Spatial restriction of AChR gene expression to …...experiments were performe on diaphragm d ,...

Development 114, 545-553 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

545

Spatial restriction of AChR gene expression to subsynaptic nuclei

ALEXANDER M. SIMON, PETER HOPPE1 and STEVEN J. BURDEN

Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA1Jackson Laboratories, Bar Harbor, Maine 04609, USA

Summary

Acetylcholine receptors (AChRs) and the mRNAs en-coding the four AChR subunits are highly concentratedin the synaptic region of skeletal myofibers. The initiallocalization of AChRs to synaptic sites is triggered by thenerve and is caused, in part, by post-translationalmechanisms that involve a redistribution of AChRprotein in the myotube membrane. We have usedtransgenic mice that harbor a gene fusion between themurine AChR delta subunit gene and the human growthhormone gene to show that innervation also activatestwo independent transcriptional pathways that areimportant for establishing and maintaining this non-uniform distribution of AChR mRNA and protein. Onepathway is triggered by signal(s) that are associated withmyofiber depolarization, and these signals act to repressdelta subunit gene expression in nuclei throughout themyofiber. Denervation of muscle removes this repression

and causes activation of delta subunit gene expression innuclei in non-synaptic regions of the myofiber. A secondpathway is triggered by an unknown signal that isassociated with the synaptic site, and this signal actslocally to activate delta subunit gene expression only innuclei within the synaptic region. Synapse-specificexpression, however, does not depend upon the continu-ous presence of the nerve, since transcriptional acti-vation of the delta subunit gene in subsynaptic nucleipersists after denervation. Thus, the nuclei in thesynaptic region of multinucleated skeletal myofibers aretranscriptionally distinct from nuclei elsewhere in themyofiber, and this spatially restricted transcriptionpattern is presumably imposed initially by the nerve.

Key words: neuromuscular synapse, human growthhormone, transgenic mice, synaptic development.

Introduction

In adult muscle fibers, acetylcholine receptors (AChRs)are virtually confined to the small patch of membranedirectly beneath the presynaptic nerve terminal (Sal-peter, 1987). This arrangement is established duringdevelopment in a series of steps that begin when thedeveloping motor nerve interacts with embryonicmyotubes and causes a redistribution of a fraction ofAChRs that were already present on the myotubemembrane (Anderson and Cohen, 1977; Ziskind-Conhaim et al., 1984; Role et al., 1985). Thereafter,AChRs are excluded from non-synaptic regions of themyofiber membrane and they remain concentrated atthe synaptic site (Burden, 1977).

The maintenance of a high concentration of AChRsat the synapse requires replacement of AChRs atsynaptic sites (Fambrough, 1979), and there is evidencethat this replacement of synaptic AChRs is ac-complished by local insertion of AChRs into thepostsynaptic membrane (Role et al., 1985). In addition,there is evidence that AChR polypeptides are syn-thesized locally in the synaptic region, since themRNAs encoding the different subunits of the AChRare highly concentrated at synaptic sites in adultmyofibers (Merlie and Sanes, 1985; Fontaine and

Changeux, 1989; Goldman and Staple, 1989; Brenner etal., 1990).

The mechanisms responsible for accumulation ofAChR mRNAs at synaptic sites are unclear, but couldinvolve selective stabilization of AChR mRNAs in thesynaptic region, transport of AChR mRNAs to thesynaptic region, or selective transcription of AChRgenes by the subset of nuclei that are within the synapticregion.

We constructed transgenic mice that contain a genefusion between 1.8 kbp of 5' flanking DNA from themurine AChR delta subunit gene and the humangrowth hormone (hGH) gene, to determine whethercis-acting elements in the AChR gene could restrictexpression of hGH to the synaptic region of skeletalmyofibers. Because hGH is processed in intracellularorganelles prior to secretion, and because these organ-elles are closely associated with nuclei, we could inferthe nuclear source of hGH transcription by studying thespatial distribution of intracellular hGH by immuno-cytochemistry. We show here that hGH in thesetransgenic mice is restricted to the perinuclear region ofnuclei within the synaptic region of the myofiber, andwe conclude that transcription of the endogenousAChR delta subunit gene is confined to nuclei that aresituated at the synaptic site. These data indicate that

546 A. M. Simon, P. Hoppe and S. J. Burden

synaptic nuclei are transcriptionally distinct from nucleielsewhere in the multinucleated skeletal myonber andthat the synaptic site provides a signal that acts locallyto activate transcription of certain genes in nuclei thatare positioned close to the synaptic site.

Materials and methods

Construction of transgenic miceC57BL/6J females were mated with LT/Sv males, and zygoticmale pronuclei were injected with a gene fusion between themurine AChR delta subunit gene (-1,823/+24) (Baldwin andBurden, 1988) and the hGH gene (Selden et al., 1986). Theembryos were transferred to B6/SJL Fl pseudopregnantfemales (Wagner et al., 1981). Five founder mice (<5GHl-5),which had integrated the transgene into their germ line, werethe source of five different transgenic lines. All integrationsare autosomal, and they range from a few copies to more thanten copies of the transgene. Mice from each transgenic lineexpress hGH in skeletal muscle, although the absolute level ofhGH expression and the degree of muscle-specific hGHexpression differs among the five lines (Table 1). Anadditional founder mouse was apparently sterile and anotherfounder mouse contained the transgene but did not expresshGH in any tissue; we did not establish lines from these mice.hGH (20-100 ng/ml) is detectable by radioimmunoassay in theserum of newborn transgenic mice.

