-
Development 114, 689-698 (1992)Printed in Great Britain © The
Company of Biologists Limited 1992
689
Cholinergic neuronal differentiation factors: evidence for the
presence of
both CNTF-like and non-CNTF-like factors in developing rat
footpad
H. ROHRER*
Max-Planck-lnstitut fiir Psychiatric Abt. Neurochemie, Am
Klopferspitz 18, 8033 Martinsried]Plancgg, FRG
•Present address: Max-Planck-lnstitut fiir Hirnforschung, Abt.
Neurochemie, Deutschordenstrasse 46, 6000 Frankfud/M. 71, FRG
Summary
Catecholaminergic sympathetic neurons are able tochange their
transmitter phenotype during developmentand to acquire cholinergic
properties. Cholinergicsympathetic differentiation is only observed
in fibersinnervating specific targets like the sweat glands in
therat footpad. A function for ciliary neurotrophic factor(CNTF) in
this process has been implied as it is able toinduce cholinergic
properties (ChAT,VIP) in culturedchick and rat neurons. We show
here that a CNTT-like,VIP-inducing activity is present in rat
footpads and thatit increases 6-fold during the period of
cholinergicsympathetic differentiation. Immunohistochemicalanalysis
of P21 rat footpads demonstrated CNTF-likeimmunoreactivity in
Schwann cells but not in sweat
glands, the target tissue of cholinergic sympatheticneurons. The
expression of this factor in footpads seemsto be dependent on the
presence of intact nerve axons, asnerve transection results in a
loss of CNTF-likecholinergic activity and immunoreactivity.
Immuno-precipitation experiments with rat footpad extractsprovided
evidence for the presence of ChAT-inducingfactors other than CNTF,
which may independently ortogether with CNTF be involved in the
determination ofsympathetic neuron phenotype.
Key words: CNTF, sympathetic neuron, Schwann cell,sweat gland,
cholinergic neuronal differentiation, rat,footpad.
Introduction
The cellular and molecular mechanisms leading to thedifferent
neuronal phenotypes of the nervous systemduring development are
largely unknown. There ishowever evidence for the pluripotentiality
of neuronalprecursor cells and for fate-determining interactions
ofprecursor cells with the environment (Sieber-Blum andCohen, 1980;
Baroffio et al., 1988; Bronner-Fraser andFraser, 1988; Turner and
Cepko, 1987; Wetts andFraser, 1988). In addition, it was shown that
postmitoticperipheral neurons in developing and even adultanimals
are able to change transmitter phenotypedepending on the innervated
target (Schotzinger andLandis, 1988, 1990; McMahon and Gibson,
1987; forreviews see Landis, 1988, 1990). The best studiedexample
is developing sympathetic neurons innervatingsweat glands in the
rat footpad. The sympathetic axonsthat reach the developing sweat
glands during the firstpostnatal week express catecholaminergic
character-istics like catecholamine histofluorescence and
tyrosinehydroxylase immunoreactivity. During the second andthird
postnatal weeks most noradrenergic propertiesare lost and
cholinergic characteristics like cholineacetyltransferase (ChAT)
activity and vasoactive intes-
tinal peptide (VlP)-immunoreactivity appear (Landisand Keefe,
1983; Leblanc and Landis, 1986; Landis etal., 1988). A similar
noradrenergic-cholinergic switchwas previously observed in vitro
when sympatheticneurons were cultured in the presence of certain
non-neuronal cells or medium conditioned by these cells(Patterson
and Chun, 1974; Furshpan et al., 1976;Patterson and Chun, 1977;
Weber, 1981; Swerts et al.,1983; Raynaud et al., 1987a) or in the
presence ofhuman placental serum and/or embryo extract (Johnsonet
al., 1976; Ross et al., 1977; Iacovitti et al., 1981).Cultured
cells from various tissues including heart, skinand gut were shown
to release several VIP- and ChAT-inducing factors (Nawa and
Patterson, 1990) and heart-cell-conditioned medium contains at
least three distinctfactors which induce a specific pattern of
transmittersand neuropeptides (Nawa and Sah, 1990). The
majorcholinergic factor from heart-cell-conditioned mediumhas been
identified, isolated and characterized as a 45 x103 MT cholinergic
differentiation factor (CDF) (Fuk-ada, 1985), which was recently
found to be identical toleukemia inhibitory factor (LIF) (Yamamori
et al.,1989). In addition, a membrane-associated factor(MANS) has
been recently purified from rat spinal cord(Wong and Kessler, 1987;
Adler et al., 1989) and a
-
690 H. Rohrer
heparin-binding activity was identified in brain extract(Kessler
et al., 1986) which also induces cholinergicproperties in cultured
sympathetic neurons.
