat glands or that gp130 signaling controls cholinergic differentiation also in other peripheral targets.
The presence of residual cholinergic, VIP-expressing sympathetic neurons
that are maintained in gp130DBHcre mice could be due to
incomplete recombination or to cytokine-independent cholinergic
differentiation. The latter explanation is supported by the finding that a
population of cholinergic sympathetic neurons is generated during embryonic
development (Ernsberger et al.,
1997; Schäfer et al.,
1997) by signals that do not involve the cytokine receptors
LIFRß and CNTFR (Stanke et
al., 2000). By analysing sympathetic stellate ganglia of
gp130DBHcre and control mice at P2, we could show that the
number of VIP-positive cells at P2 closely corresponds to the number of
VIP-positive cells observed in the adult sympathetic ganglia of
gp130DBHcre animals (compare
Fig. 4C with 4D), and to the
number of VIP- and VAChT-positive cells determined at P0 in our previous study
(Stanke et al., 2000). These
data indicate that the generation of VIP-positive neurons during embryonic
development is not controlled by gp130 signaling
(Fig. 4D). By contrast, gp130
signaling is essential for the postnatal, target-induced cholinergic
differentiation, resulting in a fivefold increase in the number of cholinergic
sympathetic neurons in the stellate ganglion.
|
Secretory responsiveness of sweat glands develops in the absence of cholinergic innervation
The development of secretory responsiveness of sweat glands, i.e. sweat
secretion in response to cholinergic agonists, depends on noradrenergic and
cholinergic neurotransmission (Tian et
al., 2000; Grant et al.,
1995). The correlation between the timing of cholinergic sweat
gland innervation and the development of secretory response, together with
inhibitory effects of cholinergic antagonists in the adult, suggested an
important function of cholinergic neurotransmission in sweat gland maturation
and functional maintenance (Grant et al.,
1995; Landis,
1999). As cholinergic innervation is lacking in
gp130DBHcre mice, it was expected that this would lead to
an impaired secretory response to cholinergic agonists. Interestingly, only a
small reduction in sweat gland activity was observed
(Fig. 6). Whereas 25±0.6
(n=12) secretory sweat glands were stimulated by pilocarpine control
footpads, 19±2.6 (n=6) sweat glands were observed in the
footpads of gp130DBHcre mice (P<0.05). These
findings suggest that secretory responsiveness can be acquired and maintained
in the absence of cholinergic neurotransmission.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Francis and Landis, 1999).
Here, we demonstrate that IL6 cytokines acting through gp130 receptors are
required for the cholinergic differentiation of sympathetic neurons
innervating sweat glands in mouse footpads. Candidate IL6 cytokines were found
to be expressed in the target tissue during the critical developmental time
period. gp130 signaling controls the differentiation of the vast majority of
cholinergic neurons in the stellate ganglion, which occurs during postnatal
development. These results implicate target-derived IL6 cytokines in the
specification of virtually all cholinergic sympathetic neurons that
differentiate postnatally.
|
;
Saadat et al., 1989;
Geissen et al., 1998;
Rao et al., 1992a). Also, in
vivo, cholinergic differentiation could be elicited by LIF overexpression
(Bamber et al., 1994). A role
for IL6 cytokines in sweat gland innervation is supported by the demonstration
of a cholinergic differentiation activity in footpad homogenates and in the
supernatants of cultured footpads that acts through LIFRß and activates
JAK/STAT pathways (Habecker et al.,
1997). It has remained unclear, however, whether these activities
reflect the physiological factor(s), as the expression of cytokines and
cytokine responsiveness can be rapidly induced in culture
(Freidin et al., 1992;
Rao and Landis, 1993;
Zigmond, 1996;
Yao et al., 1997). In
addition, factors like CNTF that are secreted from cells only at very low
levels (Stöckli et al.,
1989; Lin et al.,
1989) may represent the major active components of tissue
homogenates (Rohrer, 1992).