HistologyImmunofiuorescence

Adult mice were fixed by perfusion (4% formaldehyde in0.9% sodium chloride with 0.2 M sucrose), and dissectedmuscles were fixed further by immersion in fixative for 1 hr.The tissue was rinsed with 10 mM sodium phosphate, 150 mMsodium chloride, pH 7.3 (PBS), washed with 20 mM Tris, 150mM sodium chloride, pH 7.3, permeabilized with PBS-NP40(0.5% NP40) and incubated with antibodies to hGH (DAKO,Carpinteria, CA) (1/500 in PBS-NP40) for 3-6 hr. at roomtemperature. The tissue was washed with PBS for 0.5 hr.,incubated with fluorescein-labelled goat anti-rabbit IgG

Table 1. hGH mRNA levels in muscle and non-muscle tissue

Line #

(5GH1<5GH2<5GH3(5GH4<5GH5

Den.muscle

100100100100100

Heart

30.0<0.2

0.440.0

2.0

Liver

<0.5<0.2

0.3<6.090.0

Spleen

101

201010

In all five lines the highest level of hGH mRNA is seen indenervated muscle, but only <5GH2 shows nearly complete musclespecificity. Because there is no consistent pattern of tissueexpression among these lines, it seems likely that the site oftransgene integration and cryptic elements in the hGH gene (Lowet al., 1989) determine the level of hGH expression in non-skeletalmuscle tissue. The levels of hGH mRNA in heart, liver, spleenand denervated skeletal muscle were determined by RNaseprotection (Materials and methods). The amount of hGHmRNA/total RNA in denervated muscle from each line wasassigned 100%, and the values of hGH mRNA/total RNA in othertissues are expressed relative to denervated muscle.

(Cappel, Malvem, PA) and tetramethylrhodamine-coupledalpha-bungarotoxin (TMR-tf-BGT) in PBS-NP40 for 3-6 hr.at room temperature, washed with PBS for 0.5 hr. and fixed in100% methanol at -20°C. Muscles, or individual dissectedmuscle fibers, were mounted whole (in 20% glycerol, 80 mMsodium bicarbonate, pH 9, with 10 /̂ g/ml p-phenylenedi-amine) and were viewed with filters selective for eitherfluorescein or rhodamine (Burden, 1982). Our attempts todetect hGH with enzyme-coupled antibodies were unsuccess-ful; lack of hGH staining was probably due to poorpenetration of these reagents into muscle tissue. Mostexperiments were performed on diaphragm, intercostal,extensor digitorum longus and soleus muscles; the pattern ofhGH staining was indistinguishable among the differentmuscles. Staining for hGH in muscle that was fixed only byimmersion, and not by perfusion, was capricious; lack of hGHstaining under these conditions was probably due to secretionof hGH during the time required for dissection of deepermuscles prior to their fixation. Because of the low level ofhGH expression in lines <5GH4 and <5GH5 and the reducedfertility of line (5GH3, we restricted the immunocytochemicalanalysis to lines <5GH1 and <5GH2.

In situ hybridizationMuscles were fixed in 4% formaldehyde in PBS for 1 hr.,stained for cholinesterase and dehydrated. Single musclefibers were dissected in a drop of water on silated microscopeslides, allowed to dry overnight at 40°C and processed for insitu hybridization (Kintner and Melton, 1987). The 35S-labelled anti-sense delta subunit RNA probe (1.9 kbp long)was synthesized with SP6 polymerase from SP65-delta cDNA(LaPolla et al., 1984). Exposures were for 4-5 days.

RNA analysisRNA was isolated from tissue with guanidinium thiocyanateas described (Chomczynski and Sacchi, 1987). The levels ofdelta subunit, skeletal muscle actin and hGH mRNAs weremeasured by RNase protection (Baldwin and Burden, 1988),were quantitated with a phosphorimager (Molecular Dy-namics, Sunnyvale, CA), and were corrected for non-specifichybridization by subtracting the value for protection withtRNA. The 32P-labelled RNA probes extended from nucleo-tide 1 to 582 in the hGH gene (Selden et al., 1986), nucleotide234 to 730 in the delta subunit gene (Baldwin and Burden,1988), and nucleotide 1258 to 1661 in the actin gene (Hu et al.,1986).

Results

AChR delta subunit mRNA is highly concentrated atsynaptic sitesmRNAs encoding the four subunits of the AChR areenriched at synaptic sites (Fontaine and Changeux,1989; Goldman and Staple, 1989; Brenner et al., 1990).The distribution of delta subunit mRNA in a singleisolated myonber is illustrated in Fig. 1, which showsthat delta subunit mRNA is concentrated at thesynaptic site, whereas nuclei are present throughout themyofiber.

