Muscle differentiation: more complexity to the network of myogenic regulators

539

Muscle differentiation: myogenic regulators Hans-Henning Recent

genetic

and biochemical

our understanding myogenesis

of control

in vertebrate

combinations revealed

Arnold*

unique

and

overlapping

that establishes

Moreover,

which

within

muscle

In addition,

novel genes

myogenesis

loop-helix

(bHLH)

molecular

mechanisms

are being elucidated, factors

upstream

of myogenic or negative

interactions

most notably

between

and MEFP transcription

of that

i/l &CO that

of

basic helix-

University

the myogenic

factors.

Biology, Institute of Biochemistry of Braunschweig, Spielmannstrasse

7,

38106 Braunschweig, Germany *e-mail: [email protected] Current

Opinion

in Genetics

& Development

1998,

myoblasts

8:539-544

http://biomednet.com/elecref/0959437X00800539 Biology

Ltd ISSN

The

basic helix-loop-helix

MEFP MLP

myogenic enhancer muscle LIM protein

YACs

yeast artificial

muscle

determination

seen

when

myogenin-null

for functional

redundancy

[8-101.

0959-437X

Abbreviations bHLH MDFs

not

in vitru [h,7].

The temporal sequence of LIDF gene activations during embryogenesis and the mutant phenotypes have led to the simple view of a two-step model for skeletal myogenesis in which hlyf-5 and MyoD act early to establish myoblasts and myogenin mediates their terminal differentiation. hlRF4 was also assumed to exert late functions in the myogenic pathway, possibly similar to myogenin. This assumption is primarily based on its late activation in the embryo and in cultured muscle cells but this has not been supported by direct evidence. ‘Igrgeted inactivation of the hIRF4 gent causes only a very mild muscle phenotype bvhich argues

0 Current

is, however,

are cultured

ways. The

and cooperativity

Addresses Department of Cell and Molecular and Biotechnology,

overlap of both gents despite their markedly different onset of expression. ‘I’he functional overlap of A[\& and JQf.5 in the regulatory network of myogenesis has been confirmed by simultaneous disruption of both genes which causes a complete absence of myoblasts and differentiated skeletal muscle. The null-mutation of the myogenin gene exhibits a severe muscle phenotype with substantial but not complete reduction of skeletal muscle tissue, resulting from a differentiation defect of myoblasts

cell lineages.

have been discovered

in positive

of these

of the

the regulatory

as a key regulator

and the activity

proteins

have

of members

seems to act genetically

modulate

bHLH

factors

systematic

in mice

functions

skeletal

have advanced

underlying

In particular,

Pax3 has been identified

myogenesis MyoD.

approaches

gene disruptions

MyoD family of transcription network

and Barbara Winter

mechanisms

organisms.

of targeted

more complexity to the network of

factors factor

2

chromosomes

Introduction Specification and differentiation of skeletal muscle cells in birds and mammals is critically dependent on four muscle-specific basic helix-loop-helix (bHLH) transcription factors, frequently referred to as the family of muscle determination factors (MDFs). Targeted gene disruptions of each family member - hlyoD, blyf-5, myogenin, and hlRF4 - in mice have shown that they play central roles in vertebrate myogenesis [l-3]. Inactivation of the hlyf-5 gene - the first member of the family to be induced in muscle precursors in somites through signals from the adjacent neural tube and notochord [4] results in a delay of myotome formation and aberrant migration of the presumptive muscle precursors into the adjacent tissues, dermis and sclerotome, where they acquire non-muscle fates [S]. This early defect in myogenesis, however, is compensated by the later expression of hIyoD leading to essentially normal muscle at birth. Vice versa, disruption of the MyoD gene that is normally first expressed two days after hlyf-5 also does not perturb muscle differentiation at a gross level, suggesting substantial functional