We have previously shown that ciliary neurotrophicfactor (CNTF)
(Manthorpe and Varon, 1985; Stockli etal., 1989; Lin et al., 1989)
induces ChAT-activity andreduces tyrosine hydroxylase (TH) in
cultures of ratsympathetic neurons (Saadat et al., 1989) and that
chicksympathetic neurons respond to CNTF by an inductionof VIP
(Ernsberger et al., 1989a). In view of themultitude of cholinergic
activities described in vitro, itis important to identify the
factors present in vivo, todefine their cellular localization and
expression duringnormal development. We show here by a sensitive
VIP-induction assay combined with immunoprecipitationand
immunohistochemistry using anti-CNTF antibodiesthat CNTF is present
in developing rat footpad and islocalized in Schwann cells.
However, only about half ofthe ChAT-inducing activity in footpad
extract can beprecipitated by anti-CNTF antibodies which
indicatesthat other factors, not recognized by anti-CNTFantiserum,
are also present in developing rat footpad.
Materials and methods
Cell cultureChick sympathetic neurons were isolated from
lumbosacralsympathetic chain ganglia of E7 chick embryos and
cultivatedon a polyornithine-laminin-coated substrate in Ham's
F14medium, supplemented with 10% horse serum and 5% fetalcalf serum
as described in detail previously (Ernsberger et al.,1989a,b). CNTF
was purified from rat sciatic nerve asdescribed previously (Saadat
et al., 1989). Murine LIF wasobtained from AMRAD, Kew Victoria,
Australia and wasused in chick sympathetic neuron cultures at a
concentration,of 1000 units/ml. At this concentration, several
batches of LIFwere active as ES stem cell-maintenance factor and
inducedChAT in cultures of rat sympathetic neurons. Basic FGF
andaFGF were obtained from Progen (Heidelberg, FRG) andwere used at
a concentration of 5 ng/ml and 50 ng/mlrespectively. Acidic FGF was
assayed both in the absence andin the presence of heparin (2
/ig/ml). Acidic FGF was shownto be active as an angiogenesis factor
on chick chorioallantoicmembrane and bFGF as a survival factor for
chick motoneur-ons.
Rat sympathetic neurons were isolated from superiorcervical
ganglia of newborn rats using the procedure of Mainsand Patterson
(1973; Chun and Patterson, 1977a,b) usingmodifications described by
Schwab and Thoenen (1985) andSaadat et al. (1989). Dissociated
sympathetic neurons wereplated on 35 mm Costar tissue culture
dishes coated withpolyornithine-laminin, and cultivated in
serum-free Ham'sF12 medium supplemented with transferrin (100
/ig/ml),insulin (5 /ig/ml), putrescin (100 ^lM), progesteron (20
nM),selenium (30 nM), glutamin (500 //g/ml), Hepes (5 mM),bovine
serum albumin (0.01%) and nerve growth factor(NGF) (50 ng/ml). The
medium was changed every 3-4 days.At the end of the culture period
the cultures were washedtwice with PBS to remove serum proteins,
harvested in PBSwith a rubber policeman, collected by
centrifugation, andstored frozen at -20°C until further use.
Tissue extractsAll tissues were frozen as small pieces in liquid
nitrogen
immediately after dissection and kept frozen at — 70°C
untilfurther use. The frozen tissue was first broken to
smallerpieces using a tissue grinder cooled with liquid nitrogen
andthen homogenized in two volumes/wet weight of 30 mM NaCl,10 mM
phosphate buffer, pH 7.4, supplemented with proteaseinhibitors
aprotinin (20 ki.u./ml), benzamidin (1 mM),leupeptin (100 mM), PMSF
(0.1 mM) and EDTA (1 mM),using a glass-glass homogenizer. After a
low-speed centrifu-gation, the supernatant was centrifuged at 100
000 g for 1hour. Supernatants were sterilized by centrifugation
through0.2 jim filters (Spin X; Costar) and stored frozen as
smallaliquots. For the immunoprecipitation experiments,
footpadextracts were concentrated after the 100 000 g
centrifugation,using centriconlO microcentrators (Amicon),
sterilized andstored frozen as small aliquots. Footpad extracts
wereroutinely made from 25 rats (21-day-old), which resulted in 3-4
g tissue (wet weight). Skeletal muscle was dissected from
thehindlimb and care was taken to remove the sciatic nerve trunkas
completely as possible. For the preparation of skin extracts,an
area was shaved before dissecting the skin.
To denervate rat footpad tissue, animals were anesthetizedby
ether inhalation. The sciatic nerve and the saphenousnerve were
exposed in the region of the upper thigh of the lefthind-limb of
14-day-old animals and pieces of about 2 cm wereremoved. In order
to obtain complete denervation and toprevent reinnervation of the
denervated glabrous skin bysprouting of the saphenous nerve, which
innervates the dorsalsurface of the foot (Mills et al., 1989), both
sciatic andsaphenous nerves were sectioned. After 7 days, the
animalswere killed and footpads of operated and
unoperatedcontralateral hindlimbs were dissected. The absence
ofreinnervation was controlled by examination of the lesionednerve.
In addition, neurofilament-IR positive fibers wereabsent in
operated footpads at P21 (staining according toRohrer et al.,
1988).