Fig.

1.

AC

hR d

elta

sub

unit

mR

NA

is

conc

entr

ated

at

syna

ptic

site

s. M

ouse

diap

hrag

m m

uscl

e w

as s

tain

ed f

or a

cety

lcho

lines

tera

se,

and

indi

vidu

al m

uscl

e fi

bers

wer

e di

ssec

ted

and

proc

esse

d fo

r in

situ

hyb

ridi

zatio

n us

ing

a 35S-

labe

lled-

anti-

sens

ede

lta R

NA

pro

be (

Mat

eria

ls a

nd m

etho

ds).

(A

) T

he s

ynap

tic s

ite o

n th

e m

yofib

er i

sid

entif

ied

by c

holin

este

rase

sta

inin

g. (

B)

The

aut

orad

iogr

aphi

c gr

ains

are

con

cent

rate

dat

the

syn

aptic

site

. (C

) T

he s

ingl

e sy

napt

ic s

ite o

n an

othe

r m

yofib

er i

s id

entif

ied

byth

e ne

rve

term

inal

and

ass

ocia

ted

Schw

ann

cells

, w

hich

are

see

n cl

early

on

the

myo

fiber

sur

face

. (D

) T

he n

ucle

i of

bot

h th

e m

yofib

er a

nd S

chw

ann

cells

con

trib

ute

to a

clu

ster

of

nucl

ei a

t th

e sy

napt

ic s

ite.

Thr

ee t

o six

myo

fiber

nuc

lei

are

pres

ent

atea

ch s

ynap

tic s

ite,

and

the

dens

ity o

f m

yofib

er n

ucle

i in

non

-syn

aptic

reg

ions

is

2.5

to3-

fold

les

s. M

yofib

er n

ucle

i ar

e lo

cate

d at

the

per

iphe

ry o

f th

e ce

ll an

d ar

e fo

und

thro

ugho

ut t

he m

yofib

er (

D).

Bar

=7.

5 ft

m f

or (

A,B

) an

d 15

/m\

for

(C,D

).

Fig.

2.

Del

ta s

ubun

it ge

ne i

s tr

ansc

ribe

d pr

efer

entia

lly i

n sy

napt

ic n

ucle

i. hG

His

con

cent

rate

d at

syn

aptic

site

s of

mus

cle

from

tra

nsge

nic

mic

e th

at h

arbo

r a

gene

fus

ion

betw

een

the

AC

hR d

elta

sub

unit

gene

and

the

hG

H g

ene.

Tw

om

uscl

e fi

bers

are

sho

wn,

one

in

A a

nd B

and

the

oth

er,

at l

ower

mag

nific

atio

n,in

C a

nd D

. Si

ngle

mus

cle

fibe

rs w

ere

doub

le-l

abel

led

with

ant

ibod

ies

agai

nst

hGH

(A

,C)

and

with

TM

R-w

-BG

T t

o st

ain

AC

hRs

(B,D

), m

ount

ed w

hole

, an

dvi

ewed

with

opt

ics

sele

ctiv

e fo

r ei

ther

flu

ores

cein

(A

and

C)

or r

hoda

min

e (B

and

D).

Mos

t m

yofib

ers

have

a s

ingl

e sy

napt

ic s

ite (

B,

long

arr

ow),

and

the

pres

ence

of

two

adja

cent

syn

aptic

site

s is

unc

omm

on (

D,

long

arr

ows)

. hG

H i

sco

ncen

trat

ed a

t th

e pe

rinu

clea

r re

gion

of

nucl

ei i

mm

edia

tely

ben

eath

the

post

syna

ptic

mem

bran

e (A

,C,

shor

t ar

row

s).

Low

er l

evel

s of

hG

H a

rede

tect

able

in

the

peri

syna

ptic

reg

ion

(10-

50 f

an f

rom

the

edg

e of

the

syn

aptic

site

; A

,C,

curv

ed a

rrow

s),

whe

reas

hG

H i

s no

t de

tect

able

in

non-

syna

ptic

regi

ons.

Bar

=12

fan

for

(A

,B)

and

30 /a

n fo

r (C

,D).

Fig.

3.

Pref

eren

tial

tran

scri

ptio

n of

the

del

ta s

ubun

it ge

ne i

n sy

napt

icnu

clei

doe

s no

t re

quir

e th

e co

ntin

uous

pre

senc

e of

the

ner

ve.

The

exte

nsor

dig

itoru

m l

ongu

s m

uscl

e w

as d

ener

vate

d by

cut

ting

the

scia

ticne

rve,

and

sin

gle

mus

cle

fibe

rs, w

hich

wer

e de

nerv

ated

for

4 d

ays,

wer

e la

belle

d w

ith a

ntib

odie

s ag

ains

t hG

H (

A,C

) an

d w

ith T

MR

-<*-

BG

T (

B,D

) an

d m

ount

ed w

hole

. T

wo

mus

cle

fibe

rs a

re s

how

n, o

ne in

A a

nd B

and

the

oth

er i

n C

and

D.

