12
8/11/2019 Neuron 1995 http://slidepdf.com/reader/full/neuron-1995 1/12 Neuron, Vol. 15, 585-596, September, 1995,Copyright © 1995 by Cell Press Neu Differentiation Factor Is a Neuron-Gila Signal and Regulates Survival Proliferation and Maturation of Rat Schwann Cell Precursors Z. Dong,*t A. Brennan,*t N. Liu,~ Y. Yarden,§ G. Lefkowitz,§ R. Mirsky,* and K. R. Jessen* *Department of Anatomy and Developmental Biology University College London London WC1E 6BT England ~Amgen Center Thousand Oaks, California 91320 §The Weizman Institute Rehovot 76100 Israel Summary We show that J~ forms of Neu differentiation factor NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates pre- cursor survival and maturation is blocked by the extra- cellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These ob- servations provide important evidence that NDF is one of the hitherto elusive neuron-gila signaling mole- cules long proposed to regulate development in the Schwann cell lineage. Introduction Glial growth factor GGF) has been implicated in the regu- lation of Schwann cell development from its first descrip- tion as a mitogen for cultured rat Schwann cells Raft et al., 1978; Marchionni et al., 1993). It has now emerged that GGF belongs to a novel growth factor group, the Neu differentiation factors NDFs; Ben-Baruch and Yarden, 1994) that have been cloned from rat NDF; Wen et al., 1992), human heregulin; Holmes et al., 1992), bovine GGF; Marchionni et al., 1993), and chick acetylcholine receptor-inducing activity [ARIA]; Falls et al., 1993) tis- sues. They are encoded by a single gene but exist in an unusual variety of forms generated by alternative splicing. These forms fall into two major groups, designated ~ and I~, according to a difference in a crucial part of the mole- cule, an epidermal growth factor-like EGF-like) domain that on its own appears sufficient for receptor activation Wen et al., 1994). The NDF receptors are two related tyrosine kinases, HER-3/ErbB3 and HER-4/ErbB4 Tzahar 1Theseauthors contributed equally o this work. et al., 1994; Wen et al., 1994). In addition, the ErbB2 pro- tein, which is expressed in Schwann cells, probably acts as an NDF coreceptor in combination with ErbB3 and ErbB4 Cohen et al., 1992; Peles et al., 1993; Sliwkowski et al., 1994). In various celt types, activation of these recep- tors promotes proliferation or differentiation Peles et al., 1992; Falls et al., 1993; Goodearl et al., 1993; Pinkas- Kramarski et al., 1994; Vartanian et al., 1994). NDF may also be involved in the regulation of lineage choice in the rat neural crest Shah et al., 1994). In these experiments, crest cell clones were grown in a cocktail of known and unknown growth factors to ensure that differen- tiated neurons and Schwann cells were readily generated over time in culture. Addition of NDF strongly and selec- tively suppressed the generation of neurons in a manner consistent with the idea that NDF acted on crest cells to make the choice of the neuronal lineage less likely and that of the Schwann cell lineage more likely. NDFs were the first polypeptide growth factors shown to control Schwann cell behavior in vitro initially studied in this context as GGF extracted from brain), and they have remained prime candidate regulators of Schwann celldevelopment ever since. Nevertheless, the only known effect of these proteins on cells in the Schwan n cell lineage remains that originally described by Brockes et al. 1979), namely the stimulation of DNA synthesis in Schwann cells obtained from postnatal nerves. The idea that the embryonic period of nerve develop- ment might be a time when NDF has a particularly im- portant role to play in control of the Schwann cell lineage is supported by the cellular localization of N F mRNA in rodent embryos during the second half of the gestation period Marchionni et al., 1993; Orr-Urtreger et al., 1993; Meyer and Birchmeier, 1994). These studies have shown that the two main sources of axons in embryonic nerves, namely spinal motor neurons and dorsal root ganglion DRG) neurons, are sites of strikingly high levels of N F mRNA, whereas the glial cells within the nerve itself are negative. It is therefore p ertinent to ask wh ether neu ronally derived NDF acts to regulate glial development during this period. Analysis of glial development in the limb nerves of rat embryos Jessen et al., 1994; Gavrilovic et al., 1995) has now made it possible to address this question in some detail. In the rat hind limb, the embryonic period of nerve devel- opment spans about a week, from embryonic day 14 E14), when a significant number of nerves are first present, to birth at E21. At birth, essentially all the glial cells isolated from peripheral nerves have the well-established pheno- type of Schwann cells, with some cells being in the initial stage of myelination Jessen and Mirsky, 1992). It is, how- ever, only toward the end of the embryonic period, namely from E17 onward, that Schwann cells are present in large numbers in the major limb nerves. No such cells are pres- ent at the earliest stage of nerve development. Instead, at E14 essentially all the cells isolated from limb nerves

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Neuron, Vol. 15, 585-596, September,1995, Copyright© 1995 by Cell Press

N e u D i f f e r e n t i a t i o n F a c t o r I s a N e u r o n - G i l a S i g n a l

a n d R e g u l a t e s S u r v i v a l P r o l if e r a ti o n

a n d M a t u r a t i o n o f R a t S c h w a n n C e l l P r e c u r s o r s

Z . D o n g , * t A . B r e n n a n , * t N . L i u , ~

Y . Y a r d e n , § G . L e f k o w i tz , § R . M i r s k y , *

a n d K . R . J e s s e n *

* D e p a r t m e n t o f A n a t o m y a n d D e v e l o p m e n t a l B io l o g y

U n i ve rs i t y C o l l ege London

L o n d o n W C 1 E 6 B T

E n g l a n d

~ A m g e n C e n t e r

T hous and Oaks , C a l i fo rn i a 91320

§T he We i zma n Ins t it u te

R e h o v o t 7 6 1 0 0

Israe l

S u m m a r y

W e s h o w t h a t J~ f o r m s o f N e u d i f f e re n t i a t io n f a c t o r

N D F ) , h o m o l o g o u s t o a c e t y l c h o l in e r e c e p t o r - i n d u c i n g

a c t i v i t y , g l i a l g r o w t h f a c t o r , a n d h e r e g u l i n , p r e v e n t

a p o p t o t i c d e a t h a n d s t i m u l a t e D N A s y n t h e s i s o f t h e

E 1 4 S c h w a n n c e l l p r e c u r s o r , a n e a r ly c e l l i n t h e r a t

S c h w a n n c e l l l in e a g e . W h e n p r e c u r s o r s a r e e x p o s e d

t o N D F i n d e fi n e d m e d i u m , t h e y g e n e r a t e S c h w a n n

c e l ls w i t h o u t t h e r e q u i re m e n t f o r D N A s y n t h e s i s a n d

w i t h a t i m e c o u r s e t h a t i s s i m i l a r t o t h a t w i t h w h i c h

S c h w a n n c e l l s a p p e a r i n e m b r y o n i c n e r v e s i n v i v o .

F u r t h e r m o r e , a n e u r o n a l s i g n a l t h a t a l s o m e d i a t e s p r e -

c u r s o r s u r v i v a l a n d m a t u r a t io n i s b l o c k e d b y t h e e x t r a -

c e l l u l a r d o m a i n o f t h e E r b B 4 N D F r e c e p t o r , a p r o t e i n

t h a t s p e c i f i c a l ly b l o c k s t h e a c t i o n o f N D F s . T h e s e o b -

s e r v a t i o n s p r o v i d e i m p o r t a n t e v i d e n c e t h a t N D F i s o n e

o f t h e h i t h e r t o e l u s i v e n e u r o n - g i l a s i g n a l i n g m o l e -

c u l e s l o n g p r o p o s e d t o r e g u l a t e d e v e l o p m e n t i n t h e

S c h w a n n c e l l l i n e a g e .

I n t r o d u c t i o n

Gl i a l g row th fac to r GGF ) has b een i mp l i ca ted in the regu -

l a t ion o f S ch w ann ce l l deve l opm en t f rom i ts f ir s t desc r i p -

t i on as a m i togen fo r cu l tu red ra t S chw ann ce l l s R a f t e t

a l . , 1978; March ionn i e t a l . , 1993) . I t has now emerged

tha t GG F be l ongs to a nove l g row th fac to r g roup , the N eu

d i f fe ren t i a t ion fac to rs N D F s ; B en -B a ruch and Y a rden ,

1994 ) tha t have been c l oned f rom ra t N D F ; Wen e t a l. ,

1992), hum an heregu l in ; Ho lme s e t a l . , 1992), bov ine

GG F; March ionn i e t a l . , 1993), and ch ick acety lcho l ine

rec ept or- in duc ing ac t i v i ty [ARIA] ; F a l l s e t a l . , 1993) t i s -

sues . T hey a re en coded by a s i ng l e gene bu t ex i s t in an

unusua l va r i e ty o f fo rms gene ra ted by a l te rna t i ve sp l ic i ng .

T hese fo rms fa l l i n to tw o m a j o r g roups , des i gna ted ~ an d

I~, acco rd ing to a d i f fe rence in a c ruc ia l par t o f the mole-

cu l e , an ep i de rma l g row th fac to r - li ke E GF - l i ke ) doma i n

tha t on i ts ow n appea rs su f f i c ien t fo r recep to r a c t i va t ion

Wen e t a l. , 1994) . T he N D F recep to rs a re tw o re l a ted

ty ros i ne k i nases , H E R -3 /E rbB 3 and H E R -4 /E rbB 4 T zaha r

1These authors contributedequ ally o this work.

e t a l . , 1994; We n e t a l . , 1994) . In add i t ion , the ErbB2 pro-

te i n , w h i ch i s exp ressed i n S chw a nn ce l l s , p roba b l y acts

as an N D F co recep to r in com b i na t i on w i th E rbB 3 and

ErbB 4 Coh en e t a l ., 1992; Pe le s e t a l. , 1993; S l iwkows k i

e t a l . , 1994). In var iou s ce lt types, ac t i va t ion o f these recep-

to rs p rom o tes p ro l i f e ra t i on o r d i f fe ren t i a t i on P e l es e t a l .,

1992; Fa l l s e t a l. , 1993; G ood ear l e t a l . , 1993; P inkas-

Kramarsk i e t a l . , 1994; Var tan ian e t a l . , 1994) .

N D F may a l so be i nvo l ved i n the regu l a t i on o f l i neage

cho ice in the ra t neura l c res t Shah e t a l . , 1994) . In these

expe r i me n ts , c res t ce l l c l ones w e re g row n i n a cock ta il o f

know n and unkn ow n g row th fac to rs to ensu re tha t d if fe ren -

t i a ted neu rons and S chw a nn ce l l s w e re read i ly gene ra ted

ove r t i me i n cu ltu re . A dd i t i on o f N D F s t rong l y and se l ec -

t i ve l y supp ressed the ge ne ra t i on o f neu rons i n a mann e r

cons i s ten t w ith the i de a tha t N D F ac ted on c res t ce l l s to

make the cho i ce o f the neu rona l l i neage l ess l i ke l y and

tha t o f the S ch w ann ce l l li neag e more l i ke l y .

N D F s w e re the f ir s t po l ypep t i de g row th fac to rs show n

to contro l Schwa nn ce l l behav ior in v i t ro in i t i a l ly s tud ied

i n th i s con tex t as GG F ex t rac ted f rom b ra in ) , and they

h a v e r e m a i n e d p r im e c a n d i d a t e r e g u l a to r s o f S c h w a n n

c e l l d e v e l o p m e n teve r s i nce . N eve r the l ess , the on l y know n

e f fec t o f these p ro te i ns on ce l l s i n the S chw an n ce l l l i neage

rema i ns tha t o r i g i na l l y desc r i bed by B rockes e t a l . 1979 ),

nam e l y the s t i mu l a t i on o f D N A syn thes i s i n S chw ann ce l ls

ob ta i ned f rom pos tna ta l ne rves .

T he i dea tha t the embry on i c pe r i od o f ne rve deve l op -

men t m i gh t be a t ime w hen N D F has a p a r t i cu l a r ly i m-

po r tan t ro l e to p l ay i n con t ro l o f the S chw an n ce l l l i neage

i s suppo r ted by the ce l l u l a r l oca l iza t i on o f N F m R N A i n

roden t embryos du r i ng the second ha l f o f the ges ta t i on

per iod Ma rch ionn i e t a l. , 1993; Orr -U r t reger e t a l . , 1993;

Meye r and B i r chme i e r , 1994) . T hese s tud i es have show n

tha t the tw o ma i n sou rces o f axon s in em bryon i c ne rves ,

name l y sp i na l mo to r neu rons and do rsa l roo t gang l i on

DRG) neurons, a re s i tes o f s t r i k ing ly h igh leve ls o f N F

mR N A , w he reas the g l ia l ce l ls w i th i n the ne rve i t se l f a re

nega t i ve . I t is the re fo re p e r t i nen t to ask w h e the r neu rona l ly

de r i ved N D F ac ts to regu l a te g l i a l deve l opm en t du r ing th i s

pe r i od . A na l ys is o f g l i a l deve l opm en t i n the l i mb n e rves

of ra t em bryo s Jessen e t a l . , 1994; Ga vr i lov ic e t a l . , 1995)

has now mad e i t poss i b l e to add ress th i s ques t i on i n some

deta i l .