ChAT-assayChAT enzyme activity was assayed as described in
detailpreviously (Saadat et al., 1989). Frozen cells were
suspendedin 100 jA of homogenization buffer (5 mM Tris-acetate, pH
7.4and 0.1% Triton X-100) and homogenized by pipetting. Celldebris
was pelleted by centrifugation (2 minutes at 10 000 g).40 jA of the
supernatant was used for ChAT enzyme activityassays (Fonnum, 1969;
Raynaud et al., 1987b) and 30 /JI forprotein determination
(Bradford, 1976) using ovalbumin asstandard. The sensitivity of the
enzyme assay was increasedusing subsaturating concentrations of
acetyl-CoA as describedby Raynaud et al. (1987b).
AntiseraRabbit antisera were raised against oligopeptides that
weresynthesized according to the CNTF protein sequence.
Rabbitantiserum I was raised against a peptide corresponding
toamino acid 127-153 of the rat CN it amino acid sequence,antiserum
II was raised against the C-terminal part of CNTF(AA 186-199)
(Stockli et al., 1991). On western blots bothantisera recognize
purified rat CNTF up to serum dilutions of1/25 000 and also
identify CNTF when sciatic nerve extract isanalysed on western
blots. The mouse monoclonal antibody4-65 was raised against
recombinant rat CNTF (Stockli et al.,1991).
ImmunoprecipitationProtein A-sepharose (100 p\ of settled gel)
was incubated for 3hours at 4°C with 150-300 /il of anti-CNTF
antiserum II or acontrol immune serum using a tube rotator. After
theincubation the beads were washed 3 times with 500 jA buffer
-
CNTF expression in rat footpad 691
(10 mM Tris-HCl, pH 8, with 150 mM NaCl) and thenincubated with
300 1̂ tissue extract or extract dilutions for 3hours at 4°C. The
amount of tissue extract was adjusted toapprox. 200 VIP-inducing
units unless indicated differently.After the incubation of
antibody-coated beads with the tissueextract, the beads were
collected by centrifugation, thesupernatant was sterilized by
centrifugation through Spin-Xfilters and stored in small aliquots
at —20°C.
ImmunohistochemistryCultures of chick sympathetic neurons were
stained for VIP-IR after 4 days in culture as previously described
(Ernsbergeret al., 1989). Cells were washed, fixed for 15 minutes
with 4%paraformaldehyde in PBS, washed, permeabilized for 15minutes
with PBT1 (PBS supplemented with 1% BSA and0.1% Triton X-100) and
then incubated for 30 minutes with arabbit anti-VIP antiserum
(1:200) (Incstar Corp., Stillwater,Minnesota, USA). In some
experiments, goat anti-VIPantiserum (a generous gift of Dr. Sharp,
AFRC Edinburgh)was used, which gave identical results. After
washing,biotinylated goat anti-rabbit antiserum (1:100,
Amersham)was added for 30 minutes, followed by FTTC
streptavidin(1:100, Amersham) for 20 minutes. Then the cultures
werewashed and mounted in PBS/glycerol (l/l)- The staining forVIP
was completely abolished by preincubating the antiserumwith 40
/ig/ml VIP (Sigma) for 1 hour at room temperature.Stained cultures
were viewed with a Zeiss Axiophot fluor-escence microscope and the
proportion of VIP-IR-positiveneurons was determined. 300-600
neurons were analysed foreach experimental point, depending on the
proportion oflabelled cells.
For immunohistochemistry with footpad, the tissue wasdissected
from 21-day-old rats, fixed for 2 hours at 4°C with2%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4,washed and then
kept overnight in 0.1 M phosphate buffer,pH 7.4, supplemented with
20% sucrose. The tissue wasembedded in tissue-tec and 7 fan frozen
sections were cut.Sections were dried on gelatine-coated glass
slides andrehydrated in PGT (PBS supplemented with 0.1% gelatineand
0.2% Triton X-100). Antibody incubations were carriedout overnight
at 4°C. Rabbit antiserum I (a gift from M.Sendtner) was diluted
1:200, mouse monoclonal antibody 4-65(hybridoma supernatant,
generously provided by G. Breit-fels) was diluted 1:1 with PGT.
Control stainings were carriedout using monoclonal antibody 4-65,
which had beenabsorbed with 400 ̂ g/ml of recombinant rat CNTF
during a 1hour preincubation at room temperature. After the
incu-bation with anti-CNTF antibodies, the sections were washed(3
times for 15 minutes) and then incubated for 2 hours
withbiotinylated goat anti-mouse antibody or biotinylated
donkeyanti-rabbit antibody (1:100, Amersham). After
repeatedwashings the sections were incubated for 2 hours with
Texasred streptavidin (1:100, Amersham), washed and mounted
inPBS/glycerol (l/l).
Results
VIP-induction in cultured chick sympathetic neuronsas specific
assay for CNTF-like cholinergicdifferentiation factors in footpad
extractCNTF induces ChAT in cultures of rat sympatheticneurons and
also produces a strong increase of VIP-immunoreactivity (VIP-IR) in
cultures of chick sym-pathetic neurons (Ernsberger et al., 1989a).