The

den

erva

ted

syna

ptic

site

ism

arke

d by

TM

R-a

sBG

T (

B,D

, lo

ng a

rrow

). h

GH

is

conc

entr

ated

at

the

penn

ucle

ar r

egio

ns o

f nu

clei

im

med

iate

ly b

enea

th t

he p

osts

ynap

ticm

embr

ane

of d

ener

vate

d m

uscl

e (A

,C,

shor

t ar

row

). h

GH

is

read

ilyde

tect

able

in

the

peri

syna

ptic

reg

ion

of d

ener

vate

d m

uscl

e (C

, cur

ved

arro

w).

hG

H s

tain

ing

is m

ore

inte

nse

at s

ynap

tic s

ites

and

atpe

risy

napt

ic r

egio

ns i

n de

nerv

ated

mus

cle

than

in

inne

rvat

ed m

uscl

e.B

ar=

30 f

tm.

Fig.

5.

Non

-syn

aptic

nuc

lei

inde

nerv

ated

mus

cle

are

hete

roge

neou

s, a

nd n

ucle

ith

at e

xpre

ss t

he d

elta

sub

unit

gene

at

an e

nhan

ced

rate

are

posi

tione

d cl

ose

to n

on-

syna

ptic

AC

hR c

lust

ers.

Thr

ee m

uscl

e fi

bers

are

show

n, o

ne i

n A

and

Ban

othe

r in

C a

nd D

, an

d a

thir

d in

E a

nd F

. T

he m

uscl

efi

bers

wer

e do

uble

-lab

elle

dw

ith a

ntib

odie

s ag

ains

t hG

H(A

,C,E

) an

d w

ith T

MR

-o-

BG

T t

o st

ain

AC

hRs

(B,D

,F).

Non

-syn

aptic

AC

hRcl

uste

rs w

ere

dete

cted

with

TM

R-a

-BG

T o

nap

prox

imat

ely

one-

thir

d of

the

dene

rvat

ed m

yofib

ers

(B,D

,F,

long

arr

ows)

, an

dhG

H w

as o

bser

ved

in t

here

gion

of

each

AC

hR c

lust

er(A

,C,E

, sh

ort

arro

ws)

. N

on-

syna

ptic

AC

hR c

lust

ers

are

usua

lly f

ound

fur

ther

tha

n50

0 /m

i fro

m s

ynap

tic s

ites

(B,D

), a

lthou

gh t

hey

can

occu

r ad

jace

nt t

o th

ede

nerv

ated

syn

aptic

site

(F,

curv

ed a

rrow

). I

n so

me

case

sth

e hG

H-p

ositi

ve n

ucle

i ar

ein

the

vic

inity

of

AC

hRcl

uste

rs (

C,D

), w

here

as in

othe

r ca

ses,

hG

H-p

ositi

venu

clei

are

fou

nd p

reci

sely

unde

rlyi

ng A

ChR

clu

ster

s(A

,B a

nd E

,F).

The

dene

rvat

ed s

ynap

tic s

ite is

read

ily d

istin

guis

hed

from

non-

syna

ptic

AC

hR c

lust

ers

by i

ts c

hara

cter

istic

sha

pean

d si

ze,

and

was

ide

ntifi

edon

eac

h m

yofib

er t

hat

had

non-

syna

ptic

AC

hR c

lust

ers.

Bar

=12

/m

\ fo

r (A

,B,C

,D)

and

30 /a

n fo

r (E

,F).

AChR gene expression 547

AChR delta subunit gene is transcribed selectively insubsynaptic nucleiAccumulation of delta subunit mRNA at synaptic sitescould be caused by transcriptional and/or post-tran-scriptional mechanisms. In order to determine whetherc/5-acting elements in the delta subunit gene restrictexpression to the synaptic region of skeletal myofibers,we constructed transgenic mice that contain a genefusion between 1.8 kbp of 5' flanking DNA from'thedelta subunit gene and the hGH gene (Materials andmethods).

Growth hormone is normally expressed and secretedby somatotroph cells of the anterior pituitary (Herlant,1964), but the hormone can be expressed and secretedby a variety of non-pituitary cell types in transgenicmice (Palmiter et al., 1983; Trahair et al., 1989). Cellsthat have a regulated secretory pathway contain thehormone in secretory granules and in the golgiapparatus, whereas cells that have only a constitutivesecretory pathway contain the hormone largely in thegolgi apparatus (Trahair et al., 1989; Roth et al., 1990).Since myofibers are not regarded as secretory cells andare thought to possess a constitutive but not aregulated, secretory pathway (Kelly, 1985; Burgess andKelly, 1987), we expected intracellular hGH to befound in the golgi apparatus of myofibers. Further,because the golgi apparatus is closely associated withnuclei, and because mRNAs do not diffuse over longdistances in myotubes (Ralston and Hall, 1989a,b;Pavlath et al., 1989; Rotundo, 1990), we reasoned thatthe nuclear source of hGH transcription could beinferred by studying the spatial distribution of golgi thatcontain hGH.