I n the ra t h i nd l i mb , the emb ryon i c pe r i od o f ne rve deve l -

opm en t spans abou t a w eek , f rom em bryon i c day 14 E 14 ),

w hen a s i gn i f ican t num ber o f ne rves a re f i rs t p resen t , to

b i r th a t E21. A t b i r th , ess ent ia l l y a l l the g l ia l ce l ls i so la ted

f rom pe r i phe ra l ne rves have the w e l l -es tab l ished pheno -

type o f Schwann ce l l s , w i th some ce l l s be ing in the in i t i a l

s tag e o f m ye l ina t ion Jessen and M i rsky , 1992). I t i s, how-

eve r , on l y tow a rd the en d o f the em bryon i c pe r i od , name l y

f rom E 17 onw ard , tha t S chw a nn ce l l s a re p resen t i n l a rge

num bers i n the ma j o r l imb ne rves . N o such ce l l s a re p res -

en t a t the ea r l i es t s tage o f ne rve d eve l opm en t . Ins tead ,

a t E14 esse nt ia l l y a l l the ce l l s i so la ted f rom l imb nerves

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Neuron

586

are Schwann cel l precursors (Jessen et al. , 1994). These

cel ls represent a d ist inct intermediary stage in the genera-

t ion of Schwann cel ls f rom the neural crest. The precursors

di f fer f rom Schwan n cel ls in a n umber of ways: they die

abrupt ly by apoptosis when removed f rom axo nal contact

in v it ro; they do not express the Schwann cel l marker $100

in the cytoplasm; they are not induced to synthesize DNA

by f ibroblast growth factor 2 (FGF2) in the presence of

forskol in, a typical Schwann cel l m i togen combinat ion;

and in v i t ro they have a f lat tened morphology showing

many cel l -cel l contacts.

Thus, two separate members of the Schwann cel l l in-

eage f igure in the emb ryonic dev elopm ent of major per iph-

eral nerves: Schwann cel l precursors and Schwan n cel ls .

The t ime between E14/15 and E17/18 is cri t ical in nerve

developm ent , s ince dur ing th is per iod Schw ann cel l pre-

cursors progress to generate Schw ann cel ls . Al though th is

swi tch in g lia l phenotype involves change in a numb er of

diverse cel lular propert ies, i t occurs relat ively abruptly,

so that only dur ing a br ief wind ow at E16 can s igni f icant

numbers of both cel l types be isolated f rom the major l imb

nerves. In the present paper, we have asked wh ether NDF

might be involved in regulat ion of survival, prol i ferat ion,

and l ineage progression of Schw ann cel l precursors, s ince

these represent m ajor issues in g l ia l developme nt dur ing

the embryon ic per iod of nerve growth.

The resul ts show that NDF blocks death and, at h igher

concentrat ions, s t imulates DNA synthesis in E14 precur-

sors. Using mult iple cri teria to dist inguish prec ursors from

Schwann cel ls , we also show that , when expose d to NDF

in def ined medium , precursors genera te Schwann cel ls in

v i t ro wi th a t ime course s imi lar to that wi th which S chwann

cel ls appear in developing per ipheral nerves in v ivo. DNA

synthesis is not necessary for th is l ineage p rogression to

occur . NDF protein is not detected in E14 precursors but

is present in the cel l body an d axo ns of E14 DRG neurons.

Fur thermore, we dem onstrate that a neu ron-der ived s ignal

that mediates precursor surv ival and maturat ion can be

blocked by addi t ion of the ext race l lu lar doma in of the

ErbB4 NDF receptor , a protein that speci f ical ly b locks the

act ion of NDFs. These observat ions provide important ev i-

dence that NDF m ay be one of the hi ther to e lusive neuron -

gl ia s ignal ing m olecules long p roposed to reg ulate devel -

opment in the Schwann cel l l ineage (Bray et al. , 1981;

Ratner et al. , 1985; Jesse n et al. , 1987; Bu nge et al. , 1990;

Jessen and Mirsky, 1992).

Results

NDF Rescues Schwann Cell Precursors in a 20 hr

Survival Assay

To test wheth er NDF could p revent the abrupt apopto t ic

death that precursors undergo in def ined medium , precur-

sors were expos ed to var ious concentrat ions of NDF~-2

and the corres pond ing ~ form, NDF~-2, in a 20 hr survival

assay identical to that used pre vious ly (Jessen et al. , 1994;

Gavri lovic et al. , 1995). These and al l other experiments

in th is pape r were carr ied out in ent i re ly def ined med ium

contain ing no serum. In these in i t ia l surv ival expe r iments,

A 140

120-

-- 100,

8 0

6 ~

o~ 40

20-

0

0

• NDF~-2 .

........ ....... N D F { z - 2 ~ l r

- ' - ~ - NDF- EGF B-1 ~ . . . . . . . . .

- - - ' - ' - ND F . EG ~ I 3 ~ r . ; - : --: ' . . . . . .

10 NDF (pM)1 00 1000

Figure 1. I~ Forms of NDF P revent Death of E14 Precursors

(A) Results rom a 20 hr survival assaywith survival percentage alcu-

lated as described n ExperimentalProcedures. n this and all subse-

quent graphs, each point represents he average _+ SEM.

(B) Representative ield of E14 cultures after 20 hr in de fined medium

only.

(C and D) Representative ield of E14 cultures after 20 hr in d efined

medium plus NDF~-2 (100 pM). (D) show s L1 immunofiuorescence

view of (C).

Magnification, 800 x .

the me dium con tained a low insul in co ncentrat ion (1 nM)

that is suff icient to bind to insul in rece ptors withou t signif i-

cant bin ding to type 1 insul in-l ike grow th factor (IGF) recep -

tors (Sara and Hall , 1990). I t was found that NDFI~-2 acte d

as a potent, dose-d epende nt suppresso r of precursor

death, wherea s the a form, which var ies in the amino acid

sequence of the EGF- l ike domain, had very l i t t le ef fect

(Figure 1).

Using ident ical condi t ions and assay, we invest igated

fur ther which do mains wi th in the NDF molecule were re-

spon sible for the survival effect. NDFI3-1 and NDFI~-3,

forms that vary in the amino acid sequence of the carbox y-

term inal tai l , had effects very similar to those of NDFI3-2,

wherea s correspo nding (~ forms were inef fective (data not

shown). Surv ival was also obtained wi th a shor ter form of

NDFI3-1 (residues 177-246), which lacks both the imm uno-

globul in- l ike doma in and the spacer dom ain of NDF (NDF-

EGFI3-1 ; Figure 1), whe reas th e co rresp ond ing (~ form wa s

essent ia l ly inef fect ive (data not shown). An isolated EGF-

l ike domain of NDFI~ (residues 17 7-228), which als o lacks

the carboxy- termina l ta i l of NDF that in proND F corre-

sponds to the juxta mem bran e region, was the most potent

of al l the forms tested (NDF-EGFI3; Figure 1).

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NDF and Schwa nn Cell Precursors

587

1 ~ 2 5 j

l o

0

25-

U . I .

~_20-

¢ , ~ -

O 1 5 -

~ 1 0

~ .

al 5-

0

0

/ ~ N D F I 3 -2

400pM

-~ .INDFI3-2 120pM

~ .......... ND F~-2 40pM

'P--.~.,FGF-2 180pM+FO R

I I I I

B • FGF -2 180pM .,t

- - - - - - -

NDFI3-2 400pM

f

/

/

~ . jlt / . / /

/

17 2'0 30 4;

T I M E H o u r s )

Figure 2. NDF StimulatesDNA Synthesis n E14 Precursors

The results show the percentageof Ll-positive cells that were also

BrdU-positive ol lowing 1.5 hr long exposures o BrdU and d ouble

immunolabelingwith antibodies o L1 and B rdU. The BrdU pulses

were given to s ister cultures at thre e different imes during the first

20 hr after plating. n (B) he mediumwas changed rom one containing

NDF6-2 to one containing FGF 2 plus forskolin at 20 hr, and DNA

synthesiswas examinedusing wo further 1.5 hr BrdU pulsesas indi-

cated.

T hus , the E GF - l i ke doma i n o f N D F I3s i s su f f i c ien t fo r

essent ia l l y com ple te suppress ion o f death in E14 Schw ann

ce l l p recu rso rs , w he reas e fo rm s o f N D F su ppo r t ve ry l ow

l eve l s o f su rv i va l unde r the c ond i t i ons used i n these expe r i -

men ts .

N D F S t i m u l a t e s D N A S y n t h e s i s in S c h w a n n

C e l l P r e c u r s o r s

I n the e xpe r i me n ts desc r i bed above , su rv i va l o f ten ove r -

sho t the 100 pe rcen tage mark a t h i gh N D F conce n t ra t i ons ,

sugges t i ng tha t N D F cou l d s t i mu l a te ce l l d iv i s i on .

T h i s w as tes ted d i rec t l y by mon i to r i ng D N A syn thes i s

du r i ng the 20 h r su rv i va l pe r i od . D N A syn thes i s w as de -

t e c t e d u s i n g t h e b r o m e d e o x y u r i d i n e ( B rd U ) m e t h o d a n d

doub l e i mm uno l ab e l i ng w i th an t i bod i es aga i ns t the ce l l

adhe s i on mo l ecu l e L1 to i den t i f y p recu rso rs . S i s te r cu l -

tu res w e re exposed to th ree 1 .5 h r pu l ses o f B rdU w i th

each pu l se end i ng a t 5 , 15 , o r 20 h r a f te r p l a t i ng . T he

expe r i men t w as ca r r i ed ou t in the p resence o f F G F 2 p l us

fo rsko l in , as a nonm i togen i c con t ro l (Gav r i l ov i c e t a l. ,

1995 ) , o r va r i ous concen t ra t i ons o f N D F I~ -2 i n med i um

con ta i n i ng 1 nM i nsu li n and 13 nM IGF 1 (F i gu re 2A ). T he

5 h r pu l se gave a s ire i a r p ropo r t ion o f B rdU -pos i t ive nuc l e i ,

i r respec t i ve o f the N D F concen t ra t i on used . T h i s w as ex -

pec ted becaus e the t ime l ag gene ra l l y i nvo l ved i n m i to -

gen i c responses ensu res tha t D N A syn thes i s du r i ng the

f ir s t few hou rs i n v i t ro re f l ec ts the p rev i ous m i togen i c i npu t

seen i n v i vo ra the r than the i npu t f rom g row th fac to rs p res -

en t i n the cu l tu re d i sh . F u r the rmore , the pe rcen tage o f

p recu rso rs syn thes i z i ng D N A a t th i s po i n t (120/0 14 ) w as

i n exce l l en t ag ree me n t w ith the D N A syn thes i s ra te o f

these ce l l s in v i vo , mea su red us i ng B rdU pu l ses o f s i m i l a r

dura t ion (S tewar t e t a l . , 1993) . In the subsequent per iod ,

D N A syn thes i s in ce l l s expose d to F G F 2 p l us fo rsko l in

rap i d l y dece l e ra ted , i n acco rd ance w i th p rev i ous f i nd ings

(Gav r i lov ic e t a l . , 1995) . Ho wev er , NDFI3-2 ac te d as a

d o s e - d e p e n d e n t m i t o ge n : t h e m i t o g e n i c e f fe c t o f 4 0 p M

w as ba re l y de tec tab l e (a lthough su rv i va l a t th is N D F con -

cen t ra t i on w as 100 ; see F i gu re 3A ); the i n i t ia l ra te o f

D N A syn thes i s w as ma i n ta i ned n 120 pM; and i t i n c reased

in 400 pM (F igure 2A) .