Theproportion of VIP-positive neurons increases with
100 n A
80 -
60 -
20 -
001 0.1
CNTF I ng/ml I
100 -i B
80
6 0 -
Q .
>
40 -
2 0 -
I10
1•no
Footpad extract [ j jg /ni l
Fig. 1. (A) Induction of VLP-immunoreactivity in
chicksympathetic neurons by CNTF. Increasing concentrationsof rat
CNTF were added to cultures of E7 chicksympathetic neurons. After 4
days the cells were stainedfor VIP-IR and the proportion of
VIP-positive neurons wasdetermined. Each point represents the mean
± s.e.m. of atleast 3 independent experiments. (B) Induction of
VIP-immunoreactivity in chick sympathetic neurons by
footpadextracts. Increasing concentrations of P21 rat
footpadextract were added to cultured E7 chick sympatheticneurons.
The cultures were processed and analysed as inA. Each point
represents the mean ± s.e.m. of 5independent experiments.
increasing amount of CNTF and in the presence ofsaturating CNTF
concentrations about 60% of E7sympathetic neurons express
VIP-immunoreactivityafter 4 days in culture (Fig. 1A). Half-maximal
effectswere observed at a CNTF-concentration of 0.07 ng/ml.
Extracts of postnatal rat footpads induced a dose-dependent
increase in the proportion of VIP-expressingsympathetic neurons
(Fig. IB) and the activity thatcaused half-maximal effects was
arbitrarily defined as 1VIP-inducing unit. This biological assay
for VIP-inducing activity is very convenient because of
itssensitivity, reproducibility, and since it tolerates mosttissue
extracts even at high protein concentrations.Neuronal cell number
was unaffected by the added
-
692 H. Rohrer
extracts except for liver extracts which were slightlytoxic at
high concentrations. It should be pointed outhere that sympathetic
neurons from E7 embryos do notrequire any neuronal survival factor
(Ernsberger et al.,1989b), excluding effects of the added factors
via theselective survival of subclasses of sympathetic neurons.As
cultures of E7 sympathetic neurons are virtuallydevoid of
non-neuronal cells (Rohrer and Thoenen,1987; Rodriguez-Te"bar and
Rohrer, 1991), the VIP-induction is due to a direct action of the
added factor(s)on sympathetic neurons rather than to indirect
effectsvia non-neuronal cells. The activity in footpad extractsnot
only induced VIP in chick sympathetic neurons, butalso interfered
with sympathetic neuronal proliferationand supported the survival
of ciliary neurons (data notshown), as demonstrated previously for
purified CNTF(Ernsberger et al., 1989a).
Expression of VIP in cultured rat sympatheticneurons is affected
by several factors, like CDF/LIF,CNTF and other factors in
conditioned media andextracts whose molecular nature is still
unclear (Nawaand Patterson, 1990; Nawa and Sah, 1990). Thus it
wasimportant to examine to what extent CNTF contributesto the
VIP-inducing activity in rat footpad extracts. Weobserved that
chick sympathetic neurons, in contrast torat sympathetic neurons,
do not respond to murineCDF/LIF and thus CDF/LIF does not
contribute to thesignal obtained in the assay used. Also acidic and
basicfibroblast growth factor (aFGF, bFGF), which can actin a
CNTF-like manner as survival factor for chickciliary neurons
(Unsicker et al., 1987; Watters andHendry, 1987, Eckenstein et al.,
1990), did not induceVIP in chick sympathetic neurons. To determine
theidentity of the VIP-inducing activity, immuno-precipi-tation
experiments were carried out, using a polyclonalantiserum raised
against a synthetic peptide whosesequence corresponds to the
C-terminal end of CNTF(Stockli et al., 1991). Extracts of P21 rat
footpad wereincubated with protein A-sepharose-bound
anti-CNTFantibodies and the activity in the supernatant wasassayed.
The activity is referred to control immuno-precipitations using
unrelated antiserum. Whereasincubations with control antiserum did
not reduce theVIP-inducing activity in footpad (data not
shown),treatment with anti-CNTF eliminated 90% of the VTP-inducing
activity of footpad extracts (Fig. 2). A similarproportion of
VIP-inducing activity was eliminatedfrom rat sciatic nerve extract
which is a rich source ofCNTF (Manthorpe et al., 1986). The
antiserum seemsnot to recognize chick CNTF, as VIP-inducing
activityfrom CNTF-rich tissues, like adult chick sciatic
nerve(Eckenstein et al., 1990) or E15 chick eye (Barbin et
al.,1984) was not affected by the antiserum in immuno-precipitation
experiments (Fig. 2; eye extract andsciatic nerve extracts gave
identical results).
Developmental expression of CNTF in rat footpadThe time course
of the target-induced switch ofsympathetic neurons from
noradrenergic to cholinergicphenotype during sweat-gland
innervation has beenanalysed in detail (see Landis, 1988, 1990 for
review).