We examined the intracellular distribution of hGH inskeletal myofibers from two lines of transgenic micecontaining the gene fusion between the delta subunitand hGH genes using antibodies against hGH andimmunofluorescence. Fig. 2 shows that hGH expressionis restricted to the synaptic region of myofibers, andthat intracellular hGH is perinuclear and, by lightmicroscopy, appears to be associated with the golgiapparatus.

The perinuclear region of nuclei immediately be-neath the postsynaptic membrane are labelled in-tensely, and labelling is diminished in the perisynapticregion (10-50 //m from the synaptic site). hGH is notdetectable in non-synaptic regions of normal muscle,although nuclei are found throughout the myofiber(Fig. 1). These results show that ds-acting sequences inthe transgene are sufficient to confer synapse-specificexpression of hGH in innervated skeletal muscle.

Our data indicate that the transgene is transcribed atan enhanced rate in subsynaptic nuclei. Although thetransgene encodes 24 nucleotides of mRNA from the 5'untranslated region of the delta subunit gene, thissequence is neither conserved between delta subunitsgenes from other species (chicken 6, Wang et al., 1990;Xenopus 6, Burden, unpublished observation), norbetween genes encoding other AChR subunits (humana, Noda et al., 1983; chicken a, Klarsfeld et al., 1987;mouse y, Crowder and Merlie, 1988); therefore, these

24 nucleotides are not likely to be critical for localizingdelta subunit and hGH mRNAs to synaptic sites. Thus,our data indicate that myofiber nuclei that are pos-itioned close to the synaptic site transcribe the endogen-ous delta subunit gene at a higher rate than nucleielsewhere in the myofiber.

Synaptic activation persists in the absence of the nerveSynaptic specializations, including accumulations ofAChR and mRNAs encoding AChR subunits, persist atsynaptic sites after denervation (Fambrough, 1979;Fontaine and Changeux, 1989; Goldman and Staple,1989; Witzemann et al., 1991). In order to determinewhether synapse-specific transcription of the deltasubunit gene was maintained after denervation, weexamined the distribution of hGH in denervatedmuscle. Fig. 3 shows that hGH is concentrated atsynaptic sites in muscle that was denervated for 4 days.Like hGH at normal synaptic sites, hGH at denervatedsynaptic sites is perinuclear and is apparently associatedwith the golgi apparatus. Because hGH mRNA has ahalf-life of 2-20 hours (Diamond and Goodman, 1985),most of the hGH that is detected at denervated synapticsites was probably transcribed and translated afterdenervation. Thus, enhanced transcription of the deltasubunit gene at synaptic sites does not require thecontinuous presence of the nerve.

Denervation causes an increase in the turnover ofsynaptic AChRs and an increase in the level of AChRprotein and AChR mRNA in perisynaptic regions(Salpeter, 1987; Goldman and Staple, 1989), and thesechanges could be caused by an increase in the rate ofAChR transcription. Consistent with this idea, the levelof hGH in synaptic and perisynaptic regions appearsgreater in denervated than in innervated muscle (Figs2,3). These results are consistent with the idea thatelectrical activity suppresses transcription of the deltasubunit gene throughout the myofiber and thatenhanced transcription at synaptic sites in denervatedmuscle is caused by the removal of electrical activitydependent repression and the persistence of synapse-specific activation.

Myofiber electrical activity has an important role inrepressing AChR expression, and denervation ofskeletal muscle causes an increase in the abundance ofAChRs and mRNAs encoding AChR subunits in non-synaptic regions (Fambrough, 1979; Evans et al., 1987).Delta subunit mRNA levels increase 10- to 20-fold afterdenervation (Fig. 4), and this increase can be pre-vented, or reversed, by direct electrical stimulation ofdenervated muscle (Goldman et al., 1988; Witzemannet al., 1991). We measured delta subunit and hGHmRNA levels in innervated and denervated muscle todetermine whether electrical activity regulates the deltasubunit gene by transcriptional mechanisms. Fig. 4shows that denervation of muscle causes a 10- to 20-foldincrease in endogenous delta subunit mRNA levels anda similar increase in hGH mRNA levels. Thus, this 1.8kbp from the delta subunit gene contains the c«-actingregulatory elements that confer electrical activitydependent gene regulation.


Inn Den tRNAB

ISO

hGH

I D I D I D I D I D

LINE 5GH1 5GH2 8GH3 5GH4 8GH5

Delta

! » • '

•ft-

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AC

771I £223.