T o con f i rm tha t N D F ~-2 cou l d a l so i nduce D N A syn the -

s i s i n ce l ls tha t had fa l l en ou t o f d i v is i on , p recu rso rs w e re

ma i n ta i ned i n F GF 2 fo r 20 h r and then cha nged to a me -

d i um con ta i n i ng 400 pM N D F I3 -2 (F i gu re 2B) . T h i s caused

a rap i d s t imu l a t i on o f D N A syn thes i s tha t , a f te r 20 h r o f

exposu re to N D F ~-2 , w as s i m i l a r in m agn i tude to tha t seen

20 h r a f te r p l a t ing us i ng the sam e N D F I~ -2 concen t ra t i on

(F i gu re 2A ) . O the r fo rms o f N D F ~, i nc l ud i ng the i so l a ted

E GF - l i ke doma i n a l one , a l so s t i mu l a ted D N A syn thes i s

and show ed s i m li a r dos e - re spo nse re l a t i onsh i ps (da ta not

show n) . C om par i son o f the doses requ i red to suppo r t su r -

v i va l and s t i mu l a te D N A syn thes i s (F i gu re 2A ; a l so see

F i gu re 1 ) i nd i ca ted , how eve r , tha t rescue does no t dep end

on s t i mu l a t i on o f D N A syn thes i s , s i nce l ow concen t ra t i ons

tha t a re on l y w eak l y m i togen i c neve r the l ess suppo r t es -

sen t i a l l y fu l l su rv i va l. T h i s w a s con f i rmed i n subsequen t

expe r i m en ts ( see be l ow ) .

N D F I3s the re fo re ac t bo th to b l ock dea th and s t i mu l a te

D N A syn thes i s i n E 14 S chw ann ce l l p recu rso rs , i n con t ras t

to F GF s , w h i ch p rom o te su rv i va l on l y .

T h e I n v o l v e m e n t o f I G F1 i n N D F - D r i v e n S u r v i v a l

a n d D N A S y n t h e s i s

P rev i ous ly , w e have show n tha t F G F -med i a ted rescue o f

p recu rso rs depends on the p resence o f h i gh concen t ra -

t i ons o f insu l in o r IGF 1 (1 ~M [5 .7 ~g /m l ] and 6 .5 -1 3 nM

[50 -10 0 ng /m l ] , respec t i ve ly ) , i nd i ca t ing tha t F GF rescue

depen ds on ac t i va t i on o f type 1 IGF recep to rs (Gav r i l ov ic

e t a l. , 1995 ). T he p resen t expe r i m en ts show ed , h ow eve r ,

tha t N D F I~s cou l d c om p l e te l y p reven t p recu rso r dea th i n

med i um con ta i n i ng low i nsu l in (1 nM) and no IGF 1 (see

above ) .

T o i nves t i ga te th i s is sue fu r the r , do se - re spo nse cu rves

fo r N D F l ~ -2 -med i a ted su rv i va l w e re gen e ra ted u nde r th ree

d i f fe ren t cond i t ions : i n the abs ence o f i nsu l in and i GF 1 ;

i n the p resence o f 1 nM i nsu l in on l y , to ac t i va te i nsu li n

recep to rs ; and i n the p resence o f 1 nM i nsu l in p l us 13 nM

IGF 1 , to ac t i va te t ype 1 IGF recep to rs a l so (F i gu re 3A) .

E ven i n the absence o f bo th i nsu l i n and IGF 1 , N D F I3 -2

w as ca pab l e o f com p l e te rescue a t 20 h r. In the p resence

o f l ow i nsu l in , N D F ~-2 w as more po ten t , the E Ds0 ( the con -

cen t ra t i ons g i v i ng ha l f -max i ma l su rv iva l ) hav i ng sh i f ted to -

w a rd l ow e r concen t ra t i on by abou t 5 - to 6 - fol d . A dd i t ion

o f IGF 1 on top o f l ow i nsu l in p rom o ted su rv i va l a t the l ow es t

N D F I3 -2 concen t ra t i ons on l y , l eav i ng the E D s0 essen t i a l l y

u n c h a n g e d .

S i nce IGF 1 p rom o ted the marg i na l su rv i va l gene ra ted

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Neuron

588

1 4 0 ~

1 2 0 -

1 0 0 .

B 0 .

4 0

2 0

A /

.... -{',/ /

1 0 1 0 0 1 0 0 0

N DF~ 2 ( p M )

t

o o t

~ 4 0 t

/

o r

1 0 0 1 0 0 0

N O Fc~ 2 ( p M )

3 0 ¸

u . I 2 5 ¸

_>

~ 2 0

o

' 1 5

~ 5

0

0

i i

1 0 0 1 0 0 0

NDF~ 2

( p M )

. . . . . .. . . . N O I N S U L I N

L O W I N S U L I N

-o - L O W I N S U L I N + I G F

Figure 3. The Effec t of Insulin Growth Factorson NDF-MediatedSur-

vival and DNA Synthesis

(A and B) Results rom a 20 hr survivalassay see e gend o Figure 1).

(C) NDF stimulatesDNA synthesis n the abs enceof typ e 1 IGF recep-

tor stimulation. The results show he percentageof Ll-p ositive cells

that werealso BrdU-positive ollowingexposure o variousconcentra-

tions of NDFI3-2 n me diawith or without IGF1 as indicated.

by l ow N D F I3 -2 doses , w e tes ted w he the r IGF 1 w ou l d a l so

enha nce the ve ry l ow su rv iva l gene ra ted by e fo rms o f N D F

(see F igu re 1 ). Indeed , 13 nM IGF 1 m arked l y enhanc ed

NDFc~-2-media ted surv iva l (F igure 3B) . Un der these co nd i -

t i ons su rv i va l o f abou t 60 cou l d be ob ta i ned , a lbe i t a t

h i gh concen t ra t i ons o f N D F ~-2 . N D F ~- I and N D F ct -3 sup -

po r ted su rv i va l comparab l e to tha t seen w i th N D F ~-2 i n

the p rese nce o f 13 nM IGF 1 (da ta no t show n) .

A s a tes t o f the i nvo l vem en t o f type 1 IGF recep to r i n

N D F - regu l a ted D N A syn thes i s , dos e - res pon se cu rves fo r

NDFI3-2-s timu la ted DNA syn thes is were gene ra ted in the

p resence o f 1 nM i nsu l i n on ly , and i n the p resence o f 1

nM insu l in p lus 13 nM IGF1 (F igure 3C). DN A synthes is

w as m on i to red w i th the B rdU m e thod and an t i-L1 an t i bod -

i es as be fo re , excep t tha t the ce l ls w e re ex posed to on l y

a s ing le 1 .5 hr BrdU p u lse a t 20 hr (equ iv a len t to the las t

pu l se i n the th ree t i me po i n t assay used p rev i ous l y ) . T h i s

show ed tha t N D F I3-2 read i l y s t imu l a ted D N A syn thes i s i n

the p resen ce o f l ow i nsu l in co ncen t ra t i on (1 nM) w i thou t

IGF I . F u r the rmore , a l though add i t i on o f IGF1 enha nced

the m i togen i c response , th i s e f fec t w as ba re l y s i gn if i can t

s ta t i s t i ca l l y . In one exper iment , DNA synthes is in re -

sponse to 400 pM N D F ~-2 w as m easu red i n a s im i l a r w ay

i n the p resence o f 13 nM IG F I . Less than 1 o f p recu rso rs

w e re B rdU pos i t i ve i n th i s expe r i me n t .

T oge the r , these resu l ts i nd i ca te tha t h i gh concen t ra -

t i ons o f insu l i n o r IGF 1 (and the re fo re p resum ab l y type 1

IGF recep to r s t i mu l a t i on ) p l ay a sma l l e r ro l e i n N D F ~-

regu l a ted su rv i va l and D N A syn thes i s i n S chw ann ce l l pre -

cu rso rs than m i gh t have been expec ted f rom p rev i ous

s tud i es on F G F s and o the r g row th fac to rs on ce l l s in th i s

l i neage .

N D F b u t N o t F G F 2 S u p p o r t s L o n g - T e r m

S u r v i v a l o f P r e c u r s o r s

P rev i ous l y , tw o cond i t i ons have been show n to suppo r t

the su rv i va l o f S chw ann ce l l p recu rso rs fo r mo re than 20

h r : F GF 2 i n the p resen ce o f h i gh

co n ce n t r t i o n s

of insu l in

p l us a l ow amou n t o f se rum and , a l te rna t i ve ly , de f i ned

med i um con d i t i oned by neu rons (Jessen e t a l ., 1994 ; Ga -

vr i l ov ic e t a l . , 1995). B oth s i tua t ions inv o lve expos ure to

un i den t i f i ed fac to rs , and so fa r , de f i ned cond i t i ons tha t

a l l ow the su rv i va l o f ce l ls rem oved f rom E 14 ne rves fo r

l onge r pe r i ods have no t been i den t i fi ed .

W e t h e r e f o r e e x a m i n e d w h e t h e r N D F ~ - 2 c o u l d s u p p o r t

l onge r - te rm p recu rso r su rv i va l and com pared i t w i th F GF 2 .

B o th fac to rs w e re app l i ed i n de f i ned med i um con ta i n i ng

1 nM i nsu l in and 13 nM IGF 1 . S u rv i va l w as a ssessed as

be fo re , excep t tha t i n add i t i on to a 20 h r su rv i va l pe r i od ,

som e cove rs l i ps w e re ma i n ta i ned fo r 44 and 68 h r p r i o r

to i mm unos ta i n i ng w i th an ti-L1 an t i bod i es and coun t i ng

(F i gu re 4A ) . In N D F , ce l l num ber a t the end o f the 3 d ay

pe r i od w as som ew h a t h i ghe r than a t 3 h r, w he reas in F G F 2

ce l l num ber fe l l rap id ly a f te r the f irs t day . U s ing 1 .5 hr BrdU

pulses a t the end o f the f i rs t and second day in NDF]3-2 ,

w e found B rdU i nco rpo ra t i on in L l -po s i t i ve p recu rso rs o f

3 .0 and 3 .8 , respec t ive l y . T hus , N DF I3 -2 a t 32 pM sup -

po r ts the su rv i va l o f S chw ann ce l l p recu rso rs fo r seve ra l

days , gene ra t i ng som e i nc rease i n ce ll num ber tow a rd the

end o f the pe r i od , ow i ng to a l ow l eve l o f D N A syn thes is .

In FGF2, ho wev er , the ce l l s d ied rap id ly a f te r the f irs t day .

A l though E 14 p recu rso rs a f te r 20 h r i n F GF 2 cou l d no t

a t th is po i n t be rescued by F GF 2 , w e found tha t they cou l d

be rescued com p l e te l y by N D F . T h i s w as show n in expe r i -

men ts i n w h i ch E 14 p recu rso rs w e re m a i n ta i ned in F GF 2

fo r 20 h r and then cha nged to f resh F G F 2 o r to m ed i um

con ta i n i ng N D F I3 -2 : essen t i a l l y a ll t he ce l ls chang ed f rom

F GF 2 to N D F s u rv i ved the nex t 2 days , w he reas m os t o f

the ce l l s tha t rema i ned i n F GF 2 med i um d i ed du r i ng the

same pe r i od (F i gu re 4B ) . T h i s show s tha t , a f te r a 20 h r

exposu re to F G F 2 , p recu rso rs a re no t i r reve rs i b ly se t on

a dea th cou rse bu t have se l ec t i ve l y l os t respons i veness

to FGF2.

I n t h e P r e s e n c e o f N D F P r e c u r s o rs G e n e r a t e

S c h w a n n C e l l s o n S c h e d u l e a n d i n

t h e A b s e n c e o f D N A S y n t h e s i s

H av i ng show n tha t N D F ~-2 su ppo r ted fu l l su rv iva l o f the

L l -pos i t i ve (o r ne rve g row th fac to r recep to r [N G F R ] -po s i -

t i ve ) ce l ls , ob ta i ned as S chw ann ce l l p recu rso rs f rom E 14

ne rves , fo r a t leas t 4 days , w e aske d w he the r the deve l op -

men ta l s ta tus o f these ce l l s changed du r i ng the cu l tu re

per iod . I f NDFI3-2 , app l ied under the def ined cond i t ions

used he re , m i m i cked the cond i t i ons p reva i l ing du r ing e rn -

b ryon i c ne rve de ve l op me n t in v i vo , essen t i a l ly a ll the p re -

cu rso rs w ou l d conve r t to ce l ls w i th the S chw a nn ce l l phe -

notyp e dur ing a 4 c lay per iod (E14 + 4 = E18) , s ince, in

v i vo , ce ll s i n E 14 /15 ne rves have the p recu rso r phe no type ,

w he re as ce l ls i n E 17 /18 ne rves have a S chw a nn ce l l phe -

notyp e (Jessen e t a l . , 1994; G avr i lov ic e t a l . , 1995). A l te r -

na t i ve ly , i t w as poss i b l e tha t N D F I3 -2 b l ocked apop tos i s

i n E 14 ce l l s w i thou t p ro v i d i ng the cond i t i ons requ i red fo r

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NDF and Schwan n Cell Precursors

589

1 4 0 t A 1 2 ° t B

1 1 0 0 1 ~ N N N

~ I~. q 80 -

8

~ 60

6

40

~ 4O

20 20-

0 - , 0-20 44 68 20 44 68

T IM E h o u r s ) T IM E h o u r s )

Figure 4. Long-TermSurviva l of E14 P recursors

(A) NDF (hatched bars)supports ong-term urvival,whereas he effect

of FGF2 closedbars) s transient. Survivalwas measured see egend

to Figure 1) in sister cultures at different im es after plating and in the

presenceof NDF (32 pM) or FGF2 180 pM, a suprama ximal oncentra-

tion in a 20 hr assay).