100 -
80 -
p 60 -
20 -
rot footpad rat sciaticnerve
chick eye
Fig. 2. Effect of immunoprecipitation with anti-CNTFantibodies
on the VIP-inducing activity in extracts of ratfootpad, rat sciatic
nerve and chick eye. Tissue extractswere incubated with either
anti-CNTF antibodies, orcontrol antibodies, bound to protein
A-sepharose beads.The beads were pelleted and the VIP-inducing
activity inthe supernatants was analysed using E7 chick
neuroncultures. The activity remaining after
immunoprecipitationwith anti-CNIt--antibodies is compared to
incubations withcontrol antibodies and expressed as a percentage
ofcontrol.
100-1
> c
? °- 60-
7 ~ 4 0 -
I-2 0 -
Day 7 Day 11 Day 21Postnatal dtvttopment
adult
Fig. 3. Developmental increase of CNTF-like activity in
ratfootpad. Footpads were dissected at differentdevelopmental time
points and extracts were analysed forVIP-inducing activity.
During the first postnatal week the sympathetic fibersexhibit
noradrenergic properties like TH and dopamineyS-hydroxylase.
Cholinergic markers are absent duringearly development but appear
during the second andthird postnatal weeks. Both ChAT activity and
VIP-immunorectivity become detectable around postnatalday 11. ChAT
activity then increases about 4-fold up toP21.
We observed an 6-fold increase in the specific activityof the
VIP-inducing factor in footpad extracts betweenP7 and P21 (Fig. 3).
In adult tissue CNTF is maintained
-
CNTF expression in rat footpad 693
at reduced levels. As CNTF-dependent cholinergicdifferentiation
of rat sympathetic neurons is a slowprocess (Saadat et al., 1989),
CNTF expression in vivoshould also precede cholinergic neuron
differentiation.In agreement with this notion, we found that the
factoris expressed in footpad before the cholinergic
differen-tiation of sympathetic neurons is detectable. Since
thespecific activity was highest at P21, footpad extractswere made
routinely from this age unless indicatedotherwise.
Tissue distribution and cellular localisation of theCNTFThe
acquisition of cholinergic properties by noradren-ergic sympathetic
neurons is controlled by interactionswith specific target tissues.
In the skin, for instance,only sympathetic fibers innervating sweat
glands in theglabrous skin of rat foot pads become cholinergic,
butnot sympathetic fibers in hairy skin, innervatingvascular smooth
muscle and piloerectors (Landis,1990). Thus it was of interest to
investigate the tissuedistribution of the VIP-inducing activity.
Comparedwith rat footpad, other tissues like hairy skin, muscleand
kidney contain less, but still measurable levels ofVIP-inducing
activity (Table 1). Heart and liver extractsalso induced VIP
immunoreactivity but at levels thatwere too low to be quantified.
The highest VIP-inducing activity was found in sciatic nerve, as
expectedfrom the high CNTF levels present in this tissue.Similarly,
in chick, CNTF-rich tissues, like sciatic nerve(Eckenstein et al.
1990) and E15 eye (Barbin et al.,
Table 1. VIP-inducing activity in tissues of 21-day-oldrats
Tissue
FootpadHairy skinKidneySkeletal muscleHeartLiverSciatic
nerve
VIP-inducing activity(units/mg protein*)
98±1320±553±179±1.5
-
units
orI
VIP
100 -
80 "
60 -
20 -
J
ControlFool pad
r+-.DenervatedFooJpod
Fig. 5. Effect of nerve transection on VIP-inducing activityand
CNTF-like immunoreactivity in rat footpad. Aftersectioning both
sciatic and saphenous nerves unilaterally atP14, footpads were
analysed for CNTF-likeimmunoreactivity (A) and VIP-inducing
activity (B) at P21.(A) Frozen sections of unoperated control (a)
and nervesectioned animals (b) were stained for
CNTF-likeimmunoreactivity using a rabbit polyclonal
anti-CNTFantiserum. Nerve staining is indicated by arrows,
sweatglands by arrowheads. (B) VIP-inducing activity in extractsof
operated footpad is compared to the unoperatedcontrol. Please note
that both CNTF-like immunoreactivityand VIP-inducing activity have
disappeared virtuallycompletely 7 days after sectioning the
nerve.
To support the finding that the VIP-inducing, CNTF-like activity
in rat footpad extracts is localized in, andderived from, Schwann
cells, an attempt was made toeliminate the Schwann cell-derived
CNTF from footpadtissue (Millaruelo et al., 1986). Previous studies
havedemonstrated that nerve transection results in a
dramatic decrease of CNTF mRNA in the nerve distalto the lesion
(Sendtner, personal communication).Therefore we investigated how
much of the VIP-inducing activity could be removed from P21
footpadsby eliminating the innervation of rat footpad. To
obtaincomplete denervation of the footpad (Mills et al., 1989)both
the sciatic and the saphenous nerves weresectioned unilaterally at
P14 and one week later at P21the footpads were analysed for CNTF
immunoreactivityand VIP-inducing activity. The contralateral side
wasused as control. We found that the CNTF-IR in thenerve plexus
was strongly decreased whereas the sweatgland staining was not
affected. In addition, the VIP-inducing activity in footpad
extracts of operated legswas drastically reduced. Taken together,
these datasuggest that the majority of the VIP-inducing, CNTF-like
activity in rat footpad homogenates is localized inSchwann
cells.