JI I I D I

LINE 5GH1 5GH2 5GH3 SGH4 8GH5

Fig. 4. Denervation causesan increase in the rate oftranscription from the deltasubunit gene. (A) The levelof hGH, endogenous deltasubunit and actin mRNAsin innervated (Inn) anddenervated (Den) muscles-from line <5GH2 weredetermined by RNaseprotection (Materials andmethods). The position ofthe protected bands areindicated with arrows. (B)In four lines (SGH1-4), thelevel of hGH and deltasubunit mRNAs,normalized to the level ofactin mRNA, is 10 to 20-fold greater in denervatedthan in innervated muscle.The level of hGHexpression in lines <5GH4and <5GH5 is about 40-foldlower than in line <5GH3.Lower left leg muscles weredenervated by cutting thesciatic nerve, andinnervated and denervatedmuscles were assayed 4days later. The value fordenervated muscle from line(5GH3 was assigned 100%,and all other values areexpressed relative to <5GH3denervated muscle. Theabundance of actin mRNAwas 20% lower indenervated than ininnervated muscle.

Non-synaptic nuclei in denervated muscle areheterogeneous, and expressing nuclei are colocalizedwith AChR clustersAlthough denervation causes an increase in the level ofAChRs, most AChRs in non-synaptic regions ofdenervated myofibers are distributed diffusely and arenot detectable with TMR-a'-BGT (Fambrough, 1979).However, a small fraction of these non-synaptic AChRsaggregate, and these AChR clusters can be detectedwith TMR-tf-BGT (Ko et al., 1977). We do not detecthGH by immunofluorescence in most non-synapticareas of denervated muscle; we suspect that it ispresent, but that immunofluorescence is insufficientlysensitive to detect this low level of expression. How-ever, hGH is detectable at non-synaptic regions whereAChR clusters are found (Fig. 5). The intensity of hGHstaining at these rion-synaptic AChR clusters is similarto that observed at synaptic sites, and non-synaptichGH, like synaptic hGH, is perinuclear and is appar-ently associated with the golgi apparatus. These resultsindicate that non-synaptic nuclei in denervated myo-fibers are transcriptionally heterogeneous, since thedelta subunit gene is transcribed at different rates in

different non-synaptic nuclei (see also Fontaine andChangeux, 1987; Berman et al., 1990). Further, theseresults indicate that the processes of clustering AChRsand enhancing AChR transcription are linked, and thatcolocalization of AChR transcription and AChR clus-ters does not require a signal from the nerve.

Signal and pathway appear shortly after synapseformationAccumulation of AChRs at developing synapses is anearly event in synapse formation and occurs severaldays before birth in rodents (Frank and Fischbach,1979; Dennis, 1981; Slater, 1982). However, synapticstructure and function continue to be modified duringthe next few weeks, and the full complement of adultsynaptic properties are not acquired until about onemonth after birth (Dennis, 1981; Slater, 1982; Schuetzeand Role, 1987). In order to determine at what pointsynaptic nuclei become distinct, we examined thedistribution of hGH in muscle from newborn mice. Fig.6 shows that hGH is concentrated at synaptic sites innewborn muscle. Like synaptic staining in adult muscle,synaptic hGH staining in developing muscle is perinu-


Fig. 6. Delta subunit gene is transcribed preferentially insynaptic nuclei in newborn muscle. hGH is concentrated atsynaptic sites in muscle from newborn transgenic mice. Anindividual muscle fiber from the diaphragm muscle, whichwas double-labelled with antibodies against hGH (A) andwith TMR-u-BGT (B), was mounted whole and viewedwith optics selective for fluorescein (A) and rhodamine(B). hGH is concentrated at the perinuclear region ofnuclei immediately beneath the postsynaptic membrane(arrows); there are less nuclei at synaptic sites in newbornmuscle than in adult muscle. Bar=10 jim.

clear. However, synaptic staining in newborn muscleappears less intense than in adult muscle. Thus,synaptic nuclei become transcriptionally distinct earlyduring synapse formation and before postjunctionalfolds have developed, polyneuronal innervation hasbeen eliminated, or AChR channels with short open-times have appeared (Schuetze and Role, 1987).

Myogenin is not expressed specifically in subsynapticnucleiMyogenic basic-helix-loop-helix (bHLH) proteins areimportant regulators of muscle gene expression duringmyogenesis, and their targets include the genes en-coding AChR subunits (Piette et al., 1990). Previousstudies have shown that the levels of myogenin andMyoDl mRNA are high in muscle from newborn miceand decrease during the following two weeks (Duclertet al., 1991; Eftimie et al., 1991). Further, the levels ofmyogenin and MyoDl mRNA, like AChR mRNA, areregulated by innervation, since they increase followingdenervation (Duclert et al., 1991; Eftimie et al., 1991).We sought to determine whether myogenic bHLHproteins might be involved in synapse-specific ex-pression of the AChR delta subunit gene, and we usedan antibody against myogenin to determine whethermyogenin is concentrated in subsynaptic nuclei. Werestricted our analysis to myogenin, because antibodiesagainst MRF4 and myf-5 are not available, and wecould not detect staining in newborn or adult musclewith available antibodies to MyoDl (Tapscott et al.,

Fig. 7. Myogenin is not expressed specifically at synapticsites. Diaphragm muscles from newborn rats (A,B), oradult rats (C,D) were labelled with a monoclonal antibody(F5D) against myogenin (A,C) and TMR-O--BGT (B,D). Awhole mount of newborn muscle is shown in A and B,whereas a single adult myofiber is shown in C and D.Myogenin is expressed in nuclei throughout developingmyofibers from newborn rat muscle, but myogenin is notconcentrated at synaptic sites (A,B)- The whole mount ofthe innervated zone of newborn diaphragm muscle containsmore than one hundred myofibers, but only several dozenof the myofibers and synaptic sites are in focus. Myogeninis not detectable at synaptic sites or non-synaptic regions ofinnervated, adult muscle (C,D). Bar=30 fan.