(B) NDF (hatched bars), but not FG F2 (closed bars), rescues precur-

sors following a 1 day e xposure o FGF2. All cultures we re exposed

to FGF 2 (180 pM) for 1 da y. Some cultures were hen fixed and used

for survival assessment column at 20 hr), while other cultures were

changed to fresh med ium containing FGF2 or NDF~-2 (32 pM) as

indicated. The survival of these cultures was me asuredafter a further

1 day (column s at 44 hr) or 2 days (columnsat 68 hr). Survival was

measured as before (see egen d o Figure 1).

l i neage p r og r ess ion and S chw ann ce l l gene r a t i on . I n cu l -

t u r es t ha t i n i t i a l l y cons i s t o f p r ecu r so r s on l y , S c hw an n ce l l

g e n e r a t i o n c a n b e m o n i t o r e d q u a n t i ta t i v e l y b y e x a m i n i n g

t h e g r a d u a l a p p e a r a n c e o f c e l l s th a t e x p r e s s c y t o p l a s m i c

$ 1 0 0 a n d s u r v i v e i n d e fi n e d m e d i u m i n v i tr o w h e n p l a t e d

a t mod er a te ce l l dens i t y . Fu r the r mo r e , E 14 p r ecu r so r s ,

un l i ke neona ta l S chw ann ce l l s , do no t syn thes i ze D N A in

r e s p o n s e t o t h e c o m b i n a t i o n o f F G F 2 a n d f o r s k o li n ( s e e

above ) . Th i s sugges ted an add i t i ona l f ea tu r e d i f f e r en t i a t -

i n g t h e p r e c u r s o r a n d S c h w a n n c e l l p h e n o t y p e s . I n t h e

f o l lo w i n g e x p e r i m e n t s , w e f ir s t c o n f i r m e d t h i s b y s h o w i n g

t h a t a m i t o g e n i c r e s p o n s e to F G F 2 p l u s f o r s k o li n a p p e a r s

i n v i v o b e t w e e n E 1 5 a n d E 1 7 . W e t h e n u s e d t h e t h r e e

c r i t e r i a o f cy top lasmic $100 exp r ess ion , su r v i va l ab i l i t y

a n d m i t o g e n i c r e s p o n s e to F G F 2 a n d f o r s k o li n , to m o n i t o r

t he gen e r a t i on o f S chw a nn ce l l s f r om p r ec u r so r s i n v i t r o .

T h e D e v e lo p m e n t a l A p p e a r a n c e o f a M i t o g e n i c

R e s p o n s e t o F G F 2

T o d e t e r m i n e w h e n c e l ls f r e s h ly i s o l a t e d f r o m e m b r y o n i c

ne r ves s ta r t ed t o r espond t o FG F2 b y D N A syn thes i s , ce l l

cu l t u r es w e r e p r epa r ed f rom E 14 , E 15 , E 17 , E 18 , and new -

bo r n ne r ves and m a in ta ine d i n FG F2 p lus fo r sko l i n . D u r ing

t h e f ir s t 2 0 h r a f t e r p la t i n g , D N A s y n t h e s i s w a s m e a s u r e d

us ing t he t h r ee t ime po in t B r dU assay ca r r i ed ou t p r e -

v ious l y f o r E 14 ce l l s on l y ( F igu r e 5A ) . Th i s show ed tha t

a t 5 h r D N A s y n t h e s is w a s c o n s i d e r a b l y g r e a t e r in E 1 7

and E 18 ce l l s t han i n E 14 , E 15 , o r new bor n ne r ves , r e -

f l e c t i n g t h e p e a k o f D N A s y n t h e s i s s e e n d u r i n g n o r m a l

ne r ve de ve lo pm en t i n v i vo ( S tew ar t e t a l . , 1993 ). A l so ,

i n th e p r e s e n c e o f F G F 2 a n d f o r s k o l in , D N A s y n t h e s i s

d e c e l e r a t e d r a p i d l y in t h e c e l l p o p u l a t i o n f r o m E 1 4 a n d

E 1 5 n e r v e s . T h e s a m e r e s u lt w a s o b t a i n e d w h e n t h e F G F 2

c o n c e n t r a t i o n w a s i n c r e a s e d f ro m 1 8 0 p M , w h i c h h a s

s u p r a m a x i m a l e f f e c ts o n D N A s y n t h e s i s i n n e o n a t a l

S chw ann ce l l s , t o 600 pM ( da ta no t show n) . I n con t r as t ,

t h e c o n s i d e r a b l e i n i ti a l s y n t h e s is l e v el w a s m a i n t a i n e d in

E 17 ce l l s and e leva ted i n E 18 ce l ls . A s exp ec ted , t he t h r ee

t ime po in t assay a l so r evea led a s t imu la t i on o f D N A syn -

thes i s by FG F2 and f o r sko l i n i n new bor n ce l l s . The s im-

p les t i n t e r p r e ta t i on o f t hese r esu l t s i s t ha t FG F2 p lus f o r -

s k o l in i n t h e m e d i u m u s e d h e r e s t i m u l a t e s D N A s y n t h e s is

i n S chw ann c e l l s bu t no t i n S ch w an n ce l l p r ecu r so r s . The

m i t o g e n i c r e s p o n s e t o t h e s e f a c t o rs c a n t h e r e f o r e b e u s e d ,

t oge the r w i t h p r ev ious l y de f i ned c r i t e r ia , t o m on i t o r d i f fe r -

e n t i a ti o n in t h e S c h w a n n c e l l l i n e a g e a n d t o e x a m i n e t h e

g e n e r a t i o n o f S c h w a n n c e l ls f r o m p r e c u r s o rs .

T h e G e n e r a t i o n o f S c h w a n n C e l l s f r o m E 4

P r e c u r s o r s i n V i t ro

C e l l s w e r e p r epa r e d f r om E 14 , E 15, E 17 , E 18 , and new -

bo r n ne r ves and ma in ta ined i n N D F I3 -2 in de f i ned m ed ium .

A f t e r 1 - 4 d a y s i n v i t ro t h e i r p h e n o t y p e w a s a s s e s s e d q u a n -

t it a t iv e l y . F o r e a c h a s s e s s m e n t , t h e c e l l s w e r e r e m o v e d

f r o m t h e c u l t u r e d is h , r e p l a t e d o n c o v e r s l i p s , a n d e x a m -

ined f o r ab i l i t y t o su r v i ve f o r 20 h r i n de f i ned m ed ium on l y ;

c y t o p l a s m i c $ 1 0 0 i m m u n o r e a c t M t y a t 3 h r a f te r p la t i n g ;

and D N A syn thes i s i n r espons e to FG F 2 p lus f o r sko l in a t

5 , 15 , and 20 h r a f t e r p la t i ng . The r ep la t i ng s t r a tegy w as

used t o e l im ina te t he e f f ec ts o f g r ow th f ac to r s bound t o

the cu l t u r e subs t r a te i n t he pe r i od p r i o r t o assessmen t ,

a n d t h e m e t h o d s u s e d t o e x a m i n e p r e c u r s o r v e r s u s

S c h w a n n c e l l p h e n o t y p e w e r e i d e n t ic a l to t h o s e u s e d p r e -

v ious l y on ce l l s f r esh l y ob ta ined f r om ne r ves a t d i f f e r en t

a g e s .

I n i ti a l ly , su r v i va l w as m eas u r ed i n ce l l s f r om E 14 , E 15 ,

E 17 , and new bor n ne r ves a f t e r 1 day i n v i t r o ( Tab le 1 ) .

E 14 + 1 ( = 15 ) day ce l l s su r v i ved poo r l y , as d id ce l l s f r om

E 15 ne r ves , w he r eas E 15 + 1 ( = 16 ) day ce l l s show ed

i n c r e a s e d s u r v iv a l , i n a g r e e m e n t w i t h t h e i n c r e a s e d s u r-

v i v a l s h o w n b y c e l l s r e m o v e d f r o m E 1 6 n e r v e s w h e n c o m -

p a r e d w i t h c e l ls f r o m E 1 5 a n i m a l s . T h e a s s a y r e g i s t e r e d

a m uch be t t e r su r v i va l o f new b or n ce l l s a f t e r 1 day i n v it r o ,

i n a g r e e m e n t w i t h t h e o b s e r v a t i o n t h a t t h e s e c e l l s s u r v iv e

w e l l w h e n t e s t e d d i r e c t l y a f te r r e m o v a l f r o m t h e n e r v e.

E 17 + 1 ( = 18 ) day ce l l s show ed su r v i va l s im i l a r t o tha t

o f E 18 ce l l s . We then measu r ed su r v i va l o f E 14 ce l l s by

r ep la t i ng a f t e r 2 , 3 , and 4 day s i n v it r o ( Tab le 1 ) . E 14 +

2 ( = 16 ) day ce l l s su r v i ved s ign i f i can t l y be t t e r t han

E 14 + 1 ( = 15 ) day ce l l s . A much l a r ge r i nc r ease i n su r v i va l

w a s , h o w e v e r , s e e n b e t w e e n E 1 4 + 2 ( = 1 6) d a y c e l ls

and E 14 + 3 ( = 17 ) day ce l l s , and t he su r v i va l o f E 14 +

4 ( = 1 8 ) d a y c e l l s w a s s o m e w h a t h i g h e r th a n t h a t s e en

w i t h E 1 4 + 3 d a y c e l l s a n d c o m p a r a b l e t o t h a t s e e n w it h

new bor n + 1 day ce l l s . A l l o f t h i s i s in l i ne w i t h t he su r v i va l

s h o w n b y c e l ls r e m o v e d f ro m n e r v e s a t t h e e q u i v a l e n t

e m b r y o n i c a g e s .

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N e u r o n

59O

40-

5

3O

25

O

a. 20

Q

n- 15

rn

10

E14

lO

20

TIME (hours)

E15

E l7

10 20 10 20 0 10 20

TIME (hours) TIME (hours) TIME (hours)

NB

10 20

TIME (hours)

B ~ 20-

O 15-

10-

04 5 -

E14 + ld (=E15) E14

+ 4d =E18)

14 + 3d (=E17)

20 10 20

TIME (hours)

lO o lO 20

TIME (hours) TIME (hours)

F igu r e 5 . A M i t ogen i c R es pons e t o F G F 2 p lus F o r s k o l i n D i s t ingu i s hes S c hwan n Ce l l s fr om P r ec u r s o r s

( A ) T he r es u lt s s how t he pe r c en t age o f L l - pos i t i v e c e ll s t ha t a l s o hav e B r dU- pos i ti v e nuc le i f o l l ow ing t h r ee 1 . 5 h r l ong ex pos u r es t o B r dU du r i ng

t he f ir s t 20 h r a f t e r p la t i ng o f c e ll s r em ov ed f r om an im a ls a t d i ff e r en t dev e lopm e n t a l s t ages as i nd i c a t ed . A l l c u l tu r es we r e ex pos e d t o F G F 2 ( 180

pM) p lus forskol in (5 p.M) .

(B) The resul ts are f rom a three t ime po int BrdU a ssay as descr ibed for (A). The ass ay was car r ied out dur ing th e f irs t 20 hr af ter replating of ce lls

t ha t had been ob t a ined f r om E 14 an im a ls an d m a in t a ined f o r t , 3 , and 4 d ay s i n NDF I3 - 2 ( 40 pM ) .

E x a m i n a t i o n o f c y t o p l a s m i c $ 1 0 0 i m m u n o r e a c t i v i t y in

t h is t y p e o f e x p e r i m e n t r e v e a l e d a c o m p a r a b l e p i c t u re ( T a -

b l e 1 ). F e w E 1 4 + 1 ( = 1 5 ) d a y c e ll s w e r e $ 1 0 0 p o s i t i v e ,

w h e r e a s a b o u t h a l f o f t h e E 1 4 + 2 ( = 1 6 ) a n d E 1 5 + 1

( = 1 6 ) c e l l s b o u n d $ 1 0 0 a n t i b o d i e s . T h e g r e a t m a j o r i t y

o f E 1 4 + 3 ( = 1 7 ) a n d E 1 4 + 4 ( = 1 8 ) d a y c e l l s w e r e $ 1 0 0

p o s i t i v e . T h e s e f i g u r e s a r e i n g o o d a g r e e m e n t w i t h t h o s e

o b t a i n e d b y i m m u n o l a b e l i n g c e ll s f r o m n e r v e s a t e q u i v a -

l e n t a g e s .