precise

assessment

of the roles of individual

MFDs

has been complicated by the apparent overlap of functions observed between the \-arious myogenic bHI,H genes. A unicluc role of myogenin in skeletal muscle differentiation it1 .u& \\ith no functional redundancy to either hlyol) or hlyf-S has been demonstrated by formal genetics with crosses of myogenin and hlyoD null mutants, and myogenin and hlyf-5 mutants [ll]. Recent investigations which further address the individual roles of myogenic bHLH genes in the complex network of skeletal myogenesis are rcvicwed. \Ve also refer to the recent discovery that the transcription factor Pax3 may be a key player in myogenesis. Despite the fundamental importance of hlDFs, they are by no means the only factors affecting muscle cell differentiation. A number of other transcription factors that either positively or negatively modulate MDF activity and muscle-specific gene expression are also discussed.

How unique are the intrinsic properties of the individual myogenic bHLH genes? Overexpression of individual hlI)Fs in a variety of nonmuscle cells results in comparable activation of the program of skeletal muscle differentiation. Nevertheless, inactivation of these genes in mice leads to different myogenie phenotypes, raising the important issue of whether the different myogcnic bHLH factors have unique

540

Differentiation

intrinsic

and gene regulation

or whether

propcrtics

pttcrns

their

distinct

expression

mutant

i71 .~Ycocawc the difference.

mice

direct

lacking

cI.idcncc

eGzw. as they \Vang d d. scclucnw the

[ 121 replaced

of myogcnin

transcriptional

carrying

this

control

myogcnin

lack the rib defect mutation.

‘I‘his

of

normally

result

leads

to

mice

hlyf-5.

t)), the

associated

under

Ilomozygous

mice

are 1 iable

establishment

of

and

m)ogcnic

mice Il6’). fib-s

mrisclc mice

ahsolritcly

rcqliircd

to activate

cells

in

the

stems

indicating

iI/ fvzv and

conditions.

not very

muscle

the

myogenin

knock-in

mutant

background,

hlyoI>-null homozygotcs skclctal

die shod)

to fully

the

myogcnin

knock-in

knock-in

iirc

with

also

allclc.

;1s they

simihr

allclc

lack

;I

double

of

I13’].

\ic\l,

hIiIJIi

skeletal

the

.1!,;{-,5

promoter

ib

cmbryogcncsis.

the results

cxprcssion

the myogenin

fic,icnt

from

to either

establish

downrcp~lated

mu)

Ix

during

cxpl:iincd

knod-in

Iq

allele

the frrll complcmcnt

c.cIIs or to support

diffcrcntiation

of inyol)lnsts.

addition,

transacti\ ation

propcrtics

ilniquc

that arc distinct contribiltc

from

that 1lyolI

of target genes

niyogenii~

is

10-fold

Rlyol>‘s abilit> to rcmodcl rcK:ions

of tlic

protein

traiisac.ti\,;(tion constwd

for

prcc’iously.

eqdain

\rhy

hlyol)

early step

is tlir less cfficicnt more

Ii\ idcncc for the abilit) fcrcntiation ol,taincti

similar

nio~isc.

to

suggesting

tcmporc,sl~~itial

nl)ogcnin

--

the

c’:in lxirtiall)

tions.

(~on\~cr~cl~, in

L ial)lc

and

gcriin

is rrprc,gulatcd

abscncc

I1a1.c

of

silent

with

t\vo

implicated regions

in arc

\\hich

Interestingly,

of

ma)

hmvcvcr.

for

h4lil~l

~c~riltl 1~ factors i5

If this Icvcl

of sc\-a-al

t\vo

riilc.

of

bIII,ll

gents.

comI~in~itions

in mice.

tIicn.

Ix

dctrinicnt3l.

IIS in:icti\ation

cxamplc.

i\

niy)gcnic

results

ot

in fairly

IX].

clifhccn

of the

[IS]. In rcscticd

in

\\.hcn

pattcrii

of

for- m);ogenin

fklnc-

mic.c

\\,hich

mtlsclcs.

compcnsatc4 ho\\ wcr.