1988). Fig. 7 shows that myogenin is expressed in nucleithroughout developing myofibers in newborn muscle,but that myogenin is not detectable in innervated, adultmuscle. These results are consistent with the data formyogenin mRNA and support the idea that myogenincould have a role in coupling changes in myofiberelectrical activity to changes in AChR gene expression(Duclert et al., 1991; Eftimie et al., 1991). However,because myogenin is not concentrated at synaptic sites,either in newborn or adult muscle (Fig. 7), these datado not support the idea that myogenin has a central role


in activating AChR genes selectively in subsynapticnuclei.

Discussion

This study demonstrates that transcription of the AChRdelta subunit gene in innervated, adult muscle isconfined to nuclei that are situated within the thesynaptic region. These results indicate that synapticnuclei are transcriptionally distinct from nuclei else-where in the multinucleated skeletal myofiber and thatthe synaptic site provides a signal that acts locally toactivate transcription of a select set of genes in nucleithat are positioned close to the synaptic site.

The restricted spatial distribution of AChR geneexpression indicates that a synaptic signal, which ispresumably provided by the motor neuron, exerts itseffect over a highly circumscribed region of themyofiber. hGH is highly concentrated immediatelybeneath the postsynaptic membrane, and expression ofhGH is less abundant 10-50 /im from the synaptic siteand is not detectable further than 50 ^m from thesynaptic site. These distances are similar to thosemeasured for spread of signalling information in othertransduction systems (Lamb et al., 1981; Matthews,1986), and these data indicate that the skeletal myofiberneed not have unique mechanisms to restrict the spatialflow of information. The spatial influence of thesynaptic signal may be restricted further by repressivesignals provided by electrical activity, and this ad-ditional influence may explain why hGH is readilydetectable in the perisynaptic region of denervatedmyofibers. Although our results are consistent with theidea that transcriptional activity decreases exponen-tially from the synaptic site, our immunofluorescencemethods were not quantitative, and diffusion of RNAor rapid translocation of organelles associated with thegolgi apparatus could also produce a gradient ofintracellular hGH. Nevertheless, our data provide anestimate of the distance over which the synaptic signalexerts its effect.

Spatial restriction of delta subunit gene expressiondoes not depend upon the continuous presence of thenerve, since hGH remains concentrated at synaptic sitesfollowing denervation. Thus, either the nerve-derivedsignal that is responsible for activation of the deltasubunit gene in subsynaptic nuclei remains at synapticsites for at least several days following denervation, orthe effect of this signal persists after the nerve isremoved. In either case, the transcriptional machineryassociated with activation of the delta subunit genepersists in the subsynaptic nuclei of adult muscle anddoes not require the continuous presence of the nerve.

AChRs can cluster at non-synaptic sites on dener-vated myofibers and on embryonic myotubes (Ko et al.,1977; Vogel et al., 1972; Fischbach and Cohen, 1973),and the mechanisms that regulate the formation ofthese non-synaptic clusters have served as a model forformation of AChR clusters at synapses (Schuetze andRole, 1987). We show here that AChR accumulation at

non-synaptic sites in vivo is associated with regions ofincreased delta subunit transcription. Thus, thereappears to be heterogeneity among the non-synapticnuclei, since a small number of nuclei in non-synapticregions of denervated muscle express the delta subunitgene at an enhanced rate, similar to synaptic nuclei.Because our results show that highly expressing nucleineed not be positioned at the synaptic site, colocaliza-tion of AChR transcription and AChR clusters does notrequire a signal from the nerve.

We do not know whether there is a causal relation-ship between increased density of surface AChRs andincreased AChR transcription; however, since theexpressing non-synaptic nuclei are not randomly dis-tributed but are associated in small groups, it seemsunlikely that random activation of individual nucleicauses the appearance of surface AChR clusters. It isnot clear what triggers the formation of these synapticspecializations at non-synaptic sites; however, it ispossible that a pathway, which is normally activated bythe nerve at synaptic sites, can be activated indepen-dently of the nerve and lead to all specializationsnormally found at the synaptic site, including bothsurface clustering of AChRs and nuclear activation ofAChR genes.

At present we have little information regarding thenature of the signal from the nerve that activates AChRgene expression at synapses. The signal could bereleased by the nerve and stably maintained in thesynaptic basal lamina, like agrin, the extracellularmatrix molecule that causes AChRs to cluster atsynaptic sites (Nitkin et al., 1987). Nevertheless, agrinitself does not appear to be a likely candidate for thetranscriptional signal, since agrin causes a redistributionof surface AChRs that is not accompanied by anincrease in AChR synthesis (Godfrey et al., 1984).Alternatively, the signal may be released from thenerve and be required only transiently to activate apathway or to assemble a structure, and the signal maynot be provided constitutively (Brenner et al., 1990).Identification of the synaptic signal and its receptor willbe important steps in understanding how nerve-musclesignalling is mediated, and in understanding howtranscription is activated.