I n a f u r t h e r te s t o f w h e t h e r E 1 4 p r e c u r s o r s a c q u i r e d t h e

T ab t e 1. C hang e i n S u r v i v a l A b i l i t y and $10 0 E x p r es s ion du r i ng P r ec u r s o r / S c hwann C e l l Conv e r s ion

Ce l l s ( A ge W hen Rem o v ed f r om Ner v e P er c en t age o f Ce l ls T ha t E x p r es s $10 0 S ur v i v a l P e r c en t age

+ Day s I n V i t r o ) ± S E M 4 - S E M

E 1 4 + 0 4 _ 3 1 ___ 2

E 1 5 + 0 4 4 - 4 3 _+ 2

E 1 4+ 1 15 4 - 2 12 4 - 2

E 1 6 + 0 42 __+ 6 16 -.+- 5

E 1 4 + 2 41 _+ 4 30 -_+ 3

E 15 + 1 47 _+ 7 18 _+ 3

E 1 7 + 0 9 5 - 2 8 7 _+ 5

E 14 + 3 75 _+ 3 71 _+ 2

E 1 8 + 0 96 _+ 2 98 4 - 2

E 1 4 + 4 84 _+ 3 85 _+ 9

E 1 7 + 1 93 4- 1 92 ___ 6

New born + 0 93 4- 6 100 4- 4

New born + 1 79 _+ 5

P lus 0 day s i n v i tr o i nd i c a t es ex pe r im en t s i n wh i c h t he s u r v i v a l as s ay was c a r r i ed o u t im m e d ia t e l y a f t e r r em ov a l o f c el ls f r om ne r v es , and t he

s u r v i v a l pe r c en t ages i n t hes e ex pe r im en t s and t he pe r c en t age o f c el ls e x p r es s ing c y t op las m ic $1 00 a r e ob t a ined f r om J es s en e t al ., 1994 . T hey

a r e p r es en t ed he r e t o m ak e d i r ec t c om par i s on w i t h t he p r es en t r es ul ts eas y . P lus 1 - 4 da y s i n v i t r o ind i c a t es ex pe r im en t s i n wh i c h c e ll s we r e

m a in t a ined f o r 1 - 4 day s i n v i t ro p r i o r to t he s u r v i v a l as s ay (s ee E x per im en t a l P r ocedures ). E ac h ex pe r im e n t was c a r r ied ou t us ing d up l i c a t e o r

t ri p li ca t e c ov e r s l i ps , and eac h f i gu r e r ep r es en t s t he av e r age o f t h r ee ex pe r im en t s , ex c e p t f o r ex pe r im en t s w i t h newbor n c e l ls , w h i c h we r e c a r r i ed

out twice. In th is case, the f igure represe nts s tand ard dev iat ion f rom a pooled tota l o f 4 covers l ips .

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NDF and Schwann Ce ll Precursors

591

Sc h w a n n c e l l p h e n o ty p e o n s c h e d u le , D NA s y n th e s is i n

re s p o n s e to F GF 2 p lu s fo rs k o l in wa s e x a m in e d in c e l l s

p re p a re d f ro m n e rv e s a t E1 4 a n d m a in ta in e d in NDF fo r

1 -4 d a y s ( s e e F ig u re 5 B) . T h e p h e n o ty p e o f t h e c e l l s wa s

a s s e s s e d d u r in g th e f i rs t 2 0 h r fo l l o w in g re p la t in g , u s in g

th e th re e t ime p o in t B rd U a s s a y d e s c r ib e d p re v io u s ly .

F GF 2 p lu s fo rs k o l in wa s n o t m i to g e n ic fo r E1 4 + 1 (= 1 5 )

d a y c e l ls , a l t h o u g h th i s c o mb in a t io n s t imu la te d D NA s y n -

th e s is in E1 4 + 3 (= 1 7 ) d a y c e l l s a n d in E1 4 + 4 (= 1 8 )

d a y c e l l s. I n o th e r e x p e r im e n ts , we s h o we d th a t e x p o s u re

to 5 I~M fo rs k o l in fo r 2 0 h r i n d u c e d h ig h le v e ls o f Po mR NA

in p re c u rs o rs e x p o s e d to NDF I3 -2 fo r 4 d a y s a n d re p la te d ,

a s e x p e c te d i f t h e s e c e l l s we re Sc h wa n n c e l l s (d a ta n o t

s h o wn ) .

T o g e th e r , t h e s e o b s e rv a t io n s s h o w th a t , wh e n c u l tu re s

o f E1 4 p re c u rs o rs a re e x p o s e d to NDF I3 -2 u n d e r d e f in e d

c o n d i t i o n s , th e p h e n o ty p e o f t h e c e l l p o p u la t io n c h a n g e s

g ra d u a l l y b u t c o mp re h e n s iv e ly f r o m th a t c h a ra c te r i z in g

p re c u rs o rs to th a t o f Sc h wa n n c e l l s . T h is o c c u rs w i th a

t ime c o u rs e s im i la r t o th a t o f Sc h wa n n c e l l a p p e a ra n c e

in n e rv e s d u r in g d e v e lo p m e n t in v iv o .

D N S y n t h e s i s d u r in g S c h w a n n C e l l G e n e r a t i o n

W h e n DN A s y n th e s is wa s m e a s u re d , u s in g 1 .5 h r B rd U

p u ls e s , d u r in g th e 1 -4 d a y c u l tu re p e r io d s th a t p r e c e d e d

t h( ; p h e n o ty p ic a s s e s s me n t

d e s c r i b e d

i n th e p re v io u s s e c -

t io n , i t w a s f o u n d t o r a n g e b e t w e e n 1 0 % a n d 2 0 % , w h i c h

i~ c o m p a ra b le to th e le v e ls o f DN A s y n th e s is i n v i v o d u r in g

th e p e r io d o f E1 4 -E1 8 (S te wa r t e t a l . , 1 9 9 3 ) . W e n o w

a s k e d w h e t h e r t h e g r a d u a l

r e p l a c e m e n t

o f c e l l s w i th th e

p re c u rs o r p h e n o ty p e b y c e l l s w i th th e S c h wa n n c e l l p h e n o -

t y p e in th e c u l tu re s d e s c r i b e d a b o v e w a s d e p e n d e n t o n

th is ra te o f ce l l d iv i s io n , o r wh e th e r i t c o u ld ta k e p la c e in

c u l tu re s wh e re DNA s y n th e s is wa s in s ig n i f i c a n t .

I n th e s e e x p e r i m e n t s w e t o o k a d v a n t a g e o f t h e o b s e r v a -

t i o n th a t a t l o w c o n c e n t r a t i o n s NDFI3-2 can s upp or t sub-

s ta n t ia l s u rv i v a l wh i le i n d u c in g v e ry l i t t l e DNA s y n th e s is

( s e e a b o v e ). Ce l l s re mo v e d f ro m E1 4 n e r v e s w e r e t h e r e -

fo re ma in ta in e d in NDF ~-2 a t 2 8 p M fo r 4 d a y s (E1 4 +

4 = 18) p r io r to be ing asse ssed fo r p r e c u r s o r / S c h w a n n

cel l

p h e n o ty p e o n th e b a s e s o f s u rv i v a l, 1 0 0 e x p re s s io n ,

a n d F GF 2 re s p o n s iv e n e s s a s d e s c r i b e d a b o v e . D N A

s y n th e s is me a s u re d o n ly 1 . t% _+ 0 .4 %, 1 .9 % _ 0 .6 % ,

a n d 1 .9 % _+ 0 .8 % o n d a y s 1 ,2 , a n d 3 in v i t r o , r es pec t i v e l y .

W h e n t h e p h e n o t y p e o f t h e s e c e l ls w a s a s s e s s e d o n t h e

fo u r th d a y , 6 7 % _+ 8 % o f th e L l -p o s i t i v e c e l l s s u rv i v e d

f o r 2 0 h r in d e f in e d m e d i u m , 8 9 % + 3 % e x p r e s s e d c y to -

p la s m ic 1 0 0 , a n d DN A s y n th e s is i n th e p r e s e n c e o f F GF 2

p lu s fo rs k o l in i n c r e a s e d f r o m 3 .0 % to 1 3 .2 % d u r in g a 2 0

h r p e r io d . T h u s , a b o u t a 1 0 - fo ld d ro p in DN A s y n th e s is to

v e ry lo w le v e ls d id n o t ma te r ia l l y a l te r t h e e x te n t o f

S c h w a n n

cel l

g e n e ra t io n . T o in v e s t ig a te fu r th e r th e re la -

t i o n s h ip b e t w e e n D N A s y n t h e s is a n d S c h w a n n ce l l g e n e r -

a t io n f ro m p re c u rs o r c e l l s , E1 4 p re c u rs o rs we re ma in -

ta in e d in NDF ~-2 a t 1 6 p M fo r 3 d a y s in th e c o n t in u o u s

p re s e n c e o f B rd U in two s e p a ra te e x p e r ime n ts . T h e c e l ls

we re th e n a s s a y e d fo r p re c u rs o r /Sc h w a n n c e l l p h e n o ty p e

b y me a s u r in g 2 0 h r s u rv i v a l a s d e s c r ib e d a b o v e , e x c e p t

th a t a t th e e n d o f t h e 2 0 h r p e r io d , we c a r r ie d o u t d o u b le

i m m u n o l a b e l i n g f o r B r d U a n d L 1. T h i s s h o w e d t h a t 5 8 %

o f th e s u rv i v in g L l -p o s i t i v e c e l l s ( i. e. , Sc h wa n n c e l l s ) h a d

n o t i n c o rp o ra te d Brd U d u r in g th e 3 d a y p e r io d , wh e re a s

th e re la t i v e n u mb e r o f c e l l s th a t h a d a c q u i re d th e Sc h w a n n

c e l l p h e n o ty p e in th i s l o w ND F ~-2 c o n c e n t ra t io n wa s u n -

c h a n g e d f ro m th a t s e e n a t h ig h e r c o n c e n t ra t io n s .

T h e s e e x p e r ime n ts ma k e i t v e ry u n l i k e ly th a t th e

Sc h wa n n c e l l s g e n e ra te d in th e s e c u l tu re s a r i s e f ro m a

h y p o th e t i c a l p o p u la t io n o f ra p id l y d i v id in g s te m c e l l s , a n d

th e y s h o w th a t a t l e a s t th e g re a t ma jo r i t y o f t h e s e c e l l s

mu s t h a v e o r ig in a te d in E1 4 p re c u rs o rs th a t c o n v e r te d to

Sc h w a n n c e l l s w i th o u t th e re q u i re m e n t o f c e l l d i v is io n .

S o l u b l e E r b B 4 P r o t e i n B l o c k s S u r v i v a l A c t i v i t y i n

N e u r o n C o n d i t io n e d M e d i u m

Pre v io u s ly , d e f in e d m e d iu m c o n d i t i o n e d b y p u r i f ie d c u l -

t u r e s o f D R G n e u r o n s w a s s h o w n t o c o n t a i n p r o t e i n a c e o u s

s u rv i v a l a g e n t (s ) th a t s u p p re s s e d a p o p to s is o f p re c u rs o rs

i n a d o s e - d e p e n d e n t w a y a n d a l l o w e d E 1 5 p r e c u r s o r s t o

p ro g re s s to Sc h w a n n c e l l s d u r in g a p e r io d o f 5 d a y s w i th -

o u t th e re q u i re m e n t fo r c e l l d i v i s io n ( J e s s e n e t a l . , 1 9 94 ).

S in c e th e s e e f fe c ts a re s im i la r t o th o s e o f NDF , we n o w

a s k e d w h e t h e r t h is a c t i v i ty w a s N D F . T h i s w a s d o n e b y

te s t in g wh e th e r th e a c t iv i t y c o u ld b e b lo c k e d b y a d d i t i o n

o f a s o lu b le h y b r id p ro te in c o n ta in in g th e NDF b in d in g s i te

o f th e Erb B4 re c e p to r .