CNTF accounts only for part of the cholinergicactivity in rat
footpadHaving established the presence of CNTF as
cholinergicdifferentiation factor in rat footpad during the period
oftarget-induced switch of transmitter phenotype, it wasof interest
to determine if CNTF accounts for all, oronly part, of cholinergic
activity in the extracts.
To try to detect all cholinergic activities present in
ratfootpad homogenates we used a homologous system,i.e. cultures of
rat sympathetic neurons and analysedthe effect of footpad extracts
on the ChAT levelsexpressed by the cells. Serum-free culture
conditionswere used to exclude possible interactions with
ChAT-inducing factors in serum (Iacovitti et al., 1982;Wolinski and
Patterson, 1985a,b).
Footpad homogenates induced ChAT in a dose-dependent way in
cultures of newborn rat sympatheticneurons. After 7-11 days in
culture in the presence of 10VTP-inducing units, the specific ChAT
activity wasincreased about 20-fold as compared with
controlcultures in the presence of NGF alone. ChAT inductionin
cultures of rat sympathetic neurons required higherconcentrations
of footpad extract (Fig. 6,7) as com-pared with VIP induction in
chick sympathetic neuroncultures (Fig. 1). This may be either due
to differencesin the stability of CNTF in chick and rat
sympatheticneuron cultures, because of different
receptors/recep-tor occupancies required, or due to negative
orpotentiative interactions with other factors present inthe tissue
extracts. Previous experiments, using purifiedCNTF, demonstrated a
similar, but smaller differencebetween chick and rat neuron
cultures with respect tothe CNTF concentration required for their
specificdifferentiation effects (Ernsberger et al., 1989a; Saadatet
al., 1989).
To determine if the induction of ChAT is due to theCNTF in the
footpad, CNTF was removed by immuno-precipitation. Interestingly
only about 55% of theChAT-inducing activity of footpad extracts was
elimi-nated, whereas virtually all ChAT-inducing activity insciatic
nerve homogenates was precipitated (Fig.6A,B). When
immunoprecipitation supernatants of
-
CNTF expression in rat footpad 695
- 4 0 - 1
oQ. 3 0 -
J 20 io2uoo"5Q.
10 -
r i^ r i i1/10001/500 1/200 V100 1/50Footpad extract ( Fold
dilution )
r1/1000 V500 1/200 V100 V50Sciatic nerve extract (Fold
dilution)
Fig. 6. Induction of ChAT in cultures of rat sympatheticneurons
by footpad and sciatic nerve extracts: Effect ofanti- CNTF
antibodies. Tissue extracts were incubated withprotein A-
sepharose-bound anti-CNTF antiserum (x) orcontrol immune serum (O).
After centrifugation, theCNTF-depleted extracts were assayed for
ChAT-inducingactivity. Effect of anti-CNTF antibodies on
ChAT-inducingactivity in footpad (A) and sciatic nerve (B). Please
notethat only part of the ChAT-inducing activity in rat footpad,but
virtually all ChAT-inducing activity in sciatic nerve iseliminated
by anti-CNTF antibodies.
footpad extracts were tested in parallel experiments forVIP and
ChAT-inducing activity using cultures of chickand rat sympathetic
neurons, respectively, 90% of theVIP-inducing activity, but only
55±8% of the ChAT-inducing activity was eliminated. These results
suggestthat at least two cholinergic factors are present in
ratfootpad homogenates, CNTF and a second factor(s)which is not
recognized by antibodies against CNTF.
As CNTF is localized in Schwann cells and is reducedupon nerve
transection, it was of interest to investigateto what extent
transection would affect the ChAT-inducing activity. Since CNTF
accounts for about halfof the ChAT-inducing activity in rat footpad
homogen-ates, transection was assumed to reduce the levels
ofChAT-inducing activity by 50%. Surprisingly, the
•5a.
~ E
30-
r> | 20 Ho 95 2-
10 -
•)i
20 40 80100
Footpad extract [_ug/ml]
Fig. 7. Denervation results in a loss of ChAT-inducingactivity
in rat footpad tissue. After unilateral sectioning ofboth sciatic
and saphenous nerves at P14, footpads wereanalysed for
ChAT-inducing activity. Increasingconcentrations of operated (x)
and unoperated control (O)footpad extracts were added to rat
sympathetic neuroncultures. (•) Cultures without added extract.
ChATactivity was assayed after 11 days in culture. The datashown
are from a representative experiment. In twoadditional experiments
similar results were obtained.