Myogenin is a transcription factor that has animportant role in initiating muscle differentiation(Wright et al., 1989), and the abundance of myogeninmRNA is regulated by innervation (Duclert et al., 1991;Eftimie et al., 1991). Myogenin is not detectable atsynaptic sites in adult muscle, but is present in nucleithroughout newborn muscle. These data do not supportthe idea that myogenin has a central role in activatingAChR genes selectively in subsynaptic nuclei. How-ever, these data are consistent with the idea thatmyogenin has a role in initiatiating AChR geneexpression during myogenesis and that myogenin couldbe involved in coupling changes in electrical activity tochanges in AChR gene expression (Piette et al., 1990;Duclert et al., 1991; Eftimie et al., 1991). It will beimportant to determine whether any of the othermyogenic bHLH proteins are concentrated in subsy-


naptic nuclei. In any case, the identification of thetranscription factors that activate gene expressionselectively in subsynaptic nuclei will be facilitated byanalysis of additional transgenic lines and identificationof the important cts-acting regulatory elements in thedelta subunit gene that confer synapse-specific ex-pression.

mRNAs encoding the alpha, beta and epsilonsubunits of the AChR are also highly concentrated atsynaptic sites in adult myofibers (Merlie and Sanes,1985; Fontaine and Changeux, 1989; Goldman andStaple, 1989; Brenner et al., 1990), and it would seemlikely that the genes encoding these subunits are alsotranscriptionally activated in the subsynaptic nuclei ofadult muscle. A previous report has shown that atransgenic mouse line that harbors a gene fusionbetween the chicken AChR alpha subunit 5' flankingregion and a /3-galactosidase reporter gene, expresses/S-galactosidase in newborn muscle (Klarsfeld et al.,1991). Expression is enriched in the central region ofthe muscle, where most synapses are located; however,staining extends far beyond synaptic sites in newbornmuscle (Klarsfeld et al., 1991). Therefore, it is unclearwhether the expression pattern in newborn musclereflects preferential expression of the transgene bysynaptic nuclei as proposed (Klarsfeld et al., 1991), orwhether the pattern reflects the developmental historyof myotubes, in which myoblast fusion begins in thecentral region of the muscle (Kitiyakara and Angevine,1963; Bennett and Pettigrew, 1974; Braithwaite andHarris, 1979). Since expression of/3-galactosidase is notdetectable after postnatal day 4, the authors were notable to evaluate this latter possibility. The absence ofexpression in adult muscle could be due to silencing ofthe transgene, but it is also possible that the transgene,while it contains the elements for muscle-specific andelectrical activity-dependent regulation (Merlie andKornhauser, 1989), lacks an element necessary forsynapse-specific expression.

Like synaptic nuclei in vertebrate skeletal myofibers,nuclei in the syncytial blastoderm of Drosophila aretranscriptionally distinct (Nusslein-Volhard, 1991). Forexample, expression of the tailless gene is restricted tonuclei in the terminal regions (Pignoni et al., 1990), andexpression of the twist gene is restricted to nuclei in theventral region of the syncytial blastoderm (Thisse et al.,1988). Polarity is established in the dorsoventral andterminal systems by signals that are synthesized indistinct follicular cells which ensheathe the developingoocyte (Stevens et al., 1990; Stein et al., 1991).Although the dorsoventral and terminal systems usedifferent pathways to interpret the spatial informationprovided to the oocyte (Anderson et al., 1985; Sprengeret al., 1989; Casanova and Struhl, 1990), in each systempolar information is maintained in the egg, and thecontinuous presence of follicular cells is not required toestablish distinct transcriptional activity in subsets ofnuclei (Stevens et al., 1990; Stein et al., 1991). Theresults presented in this study indicate that thepresynaptic nerve terminal is the source of a signalwhich provides spatial information to the myofiber, and

that this signal acts to activate transcription of a selectset of genes in nuclei that are situated near the synapticsite. It will be interesting to determine whether similarcomponents and mechanisms are used to establishtranscriptionally distinct nuclei in the syncytial blasto-derm and in the syncytial myofiber and whether stepsinvolved in pattern formation in Drosophila are sharedwith steps involved in neuromuscular synapse forma-tion.

This work was supported by research grants from the NIH(NS27963) and the Muscular Dystrophy Association. Wewould like to thank Dr. W. Wright for kindly providing uswith the monoclonal antibody (F5D) to myogenin, Dr. A.Lassar for kindly providing us with the antiserum (160-307)against MyoDl, and Drs. M. Hu and N. Davidson for themouse skeletal actin gene. We also thank Dr. C. Jennings, Dr.E. Dutton and Dr. R. Lenmann for their comments on themanuscript.

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(Accepted 15 November 1991)

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