The difficulty to draw valid conclusions from in vitro studies for the in vivo
situation is also illustrated by the finding that noradrenergic
neurotransmission, which is essential for the expression of the cholinergic
differentiation factor in sweat gland cultures
(Habecker et al., 1995b),
seems not to be relevant in the in vivo situation
(Tian et al., 2000).
The present findings demonstrate that cytokines acting through gp130 in
sympathetic neurons are essential for the cholinergic differentiation of sweat
gland innervation, affecting the expression of VAChT, ChT1 and VIP. The
reduced number of VIP- and VAChT-positive cholinergic neurons in the stellate
ganglion supports the conclusion that retrograde gp130 signaling from the
target controls the neurotransmitter phenotype of sympathetic neurons. In
addition, the large, 70-80% decrease indicates that cholinergic
differentiation of neurons innervating other targets may also depend on gp130
cytokines. For the periosteum, there is indeed in vitro evidence that this
target tissue also produces a cholinergic differentiation signal acting
through LIFRß (Asmus et al.,
2001). Whether skeletal muscle vasculature receives cholinergic
sympathetic innervation is controversial in rodents
(Schäfer et al., 1998;
Guidry and Landis, 2000;
Dehal et al., 1992), but there
is clear evidence for other species, such as cat, guinea pig and chick (see
Ernsberger and Rohrer, 1999).
Previous knockdown studies in the chick have also shown essential roles of
gp130 (Geissen et al., 1998)
and LIFRß (Duong et al.,
2002) for VIP expression in cholinergic sympathetic neurons. VIP
expression in chick sympathetic neurons, in contrast to ChAT and VAChT
expression, is observed at late stages of development and is thought to be
induced by signals produced by the innervated vascular targets
(Geissen et al., 1998;
Duong et al., 2002). Taken
together, these findings suggest that all known targets of cholinergic
sympathetic neurons, i.e. sweat glands, periosteum and vasculature may control
the neurotransmitter phenotype of their innervation through gp130
cytokines.
|
|
;
Ernsberger et al., 1997;
Stanke et al., 2000). VIP is
transiently expressed in the embryonic rat superior cervical ganglion in up to
30% of the cells (Tyrrell and Landis,
1994). As the number of VIP- and VAChT-expressing cells at P0 is
not affected in mice deficient for gp130, LIFRß or CNTFR,
embryonic cholinergic differentiation seems not to be controlled by IL6
cytokines (this study). A similar conclusion was reached from the LIFRß
and gp130 knockdown in the chick, which did not affect the early expressed
cholinergic markers ChAT and VAChT
(Geissen et al., 1998;
Duong et al., 2002).
|
;
Francis and Landis, 1999). The
present results demonstrate that the majority of cholinergic sympathetic
neurons in the stellate ganglion are generated postnatally and by a
gp130-dependent mechanism. This correlates with the timing of the transmitter
phenotype switch during sweat gland innervation. The postnatal increase in the
number of VIP- and VAChT-positive cells suggests that the cholinergic
sympathetic neurons have not already become committed during embryonic
development (Schäfer et al.,
1997), and supports the notion that the gp130-dependent population
is generated by target-induced differentiation
(Guidry and Landis, 1998).
Conversely, the gp130-independent population represents cells that acquired
cholinergic properties during embryonic stages, most likely independently of
target innervation. Differentiation factors of the GFL family, acting through
the RET tyrosine kinase receptor, are not essential for the initial expression
of VAChT and ChAT in vivo, but are required for the maturation of cholinergic
sympathetic neurons during prenatal development
(Burau et al., 2004). It will
be interesting to analyse whether NT3
(Brodski et al., 2000) and
activins (Fann and Patterson,
1995) influence embryonic cholinergic sympathetic differentiation
in vivo.
Whereas detectable stores of endogenous catecholamines disappear in neurons
innervating sweat glands in rats and mice, TH- and DBH-IR were reported to
decrease to low levels in rats (Landis et
al., 1988) but to be maintained in mice
(Rao et al., 1994;
Guidry and Landis, 1995). The
lack of catecholamine production in mouse sweat gland innervation was
explained by a loss of the TH cofactor tetrahydrobiopterin and the
tetrahydrobiopterin synthetic enzyme GTP cyclohydrolase (GCH)
(Habecker et al., 2002). In
the present study, we confirm that TH-IR is present in the adult mouse sweat
gland innervation and that the extent of TH expression is not affected by the
lack of gp130 signaling. This finding, together with the maintenance of TUJ1-
and NF160-positive sweat gland innervation, demonstrates that IL6 cytokines do
not affect target innervation or sympathetic neuron survival.