:irc ha\,e

iirc

e\ idcncc

functions

probably

\llIl;s

of

associated

tllat in \cr-

v ith

thcil

I)tit it i5 al\0 clear that thew

distinct

potential

intrinsic

~bhich

most

propcrtios

likely

anti

contrit>iltcs

to

~lSl’“C’S of nl)o~:cncsls.

What lies upstream of the myogenic bHLH genes? ‘I‘hc a\ailablc and .l!yo/1 cells

c\idcncc

constitlltcs

to the

m)-ojicnic

must

these

gene acti\.itics

of

cmh\o.

the

signals and

t,f dcm

from

surfilcc

lincagc muscle

the

and location

all ciiscs,

howocr,

(neural

tube

[20],

iii

arc also in\ol\d

it i5 tinclcar bH1,l

I Ictlgcho,~ notochcdi

of ni>cqznic I)ar;ixial

it was shcnvn

of nlyogcncsis 1 gcnc

mcso-

that

IShll’\

in the control

u ithin

hoa thee

01‘

control rcKions

and

csprc5sicJn

myogcncsia

noggin

to control

which

m~is~lc-ti)rming

can indttcc

acti\ ;I-

c 1 iv\\

of \\:nt and Sonic

rcccntly

:intagonist

csprcwion \\+th

mechanisms

various

somitic

of timin!:

of .l!r/~.;

;\ comprchcnsi\

the

btriicturc4

cctodcrm

3nd their

intcgratcd

gcncs. in

activation

c step that commits

anal sut)scqucnt

:I umil)in;ltic)n

;i*ial

that

cell ciiffcrcntiatic~n

inclLlde

11,11 factors and I17-191. Jlorc

ally

suggests

the determinati\

of miisclc-specific

m)og:enc5is

recentl)

-

some

is accirmulating

unique

c~t‘cuprc‘;sion

factors

tranucti\.:ition

tion

cell linugc

mtisclc

thcrc

01 the apparcntl~

o~‘N~~I!,“(‘NIII triggcrs

the control

skeletal

and probtibl\[IO].

in

and mygcnin

diffcrcnti~ltion.

knock-oilt

normal

sunimar~.

transcription

chromatin.

that hlKI:4

sulxtitlltc

.MR/.‘4

cwentially

hlKIC-l

chroniatin. in activating

is partially

the abhcncc of m)og;enin,

In

some

niygcncsis

tr;illscril)-

hckgrcbiind

diffcrcntiation

cxprcsscd

in

ma) 31~0

patterns

has

tin&r

of myogcnic

mtisculaturc

spccitic

to promote

myogcnin

transgcnc

hI\;f-.5

roles pathwa!

strtictur;iI

;I gcncral

tehratc

m!oficnic

in 3 ~/~//‘~~,,/i//-iiiiII

muscle

skclctal

thcsc

terminal

of \lK124

from a .lIKb’J

I~J~~I~BN;Npr0motcr

norrn:il

and Jl~d-.? arc niol-c cfficicnt

in triggci-ing

cffccti\c

compensator! differentiation

elimination

mcdi:ltcs

of ~kclctal myogencsis tlic

factors,

is

one can

mutants,

normal

Ic\.cI

by

can not Ix

of myogcnic

t,tlt not in myogcnin

31 cstablishin~

blHI,l-l ‘l’his

and

of m\cqcnin

Iu3i

Significantly,

protein

achiacti

douhlc

muta-

As myogcnin

[ lh’l.

cxcpluna-

to actir,atc muscle

X1KF-l

i\ associatccl not

threshold

a

ii/ E~iev. ‘l’he simplest

in the miiscle

that is insuf-

in rcprcssi\c

II~I~C

latc

display

alone is rrnablc to support

the unanticipated

a critical

mutants

to that seen in ni~o,q3iin

or both. In

Ics5 efficient

chromatin

that

in the \l).f-5

mediating

tlii\

able

although

both the .I//?/+‘4 and .1!,~0/1 single

that myopiin

In frict. Gerber O/N/.