ChAT-inducing activity in rat footpad homogenates wasdrastically
reduced after nerve transection, whichindicates that all
ChAT-inducing activities detectable inrat footpad homogenates are
dependent on the pres-ence of intact innervation. Since aFGF is
present insciatic nerve and rapidly disappears in the nerve
distalto a lesion (Eckenstein et al., 1991), we investigatedwhether
FGF is able to induce ChAT in cultures of ratsympathetic neurons.
Neither bFGF nor aFGF had anysignificant effect on ChAT levels
(100% and 112% ofcontrol levels, respectively), excluding FGF as
acandidate for the non-CNTF-like cholinergic factor inrat
footpad.
Discussion
After our previous demonstration that CNTF specifi-cally affects
the cholinergic differentiation of culturedchick and rat
sympathetic neurons (Ernsberger et al.,1989a; Saadat et al., 1989),
the present paper providesevidence that a CNTF-like activity is
indeed present invivo at the time of cholinergic differentiation.
Thebiological and immunological characteristics of theVIP-inducing
activity detected in footpad homogenatesstrongly suggest that this
activity is CNTF or a relatedmolecule. The cholinergic activity not
only induced VIPin cultured chick sympathetic neurons but also
inter-fered with sympathetic neuronal proliferation andsupported E8
ciliary neuron survival. Other factors likeCDF/LIF (Yamamori et
al., 1989), aFGF, bFGF(Watters and Hendry, 1987; Unsicker et al.,
1987;Eckenstein et al., 1990) which in other biological assays
-
696 H. Rohrer
produce CNTF-like effects, did not induce VIP in ourchick
sympathetic neuron assay and thus did notcontribute to the signal
obtained in footpad extracts.Virtually all of the VIP-inducing
activity detected byour assay can be attributed to CNTF as 90% of
theactivity is recognized by the anti-CNTF antiserum. In aprevious
study, a cholinergic activity in rat footpadhomogenates was
described which displayed similarbiological properties and
developmental expression asthe CNTF-like factor, but was not
recognized by anti-CNTF antibodies (Rao and Landis, 1990).
Althoughthe negative finding may simply be due to a loweraffinity
of the antibodies used, it may be taken asindication that the
CNTF-like activity in footpadextracts is not identical to CNTF and
thus is notrecognized by all antibodies that recognize CNTF.
The amount of VIP-inducing activity present in ratfootpad is
considerably higher than the activity in hairyskin, skeletal
muscle, heart and liver. Elevated levelswere also observed in
kidney homogenates. Due to thesmall and only partly quantifiable
amounts of activity,no attempt was made to identify the nature of
the VIP-inducing activity in those tissues by immunoprecipi-tation.
In previous studies, no CNTF mRNA wasdetected in these tissues
(Stockli et al., 1989). VIP-inducing activities are produced by
primary cultures ofdifferent tissues analysed, i.e. heart, gut and
skin, andthe conditioned medium of heart muscle cells
containsseveral VIP-inducing activities with prominent
contri-butions by factors with an apparent relative molecularmass
of 85 x 103 and 45 x 103 (Nawa and Patterson,1990; Nawa and Sah,
1990). Thus it is unclear whetherthe low level expression is due to
non-CNTF VIP-inducing factors or to CNTF, produced either by
tissuecells or by Schwann cells.
In rat footpad, the majority of the CNTF-like VIP-inducing
activity extract is derived from Schwann cells.This conclusion is
supported by the specific Schwanncell staining using monoclonal and
polyclonal anti-bodies against CNTF. In addition, VIP-inducing
ac-tivity and Schwann cell staining both disappear aftertransection
of sciatic and saphenous nerves and thedevelopmental increase in
the levels of VIP-inducingactivity in rat footpad during the first
3 postnatal weeksis paralleled by the increase in CNTF mRNA
(Stockli etal., 1989) in the sciatic nerve. Although the existence
ofadditional sites of low level CNTF production cannot beexcluded,
the available evidence suggests a specific glialorigin of CNTF in
footpad.
The postnatal increase in the amount of CNTFcorrelates with the
increase in expression of cholinergicproperties in sympathetic
fibers innervating the sweatglands in the footpad (Landis and
Keefe, 1983; Leblancand Landis, 1986; Landis et al., 1988). As
cholinergicsympathetic differentiation in vitro proceeds with
aconsiderable delay (Saadat et al., 1989), it is expectedthat
cholinergic factors must be present several daysbefore the
detection of cholinergic properties. How-ever, CNTF is a cytosolic
protein as indicated by theabsence of a hydrophobic leader sequence
and its non-release from transfected Hela or COS-cells (Stockli
et
al., 1989; Lin et al., 1989). A physiological role ofCNTF in
vivo thus either involves a release during celldeath or a novel
release mechanism as described for IL-1 or for plasminogen
activator inhibitor (Rubartelli etal., 1990; Belin et al., 1989).