Candidate IL6 cytokines in cholinergic differentiation
What is the identity of the sweat gland-derived cytokine? The cytokines
CNTF (Saadat et al., 1989),
LIF (Yamamori et al., 1989),
OSM (Rao et al., 1992a) and
CT1 (Pennica et al., 1995b;
Habecker et al., 1995a;
Geissen et al., 1998) induce
cholinergic function and VIP production while decreasing catecholamine
content. The present study extends this list of cytokines by including CLC/CLF
and NP as candidate cholinergic differentiation factors. Previous studies
concluded that LIF (Rao et al.,
1993) and OSM do not appear to be produced by sweat glands in vivo
(Habecker et al., 1997). This
is confirmed by the RT-PCR analysis of LCM-isolated sweat gland tissue. From
the cytokines expressed in sweat gland tissue, CT1, NP and CLC/CLF are the
most likely candidates, as, in contrast to CNTF, they are secreted proteins.
In addition, CNTF expression is restricted to Schwann cells rather than sweat
glands (Rohrer, 1992). CT1 and
the newly discovered NP are both efficiently secreted
(Pennica et al., 1995a;
Derouet et al., 2004). CLC is
also secreted when co-expressed with either the soluble receptor CLF
(Elson et al., 2000;
Lelièvre et al., 2001)
or with CNTFR (Plun-Favreau et al.,
2001). CLC/CLF and NP act only on cells expressing the tripartite
CNTF receptor (Elson et al.,
1998; Elson et al.,
2000; Derouet et al.,
2004). CT1 binds to and activates a heterotrimeric receptor
composed of LIFRß, gp130 and an hypothetical CT1-specific
-receptor (Robledo et al.,
1997). As the postulated CT1 -receptor has not been
identified so far, it is unclear whether it is expressed during sweat gland
innervation. In view of the biological effects elicited by CT1 in cultured
sympathetic neurons from different species
(Pennica et al., 1995b;
Habecker et al., 1995a;
Geissen et al., 1998), it
seems very likely that CT1-receptors are present in vivo during target
tissue innervation. As NP is expressed at much lower levels than
CT1 and CLC/CLF, apparent from both the RT-PCR and in situ
hybridisation analysis, CT1 and CLC/CLF are the most likely candidates for the
sweat gland cholinergic differentiation factor.
The co-expression of several secreted cytokines with cholinergic
differentiation activity indicates an unexpected redundancy in the
target-dependent control of this neurotransmitter phenotype. It should also be
kept in mind that the involvement of additional, unknown cytokines cannot be
excluded. To define the relevant factors would thus require the combined
elimination of CT1, CLC, CLF and possibly NP, involving conditional knockouts,
as mice deficient for CLF die around birth
(Forger et al., 2003). The
cytokine redundancy explains the difficulty in defining physiological relevant
factors by loss-of-function approaches in sweat gland homogenates; for
example, antibodies against CNTF and CT1 were unable to deplete the
cholinergic activity of footpad homogenates
(Rao et al., 1992b;
Rohrer, 1992;
Habecker et al., 1997).
The in situ hybridisation analysis suggests that CT1 and
CLC/CLF may be expressed at much higher levels by myoepithelial cells
than by secretory cells. During development, both of these cells are generated
from invaginating ectodermal cells and both cell types seem to express
muscarinic acetylcholine receptors (Landis
and Keefe, 1983; Grant et al.,
1991). The myoepithelial cells of exocrine glands are highly
contractile and have a central role in the ejection of liquids produced by the
luminal secretory cells. They are located at the circumference of the glands,
in direct contact with the basal lamina. This position is well suited for the
production of factors that influence the innervating axons, which are present
within a distance of about 1-2 µm of the basal lamina
(Landis and Keefe, 1983).
