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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(Accepted 5 December 1991)