T o e n s u re u n a m b ig u o u s ly th a t th e a c t i v i t y in th e c o n d i -

t i o n e d m e d i u m w a s o f n e u r o n a l or i gi n , w e u s e d D R G n e u -

ro n s p u r i f ie d b y im mu n o p a n n in g to y ie ld c u l tu re s in wh ic h

> 9 5 % o f th e c e l ls w e r e n e u r o n s a t t h e ti m e o f m e d i u m

c o l le c t io n (F ig u re 6 A, u p p e r p a n e l ) . W e c o n f i rme d immu -

n o c y to c h e m ic a l l y th a t th e n e u ro n s in th e s e c u l tu re s e x -

p re s s e d NDF p ro te in (F ig u re 6 A, l o we r p a n e l ) . De f in e d

m e d i u m w i t h o u t s e r u m w a s c o n d i t io n e d b y t h e s e c u l t u re s

fo r 2 4 h r . Us in g th e 2 0 h r s u rv i v a l a s s a y d e s c r ib e d p re -

v io u s ly a n d c e l ls f ro m E1 4 n e rv e s , th is m e d iu m w a s th e n

te s te d a lo n e o r fo l l o w in g a d d i t i o n o f 1 o r 3 l~g/m l E rb B4

p ro te in . Co n t ro l e x p e r ime n ts in v o lv e d th e a d d i t i o n o f

E r b B 4 t o s u r v i v a l m e d i u m c o n t a i n i n g F G F 2 t o g e t h e r w i th

IGF1 and fo rsko l in to tes t fo r nonspec i f ic e f fec ts , and the

a d d i t i o n o f E rb B4 to s u rv i v a l me d iu m c o n ta in in g re c o mb i -

n a n t NDF ~-2 to v e r i f y th a t th e re c e p to r p ro te in b lo c k e d

th e e f f ec t s o f N D F u n d e r t h e p r e v a i l in g e x p e r i m e n t a l co n -

d i t io n s . E rb B 4 h a d n o e f fe c t o n F GF -m e d ia te d s u rv iv a l o f

E1 4 c e l l s b u t s t ro n g ly i n h ib i te d th e e f fe c t b o th o f NDF I~-2

a n d n e u ro n -c o n d i t i o n e d me d iu m (F ig u re 6 B). I n o th e r e x -

p e r ime n ts , we fo u n d th a t th e Erb B4 p ro te in a l s o c a u s e d

a 6 5 % re d u c t io n o f p re c u rs o r s u rv i v a l in a 2 0 h r s u rv i v a l

a s s a y o f n e u ro n -p re c u rs o r c o c u l tu re s . I n th i s s y s te m, p re -

c u rs o rs a re s e e d e d o n to p o f e x t re me ly s p a rs e c u l tu re s

o f p u re n e u ro n s , a n d s u rv i v a l o f p re c u rs o rs i s me d ia te d

b y c o n ta c t w i th a x o n s , i n th e a b s e n c e o f a n y d e te c ta b le

s u rv i v a l a c t i v it y in th e c u l tu re me d iu m (Z . D . u n p u b l i s h e d

data) .

T h e Erb B4 re c e p to r h a s n o t b e e n fo u n d to b in d to fa c to rs

o th e r th a n th o s e o f t h e N DF fa m i l y . I t i s th e re fo re v e ry l i k e l y

th a t th e ma jo r a c t i v e in g re d ie n t i n th e n e u ro n -c o n d i t i o n e d

me d iu m i s NDF . Erb B4 e x e r te d a s t ro n g e r i n h ib i to r y e ffe c t

o n 4 0 p M NDF I~-2 th a n i t d id o n th e

n e u r o n c o n d i ti o n e d

m e d i u m . T h i s c o u l d b e d u e t o t h e c o n c e n t r a t i o n o f N D F

in the n e u r o n c o n d i ti o n e d m e d i u m b e i n g h i g h e r th a n 4 0

p M . A l t e r n a ti v e ly , th e c o n d i t i o n e d m e d i u m m a y c o n t a in

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B NEURON

CONDITIONED MEDIUM

Neuron

592

100-

80.

--I

60-

t.~ 40-

20-

C

0

N F

i

0 1 3

ErbB4

(p-g/ml )

NDF~-2 FGF-2

0 1 3 0 1 2 3

ErbB4 l~g/ml) ErbB4 t~g/ml)

Figure 6. The Neuron-Derived Precursor Survival Signal Is Likely to be NDF

(A) The upper panel is a representative phase-contrast view showing the ap pearance of the highly puri f ied, immunopanned DRG cultures used

to generate conditioned medium. The ph otograph was taken 48 hr after plating; over 95 of the cel ls in these cultures were neurons (magnification,

400 x ). The lower panel shows NDF immunoreactivity in a DRG culture, simi lar to the one shown in the upper panel. The cel ls were labeled using

the 5D6A anti-NDF antibody. Note immunolabel ing of neuronal cel l bodies and bundles of naked neuri tes (magnification, 600 x ).

(B) Soluble ErbB4 protein blocks the survival activity in DRG-conditioned medium and the survival effect of NDFIf,-2; the survival effect of FGF2

is unaffected. The results are from 20 hr survival assays.

(C) Immunotabeling of E14 DRG neurons and precursors using antibodies against NDF, ErbB4, and ErbB2, as indicated on the immunofluorescence

micrographs in the upper panels. The lower pane ls show the corresponding phase-contrast views. Note that both celt bod ies and processes of

the two DRG neurons are NDF positive (antibody 5D6A), whereas E14 precursors are NDF negative. ErbB4 immunolabel ing is uniform and

present in essentially all precursors, whereas ErbB2 labeling varies more from cell to cell (controls were negative; see Experimental Procedures).

Magnification, 700-800 x.

o t h e r n e u r o n - d e r i v e d f a c t o r s t h a t s y n e r g i z e w it h N D F t o

p r o m o t e p r e c u r s o r s u r v i v al .

Localization of NDF and NDF Receptors

in E14/15 Cells

T o l o c a l iz e N D F p r o t e i n i n E 1 4 D R G n e u r o n s , w e u s e d

a n t i - N D F a n t i b o d i e s a n d im m u n o h i s t o c h e m i c a l m e t h o d s .

W h e n n e u r o n s d i s s e c t e d f r o m E 1 4 a n i m a l s w e r e e x a m -

i n e d a f t e r 1 - 2 d a y s i n v i t ro , s t ro n g i m m u n o l a b e l i n g w a s

s e e n i n b o t h t h e c e ll b o d i e s a n d n e u r o n a l p r o c e s s e s ( F i g -

u r e 6C ) . N o s i g n i f i c a n t a n t i b o d y b i n d i n g w a s d e t e c t e d i n

E 1 4 p r e c u r s o r s i n s i m il a r i m m u n o l a b e l i n g e x p e r i m e n t s

( F i g ur e 6 C ). C o m p a r a b l e r e s u lt s w e r e o b t a i n e d u s i n g

t h r e e d i f f e re n t a n t i - N D F a n t i b o d i e s ( s e e E x p e r i m e n t a l P r o-

c e d u r e s ) .

E x p r e s s i o n o f t h e E r b B 4 N D F r e c e p t o r a n d o f t h e E r b B 2

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N D F c o r e c e p t o r w a s a l so e x a m i n e d b y i m m u n o h i s t o c h e m -

ist ry ; s u i ta b le a n t ib o d ie s a g a in s t t h e ra t E rb B3 re c e p to r

we re n o t a v a i la b le (F ig u re 6 C). Es s e n t ia l l y a ll E1 4 p re -

c u r s o rs s h o w e d s p e c k l e d a n d a p p a r e n t l y m e m b r a n e -

a s s o c i a te d i m m u n o l a b e l i n g w h e n e x a m i n e d w i t h a n t i b o d -

ie s a g a in s t E rb B4 . T h e la rg e ma jo r i t y o f p re c u rs o rs a l s o

b o u n d a n t ib o d ie s a g a in s t E rb B2 , b u t th e in te n s i t y o f t h e

imm u n o la b e l in g wa s m o re v a r ia b le in th i s c a s e .

D i s c u s s i o n

T h e p re s e n t re s u l t s s h o w th a t 13 fo rms o f NDF re g u la te

s u rv i v a l a n d DNA s y n th e s is o f E1 4 Sc h w a n n c e l l p re c u r -

s o rs . NDF a ls o s u p p o r t s o rd e r l y l i n e a g e p ro g re s s io n o f

th e s e c e l l s s in c e , i n th e p re s e n c e o f NDF , th e y fo rm

Sc h wa n n c e l l s i n v i t r o w i th a t ime c o u rs e th a t i s s im i la r

t o t h a t o f S c h w a n n c e l l a p p e a r a n c e in m a j o r e m b r y o n i c

n e rv e s in v iv o . Un d e r d e f in e d c o n d i t i o n s in m e d ia w i th o u t

s e ru m, n o o th e r g ro wth fa c to r i s p re s e n t ly k n o wn to e x e r t

c o m p a ra b le e f fe c ts o n th e s e c e l ls . A p ro te in th a t re g u la te s

p r e c u r s o r d e v e l o p m e n t i n a s i m i la r w a y w a s p r e v i o u sl y

d e s c r i b e d in d e f i n e d m e d i u m c o n d i t i o n e d b y D R G n e u r o n s

(Jessen e t a l . , 1994). H ere we re por t tha t the e f fe c t o f th is

p ro te in i s a b o l i s h e d b y e x p o s u re to a s o lu b le fo rm o f th e

e x t ra c e l lu la r d o m a in o f t h e Erb B 4 ND F re c e p to r , a p ro te in

th a t b in d s to NDF w i th a v e ry h ig h d e g re e o f s p e c i f i c i t y

(Cu lo u s c o u e t a l . , 1 9 9 3 ; P lo wma n e t a l . , 1 9 9 3 a , 1 9 9 3 b ;

T z a h a r e t a l . , 1 9 94 ). T o g e th e r , t h e s e o b s e rv a t io n s id e n t i f y

NDF a s a n e u ro n - g l i a s ig n a l t h a t c o n t ro l s s u rv i v a l a n d

d i f f e re n t ia t i o n o f Sc h w a n n c e l l p re c u rs o rs in a s imp le a n d

we l l - d e f in e d n v i tr o mo d e l o f n e u r o n -g l i a i n te ra c t io n s d u r-

in g e mb ry o n ic n e rv e d e v e lo p m e n t . NDF i s l i k e l y to c a r r y

o u t a s im i la r f u n c t io n in v i v o , p a r t i c u la r l y i n v ie w o f th e

s t r i k in g ly h ig h N F m R N A e x p r e s s i o n in t h o s e n e u r o n s

th a t fo rm e m b ry o n ic p e r ip h e ra l n e rv e s (Ma rc h io n n i e t a l. ,

1 9 9 3 ; Or r -Ur t re g e r e t a l. , 1 9 9 3 ; Me y e r a n d B i r c h me ie r ,

1 9 9 4 ) a n d in v ie w o f th e p re s e n t o b s e rv a t io n th a t th e s e

c e l l s e x p re s s ND F p ro te in s in c e l l b o d ie s a n d p ro c e s s e s

wh i le Sc h wa n n c e l l p re c u rs o rs s h o w lo w o r n o NDF e x -

press ion .

A l th o u g h th e e x is te n c e o f n e u ro n a l s ig n a ls re s p o n s ib le

fo r d r i v in g d i f f e re n t ia t io n in th e S c h wa n n c e l l l i n e a g e h a s

b e e n a c e n t ra l i d e a in s tu d ie s o n PN S d e v e lo p m e n t fo r a

lo n g t ime , th e s e s ig n a ls h a v e re ma in e d u n k n o wn a t th e

mo le c u la r l e v e l . T h e mo le c u la r i d e n t i f i c a t io n o f a n im -

p o r ta n t c o m p o n e n t o f t h e s e s ig n a ls i s th e re fo re a s ig n i f i-

c a n t s te p fo rwa rd .

N D F a n d t h e S u p p r e s s i o n o f C e l l D e a t h

N D F p o t e n t ly a n d c o m p l e t e ly b l o c k e d t he a c u t e a p o p t o t ic

d e a th o f E1 4 p re c u rs o rs . T h is e f fe c t wa s s e e n in c o m-

p le te l y d e f in e d m e d ia u s in g c e l l s th a t h a d n e v e r b e e n e x -

p o s e d to s e ru m o r o th e r u n id e n t i f i e d fa c to rs , a n d th e re s -

c u e re q u i re d n e i th e r i n s u l in n o r IGF I .