Functions of cholinergic sympathetic innervation
Sweat secretion is elicited by cholinergic agonists, acting through
muscarinic acetylcholine receptors of the M2 glandular (m3 molecular) subtype,
in developing and adult sweat glands
(Stevens and Landis, 1987).
The morphological development of sweat glands, as well as the molecular and
pharmacological properties of muscarinic cholinergic and adrenergic receptors
of sweat glands are independent of innervation
(Grant and Landis, 1991;
Grant et al., 1991;
Habecker et al., 1996).
However, innervation is essential for a late step of sweat gland maturation,
the development and maintenance of secretory responsiveness, i.e. the ability
of glands to produce sweat after nerve stimulation or agonist treatment
(Stevens and Landis, 1987;
Stevens and Landis, 1988).
Both catecholaminergic (Tian et al.,
2000) and cholinergic (Grant
et al., 1995) neurotransmission are required for the acquisition
of secretory responsiveness during development. Cholinergic neurotransmission
is also necessary for the maintenance of secretory responsiveness in adult
sweat glands (Grant et al.,
1995). These findings, showing an essential role of cholinergic
neurotransmission for the functional maturation of sweat glands, predicted
that mice devoid of cholinergic sweat gland innervation would not acquire and
maintain secretory responsiveness. The present study demonstrates, however,
that the sweating response of glands to cholinergic agonists is maintained in
adult animals in the absence of cholinergic innervation. Adrenergic
neurotransmission, essential to induce the sweating response during
development (Tian et al.,
2000), seems to be sufficient to keep the vast majority of sweat
glands in a functional state that allows their response to experimentally
administered cholinergic agonists. To explain the finding that the induction
of the sweating response depends on both cholinergic and adrenergic
neurotransmission (Tian et al.,
2000), it has been suggested that a specific step in
stimulus-secretion coupling, downstream of second messenger generation, would
require acetylcholine and catecholamine signaling, and that both transmitters
may control the same step. According to this notion, the lack of cholinergic
neurotransmission in gp130-deficient mice would be compensated by the action
of catecholamines. Such a compensation is not possible after the disruption of
muscarinic neurotransmission in adult rodent sweat glands as their innervation
is purely cholinergic (Grant et al.,
1995).
In contrast to their ability to induce and maintain the secretory response,
adrenergic agonists are very ineffective in eliciting sweat secretion
(Stevens and Landis, 1987;
Stevens and Landis, 1988). The
inability of sweat glands to respond to adrenergic agonists requires the
switch of the sympathetic innervation from adrenergic to cholinergic
neurotransmission. The present findings demonstrate that IL6 cytokines are the
essential, physiologically relevant signals for the specification of the
appropriate cholinergic neurotransmitter phenotype during sweat gland
innervation.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Asmus, S. E., Parsons, S. and Landis, S. C.
(2000). Developmental changes in the transmitter properties of
sympathetic neurons that innervate the periosteum. J.
Neurosci. 20,1495
-1504.
Asmus, S. E., Tian, H. and Landis, S. C. (2001). Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms. Dev. Biol. 235,1 -11.[CrossRef][Medline]
Bamber, B. A., Masters, B. A., Hoyle, G. W., Brinster, R. L. and
Palmiter, R. D. (1994). Leukemia inhibitory factor induces
neurotransmitter switching in transgenic mice. Proc. Natl. Acad.
Sci. USA 91,7839
-7843.
Betz, U. A., Bloch, W., van den Broek, M., Yoshida, K., Taga,
T., Kishimoto, T., Addicks, K., Rajewsky, K. and Müller, W.
(1998). Postnatally induced inactivation of gp130 in mice results
in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary
defects. J. Exp. Med.
188,1955
-1965.
Brodski, C., Schnürch, H. and Dechant, G.
(2000). Neurotrophin-3 promotes the cholinergic differentiation
of sympathetic neurons. Proc. Natl. Acad. Sci. USA
97,9683
-9688.