An interesting character-istic of these unconventional release
mechanisms is thepossibility of release under certain conditions
i.e. byinducing cellular differentiation (Belin et al., 1989)
orunder stress conditions (Rubartelli et al., 1990). Thereare an
increasing number of proteins without signalsequence but with known
extracellular functions andcell surface receptors for these
factors, such as bFGF(Abraham et al., 1986) and ADF (Tagaya et al.,
1989).If the CNTF-like factor expressed by Schwann cells isinvolved
in cholinergic sympathetic neuron differen-tiation, the
availability of this factor must be restrictedto those fibers that
acquire cholinergic properties.There is considerable evidence to
indicate that thecholinergic differentiation of sympathetic fibers
in therat footpad is due to interaction with its target, thesweat
glands (Landis, 1988, 1990). The clearest evi-dence came from
transplantation experiments wheresweat glands were transplanted
into areas of hairy skin.Cholinergic sympathetic fibers are not
present in hairyskin; however, in the transplant situation, sweat
glandinnervating fibers became cholinergic (Schotzinger andLandis,
1988). As the innervating fibers are separatedfrom the sweat glands
by a basal lamina (Landis andKeefe, 1983), cholinergic sympathetic
differentiationimplicates a diffusible cholinergic differentiation
factorproduced by sweat glands. The virtually complete andparallel
reduction of VIP-inducing activity and CNTF-immunoreactivity upon
nerve transection suggests thatthe majority of the activity is
localized in Schwann cells.Thus, the CNTF-like activity in footpad
extracts seemsto be a less likely candidate for the sweat
glandcholinergic differentiation factor. However, a role forSchwann
cell-derived CNTF in cholinergic differen-tiation, implicating a
diffusible signal from sweat glandswhich would cause a local and
specific release of CNTF,is not excluded.
The analysis of the effects of footpad extracts onChAT-induction
in rat sympathetic neurons providedevidence for a second,
non-CNTF-like cholinergicactivity. The inability to eliminate all
ChAT-inducingactivity from rat footpad homogenates is not due
tolimitations of the immmunoprecipitation conditions:increasing the
amount of anti-CNTF antibody did notprecipitate more ChAT-inducing
activity. In addition,in sciatic nerve homogenates much higher
concen-trations of ChAT-inducing activity than those present
infootpad homogenates were completely precipitatedunder identical
conditions. One candidate for such anon-CNTF cholinergic factor is
CDF/LIF (Yamamori etal., 1989; Yamamori, 1991). However, carefully
con-trolled immunoprecipitation experiments with anti-CDF/LEF have
shown that the ChAT-inducing activityin rat footpad homogenates is
not immunologicallyrelated to CDF/LIF (Rao and Landis, 1990).
It is known that ciliary survival activity and CNTFmRNA in
peripheral nerves of adult mammals is
-
CNTF expression in rat footpad 697
decreased distally to a lesion site within a time period ofa few
days (Millaruelo et al., 1986; Sendtner, unpub-lished results).
Therefore we investigated whether non-CNTF ChAT-inducing activity
could be identified infootpads after lesion of the sciatic and
saphenousnerves. Surprisingly, the ChAT-inducing activity infootpad
extracts was drastically reduced, suggestingthat both CNTF and
non-CNTF-like factors are lostupon nerve transection. This may
indicate an inner-vation-dependent synthesis of cholinergic factors
in thetarget or a CNTF-like expression and localization of
thesecond factor. Acidic FGF is present in sciatic nerveand
transection results in dramatic decrease of aFGFcontent distal to
the lesion (Eckenstein et al., 1990). Asneither aFGF nor bFGF had
any effect on VIP- orChAT-induction, a contribution of FGF to
non-CNTF-like cholinergic activity in footpad can be excluded.
The existence of a multitude of different cholinergicfactors,
detected by in vitro studies, may be explainedby the assumption
that different factors are responsiblefor the induction of specific
neuronal characteristics indifferent cholinergic neurons. In this
context it shouldbe noted that the transcription of the VTP gene
isregulated by two different signal transduction pathways(Fink et
al., 1991). Although some factors affect only asingle neuronal
property (Nawa and Patterson, 1990),most of the factors described,
like CDF/LIF and CNTF,are able to simultaneously influence a wide
variety ofneuronal characteristics. Cholinergic
differentiationfactors may be necessary not only for the induction,
butalso for the maintenance of the cholinergic phenotype,and it is
not known whether induction and maintenanceof specific neuronal
traits are due to the same ordifferent factors in vivo. Thus
induction and/or main-tenance of a specific neuronal phenotype may
dependon the combinatorial interaction of different factorswhose
contribution to the final phenotype needs to beanalysed by
experiments where the function of indi-vidual factors is interfered
with, i.e by the application ofactivity-blocking antibodies or by
altered CNTF ex-pression in transgenic mice.
Thanks are due to H. Thoenen for continuous support andcritical
comments on the manuscript. I want to thank M.Sendtner for
collaboration and discussions, K. Stockli forhelpful suggestions
and C. Krieger, C. Ronnacker and C.Muller for excellent technical
assistance. H.R. was supportedby the Deutsche
Forschungsgemeinschaft (SFB 220).
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