T h e e f fe c t o f NDF d i f f e r s f r o m th e s u rv i v a l e f fe c ts o f

F GF 2 d e s c r ib e d p re v io u s ly ( J e s se n e t a l . , 1 9 9 4 ; Ga v r i l o v i c

e t a l . , 1995) in a t leas t two respec ts . F i rst , ND F suppo r ts

p re c u rs o r s u rv i v a l fo r s e v e ra l d a y s in d e f in e d me d iu m w i th -

o u t s e ru m, wh e re a s th e e f fe c t o f F GF 2 tu rn e d o u t to b e

s u rp r i s in g ly t ra n s ie n t u n d e r th e s e c o n d i t i o n s , l a s t in g e s -

s e n t ia l l y fo r 1 d a y o n ly . Ev id e n t l y , l o n g e r - te rm s u rv i v a l i n

F GF 2 d e p e n d s o n c o -o p e ra t i v i t y b e twe e n F G F 2 a n d s e -

rum fac to rs (Jessen e t a l . , 1994) . Second, NDF b locks

d e a th in me d iu m c o n ta in in g a lo w c o n c e n t ra t io n o f i n s u l in

(1 0 n M) a n d n o IGF 1 , i . e . , i n th e a p p a re n t a b s e n c e o f

t y p e 1 IGF re c e p to r s t imu la t io n (Sa ra a n d H a l l , 1 99 0 ). T h e

a c t io n o f F GF 2 , i n c o n t ra s t , c ru c ia l l y d e p e n d s o n a h ig h

c o n c e n t r a ti o n o f I G F I .

W h e n a h a l f -m a x ima l c o n c e n t ra t io n o f ND F wa s u s e d ,

in c o mb in a t io n w i th lo w d o s e s o f i n s u l in fo r a c t i v a t io n o f

th e in s u l in re c e p to r o n ly ( I n M) , t h e two fa c to rs s y n e rg iz e d

s t ro n g ly i n b lo c k in g c e l l d e a th . Ho w e v e r , e v e n in th e c o m-

p le te a b s e n c e o f b o th in s u l in a n d IGF 1 , h ig h e r c o n c e n t ra -

t i o n s o f NDF s u p p o r te d s u rv i v a l s im i la r t o th a t s e e n in

me d ia c o n ta in in g lo w in s u l in o r l o w in s u l in p lu s a h ig h

c o n c e n t ra t io n o f IGF 1 . Su rp r i s in g ly , t h e re fo re , n e i th e r i n -

s u l in re c e p to r n o r IGF re c e p to r a c t i v a t io n i s n e c e s s a ry fo r

ND F -me d ia te d b lo c k a g e o f a p o p to s is i n th e s e c e l ls .

R e g u l a t i o n o f D N S y n t h e s i s D i f fe r s b e t w e e n

S c h w a n n C e l ls a n d P r e c u r s o r s

I n th e p re s e n t e x p e r im e n ts , we h a v e e s ta b l i s h e d a n a d d i -

t i o n a l p h e n o ty p e th a t d i s ti n g u is h e s E1 4 /1 5 p re c u rs o rs

f r o m E 1 7 a n d o l d e r S c h w a n n c e ll s , n a m e l y th e a b s e n c e

o r p re s e n c e o f a m i to g e n ic re s p o n s e to F GF 2 . W e fo u n d

prev io us ly tha t the p75NOFR/L1-posi tivep re c u rs o rs in E1 4

n e rv e s d o n o t re s p o n d to F GF 2 p lu s fo rs k o l in b y s t imu la -

t ion o f DN A syn the s is (Gavr i lo v ic e t a l ., 1995). The se ce l ls ,

wh ic h a re d i v id in g a t a n a p p re c ia b le ra te in th e n e rv e

(Stewar t e t a l . , 1993) , fa l l ou t o f d iv is ion dur ing the f i rs t

2 0 h r i n th e c u l tu re d i s h , e v e n in th e p re s e n c e o f F GF 2

a n d fo rs k o l in , a l t h o u g h th e s e fa c to rs e f fe c t i v e ly b lo c k

a p o p to s is d u r in g th e s a me p e r io d . W e s h o w h e re th a t th e

s a me a p p l ie s to p re c u rs o rs f ro m E1 5 n e rv e s , wh i le th e

L l -p o s i t i v e p o p u la t io n f ro m E1 7 n e rv e s re s p o n d s d i f fe r -

e n t l y . Ov e r 8 0 % o f th e s e c e l l s a re Sc h wa n n c e l l s o n th e

b a s is o f s u rv i v a l a b i l i ty a n d 1 0 0 e x p re s s io n . T h e y a re

d iv id in g rap id ly in v ivo (Stewa r t e t a l. , 1993), a nd th is ra te

i s ma in ta in e d n th e p re s e n c e o f F GF 2 p lu s fo rs k o l in d u r in g

th e f i r st 2 0 h r i n v i t r o . W i th o u t F GF 2 a n d fo rs k o l in , t h e s e

c e l l s s u rv iv e b u t fa l l o u t o f d i v i s io n d u r in g th e s a me p e r io d .

Ce l l s re mo v e d f ro m E1 8 a n d n e w b o rn n e rv e s a ls o re s p o n d

to F GF 2 p lu s fo rs k o l in b y s t imu la t io n o f DNA s y n th e s is .

T h e mo le c u la r b a s is fo r th i s d i f f e re n c e in F GF re s p o n -

s i v e n e s s b e twe e n E1 4 /1 5 Sc h wa n n c e l l p re c u rs o rs o n th e

o n e h a n d a n d E 1 7 a n d o l d e r S c h w a n n c e l ls o n t h e o t h e r

n o w r e m a i n s to b e d e t e r m i n e d .

S c h w a n n C e l l G e n e r a t io n

1 0 0 e x p re s s io n , s u rv i v a l r e q u i re me n ts , a n d m i to g e n ic

re s p o n s e to F GF 2 re p re s e n t a d i v e rs e c o l le c t io n o f a p p a r -

e n t l y u n re la te d p h e n o ty p ic fe a tu re s . Ne v e r th e le s s , a l l

t h re e a re s u b je c t t o s t ro n g d e v e lo p m e n ta l r e g u la t io n in

th e Sc h w a n n c e l l l i n e a g e . T h e y c h a n g e w i th a s im i la r t ime

c o u rs e a n d d u r in g th e s a me p e r io d o f e mb ry o n ic n e rv e

d e v e lo p me n t , i . e . , b e twe e n E1 4 a n d E1 8 w i th th e ma jo r

s h i f t o c c u r r in g b e twe e n E1 5 a n d E1 7 . T h is c o m p re h e n s iv e

p h e n o t y p i c c h a n g e c a n n o w b e r e p r o d u c e d u n d e r d e f i n e d

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Neuron

594

c o n d i t io n s i n v i t ro : L l / N G F R - p o s i t i v e p r e c u r s o r s r e m o v e d

f r o m E 1 4 n e r v e s a n d e x p o s e d t o N D F I 3- 2 f o r 1 - 4 d a y s

s h o w a g r a d u a l a l t e r a t i o n i n a ll t h r e e p a r a m e t e r s t h a t fo l -

l o w s a t im e c o u r s e v e r y s i m i l a r t o t h a t s e e n i n v i vo . T h i s

s u g g e s t s t h e e x i s t e n c e o f a p r o g r a m i n E 1 4 c e l l s fo r c o o r d i-

n a t in g t h e e x p r e s s i o n o f a s u b s t a n t i a l n u m b e r o f fu n c t io n -

a l l y d i v e r s e g e n e s . I t i s l i k e l y t h a t t h i s p r o g r a m i s d r i v e n

b y N D F . A l t e r n a t i v e l y , it m a y u n f o l d c e ll a u t o n o m o u s l y i n

c e l ls in w h i c h N D F h a s b l o c k e d a p o p t o s i s . T h e r e s o l u t i o n

o f t h e s e i s s u e s a n d t h e m o l e c u l a r i d e n t i f i c a t io n o f t h e u n -

d e r ly i n g g e n e r e g u l a t o r y m e c h a n i s m s r e p r e s e n t i m p o r t a n t

f u t u r e l i n e s o f w o r k .

Experimental Procedures

ntibodies

Rabbi t ant iserum to bov ine $ 100 prote in (Dak opat ts A/C) was used

at a d ilu tion o f 1:10,000. Mo noc lona l ant ibody (MAb) 192 IgG, which

recognizes the low a ff ini ty p75 NG~R, wa s a gi f t fro m E. Johnson, Jr .,

and was used in the form o f asc i tes f lu id a t a d ilu tion of 1:100. MAb

ASCS4, which in the rat recognizes the cel l adhes ion molecule L1

(Sweadner, 1983) , was a g i f t from Dr . A. Fur ley . I t was used in the

form of cell cu l ture supernatant . MAb agains t BrdU w as a g if t f rom

M. Jones and Dr . D . Mason an d wa s used at a d i lu t ion of 1:100. Rabbi t

ant i serum to recombinant rat NDF (Wen et a l . , 1994) was used at a

concentration of 1:50. Aff ini ty-pur i fied rabbi t antiseru m 1915 to reco m-

b i nan t hum an N D F w as m ade and c har ac te r iz ed by D r. D . Wen , Am ge n

C en te r , and w as us ed a t a c onc en t r a ti on o f 5 - 10 g /m l . M ous e M Ab

5D6A agains t recomb inant huma n NDF wa s made and charac ter ized

by Dr . D . Chang, Amg en Center , and w as used at a d i lu t ion of 5-10

~g/ml . MAb K -15 to ErbB2 rece ptors and M Ab C-18 to ErbB4 receptors

were f rom Sa nta Cruz Biotec hnology (used at 1 ~g/m l ). OX7 hybr idoma

cel l l ine wa s f rom European Col lec t ion of Anim al Cel l Cul tures . Rabbi t

ant i-mouse immunoglobulin was f rom Dakopat ts A/C. G oat anti -human

ant ibody wa s f rom Sigm a Chemical Company. F luorescein conjugated

to goa t anti - rabbi t immun oglobul in (Cappel Labs), absorbed with

mo use immun oglobul in to rem ove cross-reacting antibodies, and tet-

ramethy l rhodamine conjugated to goat ant i -mouse immunoglobul in

(Cappel Labs) , absorbe d w i th rabbi t immunoglobulin to remove cross-

reacting antibodies, we re both used at a di lut ion of 1:100. Biotinylated

sheep ant i- rabbi t and ant i-mouse immunoglobulins and s t reptav id in-

f luorescein were f rom Am ersham Internat ional .

O t h e r

Materials

Transferr in, selenium, putrescine, tr i iodothyronine, thyroxine, dexa-

methasone, insul in, cytosine arabinoside, hyaluronidase, laminin,

poly -L- tys ine (molecu lar weight 300,000), and bov ine serum albumin

(BSA) were obta ined f rom Sigma Chem ical Com pany. T issue cul ture

pet ri d ishes and 24-wel l p lates were f rom Falcon. Prote in A Se pharose

column w as f rom P harm ac ia Biotech. Four -well p lates were f rom Nunc

Li fe Technologies . NGF was a g i f t f rom Dr . J . Winter ; bFG F was f rom

R&D Systems. Fe ta l ca l f serum was f rom Ad vance d Prote in Products.

Dulbecco's m odi f ied Eagle's medium (DMEM ) , m in imum essent ia l me-

dium (MEM) , Ham's F12 medium, an d t ryps in were f rom GIBCO Labo-

rator ies . Col lagenase was obta ined f rom Wor th ington Biochemical

Corporat ion. BrdU wa s from B oehr inger Mannheim. C i ti fl uor was ob-

tained from City Universi ty (London).

Defined Medium

Def ined medium used in th is s tudy wa s a modi fi cation of the medium

of Bot tenste in and S ato (1979) . A 1:1 m ix ture of DME M and Ham's F12

was supplem ented w i th ( fina l conce ntrat ion in parenth eses) t rans fe r r in

(100 I~g/ml) , prog este rone (60 ng/ml) , putre scine (16 i lg/ml) , insul in

(5.7 ng/ml, 10 9 M), IG F1 (100 ng /ml, 13 nM), thyro xine (0.4 t~g/ml) ,

selenium (160 ng/m l ) , t r i iodothyro nine (10.1 ng/m l ), de xam ethaso ne

(38 ng/ml) , gluc ose (7.9 mg/m l) , BS A (0.3 mg/ml) , penici l lin (100 IU/

ml) , streptomycin (100 IU/ml) , and glutamine (2 mM ). This medium is

refer red to as def ined medium or def ined med ium w i thout serum. In

som e exper ime nts insul in an d/or IGF1 were omi t ted f rom the medium;

this is indicated in the text.

Cell Cultures

Cul tures of Schwann cel l s and precursors were prepared essent ia l l y

as descr ibed prev ious ly (Jessen et a l . , 1994; G avr i lov ic et a l. , 1995) .

Af ter d issoc iat ion and cen t r i fugat ion, the cel ls were resuspended in

def ined medium, counted, and p lated (at a dens i ty of 2000 cel l s per

coversl ip unless ot her wise indicated) in a 10 or 20 ~.1 drop o n laminin-

coated covers lips . Af ter 3 hr , the cul tures were toppe d up w i th 280

I~1 of defined me dium . Coversl ips we re coate d with poly-L- lysine and

laminin as descr ib ed previously (Jessen et al. , 1994).