Brodski, C., Schaubmar, A. and Dechant, G. (2002). Opposing functions of GDNF and NGF in the development of cholinergic and noradrenergic sympathetic neurons. Mol. Cell. Neurosci. 19,528 -538.[CrossRef][Medline]
Burau, K., Stenull, I., Huber, K., Misawa, H., Berse, B., Unsicker, K. and Ernsberger, U. (2004). c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice. Eur. J. Neurosci. 20,353 -362.[CrossRef][Medline]
Burbach, G., Dehn, G., Del Turco, D. and Deller, T. (2003). Quantification of layer-specific gene expression in the hippocampus: effective use of laser microdissection in combination with quantitative RT-PCR. J. Neurosci. Methods 131, 93-91.[CrossRef][Medline]
Dehal, N. S., Kartseva, A. and Weaver, L. C. (1992). Comparison of locations and peptide content of postganglionic neurons innervating veins and arteries of the rat hindlimb. J. Auton. Nerv. Syst. 39, 61-72.[CrossRef][Medline]
Derouet, D., Rousseau, F., Alfonsi, F., Froger, F., Hermann, J.,
Barbier, F., Perret, D., Diveu, C., Guillet, C., Preisser, L. et al.
(2004). Neuropoietin, a new IL-6-related cytokine signaling
through ciliary neurotrophic factor receptor. Proc. Natl. Acad.
Sci. USA 101,4827
-4832.
Duong, C. V., Geissen, M. and Rohrer, H. (2002). The developmental expression of vasoactive intestinal peptide (VIP) in cholinergic sympathetic neurons depends on cytokines signaling through LIFRß-containing receptors. Development 129,1387 -1396.
Elson, G. C. A., Graber, P., Losberger, C., Herren, S.,
Gretener, D., Menoud, L. N., Wells, T. N. C., Kosco-Vilbois, M. H. and
Gouchat, J.-F. (1998). Cytokine-like factor-1, a novel
soluble protein, shares homology with members of the cytokine type I receptor
family. J. Immunol. 161,1371
-1379.
Elson, G. C. A., Lelièvre, E., Guillet, C., Chevalier, S., Plun-Favreau, H., Froger, J., Suard, I., De Coignac, A. B., Delneste, Y., Bonnefoy, J. Y. et al. (2000). CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex. Nat. Neurosci. 3,867 -872.[CrossRef][Medline]
Enomoto, H., Crawford, P. A., Gorodinsky, A., Heuckeroth, R. O., Johnson, E. M., Jr and Milbrandt, J. (2001). RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128,3963 -3974.
Ernsberger, U. and Rohrer, H. (1999). Development of the cholinergic neurotransmitter phenotype in postganglionic sympathetic neurons. Cell Tissue Res. 297,339 -361.[CrossRef][Medline]
Ernsberger, U., Sendtner, M. and Rohrer, H. (1989). Ciliary neurotrophic factor (CNTF) interferes with the proliferation of embryonic chick sympathetic neurons and induces the expression of vasoactive intestinal peptide (VIP). Neuron 2,1275 -1284.[CrossRef][Medline]
Ernsberger, U., Patzke, H. and Rohrer, H. (1997). The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation. Mech. Dev. 68,115 -126.[CrossRef][Medline]
Fann, M.-J. and Patterson, P. H. (1995). Activins as candidate cholinergic differentiation factors in vivo.Int. J. Dev. Neurosci. 13,317 -330.[CrossRef][Medline]
Forger, N. G., Prevette, D., DeLapeyrière, O., de Bovis,
B., Wang, S. W., Bartlett, P. and Oppenheim, R. W. (2003).
Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic
factor for lumbar and facial motoneurons in vivo. J.
Neurosci. 23,8854
-8858.
Francis, N. J. and Landis, S. C. (1999). Cellular and molecular determinants of sympathetic neuron development. Annu. Rev. Neurosci. 22,541 -566.[CrossRef][Medline]
Francis, N. J., Asmus, S. E. and Landis, S. C. (1997). CNTF and LIF are not required for the target-directed acquisition of cholinergic and peptidergic properties by sympathetic neurons in vivo. Dev. Biol. 182,76 -87.[CrossRef][Medline]
Freidin, M., Bennett, M. V. L. and Kessler, J. A.