The survival ass ay has been descr ibed in detail previously (Gavr i-

Iovic et al ., 199 5; also see Jessen et al . , 1994 ). In br ief, 20 h r after

p lating the cel l s we re f ixed and immunolabeled w i th ant ibody 192 IgG

ag ain st the p75 NQFRor ant ibody ASCS4 agains t L1 (see below) . The

num ber of f la t tened imm unopos i ti ve cel ls at th is time point was

counted in the fluoresce nce m icroscope and expres sed as a percent -

age of the n um ber o f such cel ls on s is ter covers lips 3 hr af ter p lating;

th is f igure i s refer red to as percentag e surv ival . T he ass ay as descr ibed

abov e is referred to as the 20 hr surv ival assay. In some exp er imen ts ,

surv ival was examined over longer per iods ; th is i s indicated in the

tex t .

The BrdU a ssay of DNA synthes is was car r ied out by expos ing cel l s

to BrdU (20 ~M) for per iods of 1.5 hr fo l lowed'by double immunola-

bel ing us ing ant ibodies to L1 (see below) and BrdU. The percentage

of L l -pos i t i ve cel l s that a lso had BrdU-pos i ti ve nuc le i was taken to

indicate the propo rt ion of gl ial cel ls that synthesized DNA.

For identi f ication of Sch wan n cell precursors, antibodies a ga inst L1

were used in mo st experiments, a l though the resul ts were general l y

checked using a ntibo dies a gain st the p7 5 NaFR, wh ich served as the

ant igenic m arker for precursor identi fi cation in our ear l ier expe r iments

(Jessen et al . , 1994; Gavr i lovic et al . , 1995). Schwan n cel l precursors

exp ress cel l surfa ce L1 immunoreactiv i ty (Jessen et al. , 1994). In the

present study, we carr ied out double- immuno fluorescence exp er i-

ments us ing ASCS4 ant ibody and 192 IgG ant ibody to detec t L1 and

NGFR , respectively, a nd class-speci fic f luorochrome-labeled anti -

mou se second laye r ant ibodies . A l l the L l - imm uno labeled cel ls a lso

expressed p75 NGFR. On the oth er hand, p75 N°FR wa s also de tecta ble

on a few Ll -neg at ive cel ls . In cul tures mainta ined in NDF ( the condi -

t ions used in near ly a l l the exper im ents in the prese nt paper ), 3% of

the p75NGFR-posit ive cel ls we re L1 negative after 20 h r in vi tro, and the

num ber of such cel l s af ter longer per iods in cul ture was lower (data

not shown) . Som e o f the L1-negat ive/p75NG~H-pos it ive cel ls show ed

nei ther the f la t tened mo rphology no r the group-form ing tenden cy char-

ac teris ti c of the v ery g reat m ajor i ty of ce l ls in the prec ursor cul tures ,

but ten ded instead to occu r singly on the coversl ip; fur thermore, prel im-

inary tes ts indicated th at these cel l s d id n ot acqui re s ignif icant $100

expression with t im e in culture. I t is possible th at this smal l subpopula-

tion of p75NG FR-positivecel ls d oes no t belong to the g l ia l l ineage. How-

ev er t ha t m ay be , w e hav e p r e fe r red , i n t he p r es en t w or k , no t t o m ak e

ou r observations on th e total p75NGFR-posit ive pop ulatio n but ra ther to

conf ine them, wh ereve r prac t icable, to the sl ight ly smal ler and m ore

hom ogeno us po pulat ion of cel l s tha t express both L1 and p75 ~GFRand

take par t in form ing f la t tene d cel lu lar groups. For prac ti ca l reas ons,

p75 NG~Rwas neve r theless used to ident ify precursors in som e double-

labeling expe r iments ; th is i s indicated in the tex t .

For cul tur ing neurons, a pe l le t of DRG cel l s was prepared as de-

scr ibed e lsewhe re (Jessen e t a l ., 1 994) . The cel l s we re then resus-

pended in 6 m l of DMEM plus 10% feta l ca l f serum o r defined m edium,

and the suspens ion was t rans fer red to a 90 mm plas t i c d ish coa ted

with antibodies a ga inst Thy 1.1 in DRG cel l suspension. (Thy 1.1 is

present on both ne uron s and f ibroblasts, but not on gl ial cel ls.) Afte r

2 - 3 m i n a t 37° C , t he d is h w as w as hed 4 - 5 t i m es v e r y qu ic k ly w i t h

DMEM plus 10% feta l ca l f serum or def ined med ium. The neurons

w er e t hen s t r eam ed o f f w i th de f i ned m ed i um c on ta i n ing 50 ng /m l N GF

and plated in 4-wel l poly-L-lysine-coated plastic plate s at a densi ty of

100,000-150,000 neurons/wel l. The volume of cul ture medium pe r

wel l was 300 Id, and 95% of the cel l s in the cul tures at 20 hr were

neurons . T he m ed i um w a s c hanged a t 20 h r , and c ond i t ioned m ed i um

was col lec ted at 48 and 72 hr af ter p lating only .

To coa t w i th ant i -Thy 1.1, the d ish wa s f ir s t incubated w i th 6 m l

of Tr is buf fer solut ion (50 mM; p H 9.5) conta in ing rabbit ant i-mouse

immunoglobulin (38 t~g/m l f ina l concentrat ion) for 18 hr at 4°C. I t was

then washe d 3 t ime s w i th phosphate-buf fered sal ine (PBS) and incu-

bated w i th a m ix ture of OX7 ant ibodies (Mason and W i ll iams, 1980)

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NDF and Schwann Cel l Precursors

595

i n the f o r m o f c u l t u re s uper na tan t ( 4 m l ), M EM - H EPE S ( 2 m l) , and

BSA (400 p.I at 35 mg/ml ) fo r 1-4 hr at room tem peratu re. The d ish

was washed 3 t imes w i th PBS and used immediate ly .

In replat ing assays for mon i tor ing precursor ma turat ion, cel ls f rom

nerves at var ious age s (see Resul ts ) we re p lated at a dens i ty of 12,000

cel ls per wel l in 50 p.I of defined me dium o n laminin-coated, poly-L-

lysine-coated wel l s . Af ter 3 hr, the m edium w as w i thdrawn and re-

p laced w i th 300 I~1 of def ined me dium conta in ing the ap propr iate con-

cent rat ion of NDF~-2. Af ter 1-4 days in v i t ro, the cel l s were r insed

once w i th verse ne and incubated for 5 m in w i th 300 ~1 of versene p lus

100 p.I of enzym e cockta il a t 37°C. The cel l s were t r i turated o f f the

wel ls and remo ved to a cent ri fuge tube conta in ing def ined m edium,

spun at 1000 rpm for 10 ra in, and resuspended in defined medium. T he

cel ls we re rep lated a t a densi ty of 2,00 0 cel ls in 20 i11 on laminin-coated

coversl ips . Af ter 3 hr , the cul tures were topp ed w i th def ined medium

for the survival assay, wi th defined medium containing 3 ng/ml bFGF

and 5 p.M forskol in for the cell d iv ision assay, or f i xed for exam inat ion

o f $100 ex p r es s i on .

Construction and Expression of a Secreted Soluble

ErbB Receptor

T o c ons t r uc t a s o l ub l e E r bB4 r ec ep to r - im m unog tobu l i n c h i m er i c D N A,

the express ion vec to r CDM7 ( Inv it rogen) bear ing the ex t race l lu lar por -

t ion of ErbB4 fused in f rame to an Fc por t ion (h inge, CH2, and CH3)

of a hum an immuno globul in y-1 wa s used. Essential ly, the parental

CDM 7 plasmid was d igested w i th Bam HI and EcoRI to a l low fus ion

of the Fc por t ion w i th the ex t rac el lu lar domain o f ErbB4 (denoted IgB4) .

The ex t race l lu lar domain o f ErbB4 was ampl i f ied by us ing the polymer-

ase chain reaction (30 cyc les of 1.5 m in a t 96°C, 2 m in at 52°C, and

3 min at 72°C) . The am pl ifed e x t racel lu lar domain was pur i fied a nd

digested w i th Bam HI and EcoRI and inserted into the ap propr iate s i tes

in the expres s ion vec tor . The upst ream and do wnstream ol igonuc leo-

t ide pr imers of ErbB4 had the fo l low ing sequences: 5 ' -CGCCGGGA AT-

T C C AA AAAA T GAA GC C G GC G AC - 3 ' and 5 '- C C C GGGGAT CC C T AG-

CATG TTGTG GTAA AGTG G-3', respectively. The bui l t-in cloning si tes

are under lined. Nucleotide sequen cing c on fi rme d the integr i ty of the

open rea ding f ram es of the chim er ic cDNA and a lso par t ia l ly ver if ied

cor rec t seque nces. For e lec t roporat ion of HEK 293 cel ls , 10 p.g of the

result ing IgB4 plasmid toge ther w i th 0.5 I~g of pSV 2/neo wa s mixed

wi th 2 x 106 cel ls in 0.8 m l of DM EM The elec t roporat ion was car r ied

out w i th a Bio-Rad gen e pulser us ing vol tage and ca pac i tanc e set t ings

of 270 V and 960 I~F, respective ly . Indiv idual c lone s were selec ted

wi th G418 (800 p.g/m l ) and m ainta ined in DMEM w i th 10% feta l ca l f

serum . The condi tioned med ia we re assayed w i th a goat anti -human

ant ibody to selec t pos i t i ve c lones that secreted the fus ion prote ins .

Med ia condi t ioned by a selec ted cel l c lone were then pur i fied us ing

prote in A Seph arose column. Protein concentrat ion was es t imate d by

get staining and comparison with myosin.

Immunohistochemistry

Immunolabeling of $100, L1, NGFR, and BrdU is descr ibed e lsew here

(Jessen et a l . , 1994) . F ixation for ErbB2 and E rbB4 was 4% paraform al -

dehyde (20 m in) fo l lowed by PBS wash and then 0.1% Tr i ton X-100

(20 min). ErbB2 and ErbB4 imm unorea ct iv i ty was v isual ized us ing the

biot in-s t reptav id in method. F ixat ion for NDF was 2% paraform alde-

hyde (7 min). NDF imm unoreactiv i ty wa s visual ized using the biot in -

s t reptav id in meth od. A l l ce l ls f i xed by 2% paraform aldehy de pr ior to

immunosta in ing were f i xed w i th 4% p araform aldehyd e at the end of

t he i m m uno l abe l i ng

Controls for Immunohistochemistry

In every case, omiss ion of the f i r s t ant ibody layer abol i shed any immu-

nolabeling. Abso rpt ion of the ant i-ErbB2 ant ibody w i th ErbB2 con t ro l

peptide, but not wi th ErbB4 peptide, ab ol ished the immun otabel ing.

Sim i lar ly , absorpt ion of the ant i-ErbB4 ant ibody w i th ErbB4 cont ro l

peptide, bu t not wi th ErbB2 peptide, abol ished label ing. A bso rption

of ant i serum to NDF w i th NDFJ3-2, but n ot w i th FGF2, ab ol i shed immu-

nolabel ing o f DRG neurons.

Quantification

Al l quant i ta t i ve resul ts are based on a m inimum of three separate

expe r iments (unless s tated otherw ise). Each determ inat ion w i th in an

expe r imen t is based on cou nts f rom three covers lips in the great major -

i ty of cases, bu t occas iona l l y on co unts f rom two or fou r covers l ips .

Error bars on graphs indicate SEM. In al l survival assays, survival is

expresse d as percenta ge surv ival ; th is term is def ined abov e.

Acknowledgments

This work wa s car r ied out w i th sup por t f rom th e Wel lcom e Trus t (grants

042257/Z /94/Z and 036963/1.5) an d the Medical Resea rch Counci l o f

Grea t Bri ta in. We are gratefu l to M rs . D . Bar tram for exper t typ ing and

edi t ing of the manuscr ipt . We a re indebted to Ms. M. Jone s and Drs .

E. Johnson, Jr ., D. Chang, D. Wen, and A. Fur ley for gi f t of antibodies

and to Dr . J . Winter for donat ion of NGF.

The costs of publ icat ion of th is ar t i c le were def rayed in par t by

the pay m ent o f page c har ges . T h i s a r t ic l e m us t t he r e fo r e be he r eby

m ar k ed

advert isement

i n ac c o r danc e w i t h 18 U SC Sec t i on 1734

sole ly to indicate this fact.

Received March 23, 1995; revised June 5, 1995.

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