(1992). Cultured sympathetic neurons synthesize and release the
cytokine interleukin 1ß. Proc. Natl. Acad. Sci.
USA 89,10440
-10443.
Geissen, M., Heller, S., Pennica, D., Ernsberger, U. and Rohrer, H. (1998). The specification of sympathetic neurotransmitter phenotype depends on gp130 cytokine receptor signaling. Development 125,4791 -4801.[Abstract]
Glebova, N. O. and Ginty, D. D. (2004).
Heterogeneous requirement of NGF for sympathetic target innervation in
vivo. J. Neurosci. 24,743
-751.
Goridis, C. and Rohrer, H. (2002). Specification of catecholaminergic and serotonergic neurons. Nat. Rev. Neurosci. 3,531 -541.[CrossRef][Medline]
Grant, M. P. and Landis, S. C. (1991). Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation. J. Neurosci. 11,3772 -3782.[Abstract]
Grant, M. P., Landis, S. C. and Siegel, R. E. (1991). The molecular and pharmacological properties of muscarinic cholinergic receptors expressed by rat sweat glands are unaltered by denervation. J. Neurosci. 11,3763 -3771.[Abstract]
Grant, M. P., Francis, N. J. and Landis, S. C. (1995). The role of acetylcholine in regulating secretory responsiveness in rat sweat glands. Mol. Cell. Neurosci. 6,32 -42.[CrossRef][Medline]
Guidry, G. and Landis, S. C. (1995). Sympathetic axons pathfind successfully in the absence of target. J. Neurosci. 15,7565 -7574.[Abstract]
Guidry, G. and Landis, S. C. (1998). Target-dependent development of the vesicular acetylcholine transporter in rodent sweat gland innervation. Dev. Biol. 199,175 -184.[CrossRef][Medline]
Guidry, G. and Landis, S. C. (2000). Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice. Aut. Neurosci. 82,97 -108.
Habecker, B. A., Pennica, D. and Landis, S. C. (1995a). Cardiotrophin-1 is not the sweat gland-derived differentiation factor. NeuroReport 7, 41-44.[Medline]
Habecker, B. A., Tresser, S. J., Rao, M. S. and Landis, S. C. (1995b). Production of sweat gland cholinergic differentiation factor depends on innervation. Dev. Biol. 167,307 -316.[CrossRef][Medline]
Habecker, B. A., Malec, N. M. and Landis, S. C.
(1996). Differential regulation of adrenergic receptor
development by sympathetic innervation. J. Neurosci.
16,229
-237.
Habecker, B. A., Symes, A. J., Stahl, N., Francis, N. J.,
Economides, A., Fink, J. S., Yancopoulos, G. and Landis, S. C.
(1997). A sweat gland-derived differentiation activity acts
through known cytokine signaling pathways. J. Biol.
Chem. 272,30421
-30428.
Habecker, B. A., Klein, M. G., Sundgren, N. C., Li, W. and
Woodward, W. R. (2002). Developmental regulation of
neurotransmitter phenotype through tetrahydrobiopterin. J.
Neurosci. 22,9445
-9452.
Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Müller-Newen, G. and Schaper, F. (2003). Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem. J. 374,1 -20.[CrossRef][Medline]
Hiltunen, P. H. and Alraksinen, M. S. (2004).
Sympathetic cholinergic target innervation requires GDNF family receptor
GFR2. Mol. Cell. Neurosci.
26,450
-457.[CrossRef][Medline]
Hirota, H., Chen, J., Betz, U. A. K., Rajewsky, K., Gu, Y., Ross, J., Jr, Müller, W. and Chien, K. R. (1999). Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress. Cell 97,189 -198.[CrossRef][Medline]
Honma, Y., Araki, T., Gianino, S., Bruce, A., Heuckeroth, R. O., Johnson, E. M. and Milbrandt, J. (2002). Artemin is a vascular-derived neurotrop