thcsc gcncs in regions of transcription~~ll\i

hiit

Ilnder

program.

effect on myq3v2sis

cell differentiation

and Xlyol) that

low -ICI cl

2nd RIy111

phrnot>I)cs.

ticmonstratcri

acti\stion

whcrcas

those of hlyf-5

to the ol~ser\d

recently

ma!’

diffcrcntiation.

double

similar

in these ,lfR~‘4/~ll~oD

csscntial

1l.i’j.

phenotype

muscle tion

mygcnin-null

diffcrcntiatcd

mutant

expressed conclude

pro-

hy the ‘Il,~f-.i/t~/~l?rrr’I/iN

most

deficiency

whereas

mutants,

normally

tional

.WR/*‘4

t~~~~o,gn/irn is

hl~-f-.5 are apparently

diffcrcntiation

,lIRb‘4/,lf~wl1

miisclc

se\zre

tions have little

reduced

is not sufficient

:i second

this

to the m);ogcnin

on

the diaphragm)

rcscucd

not

the

Ixcausc

for J~y~-.j uhcii

(hsistcnt

mice

are bred

hmvevcr.

birth

(including

is lackinK.

muscle.

after

formation

compcns2tc

miitant

mutants

muscle

Apprcnt1y

[l-I*]

nor

efficiently.

11nexpectedly,

As

niyogcnin-null

additional

,I/RF4

myoblast and/or

as

of the emlqw.

U’hcn

tcin

for

i/j

phcnotypc

in

1)~ the

ncithcr

no

gcncs

the amount of rcsid-

present

unaffcctrd that

of both

the same

In particular,

hlyol)

the

overlap

normally

mutation, thcsc

and rnnwg~~ir/ provide

‘L/RF4

essentially

ual

in place of

function

exhibit

my~,Xenin-null mutant

the blyf-.5-null

with

that myogenin

for early hlyf-5

coding

myqenin

mutation

suggests

can compensate the

in

to express

‘knock-in’

hlyf-5

mytomc

.wlrflS

in order

both

foi- functional

the romitc. $ign;ils

In

arc actll-

cxprcs5ion.

mvofor

the

dout3lc

(Zorrect

sl’;itiotcmpor”l

cxprcssion

animals

and acti\,ation

in diffcrcntiating

ot’~~~wy~i~~ in transgcnic m>-ot>last5 i/l r,;tu/

Muscle differentiation Arnold and Winter

dej)ends

on li-box

proxinlal scription h,IKF4

fztorh

region

clement

factors

hovcvcr, promoter

myogcnin

culture

promoter

mouse

fragment

only

timcs.

directs

probat,ly

requires

(K Zwcigcrdt,

:idditional,

II-II

more

limited

that

an X.5 kh j32utcrn [%].

control

is known this

mollsc.

about the control

gene is located

and most of the intcrgcnic in

transgcnic

branchial [‘sing

mice

arches

and.

mouse

,b($.5

uncxpcctcdly

lows.

it

distant

elements,

the .l!$.!i of the

control

results

itidiutc

regions

regulate

the \ arious bl?

Llorco\w.

e\ en lateral

nu er express .jQ&

clcmcnts

somites

the

rcccntly

that

been

regions

to different

almost certainly

or locus

the

control

.11$.? cxprcssion of the embryo.

signals.

As thcsc

lie closer to .llRP4

underlying

to

composition

investigated.

cnhanccrs

the control

uith

in

prolu-

regulator)

than to ;jl~f-.T.

of both g:rnes will

be interesting.

these

evidcncc

that

obscure. transcription

tranccrij,tion

is cxj>rcsscd paraxial migrate

and

Pos.~ constitutes

of skeletal

in the dorsal

in

somites

to the dermom)otomc

neural

where

including

one

muscle.

of

Pax3

tube,

it

in

functions

a Pus.i

of impaired

3 ~0nscq1Icn~~

mutation migration

the lateral d~rmom);otome.

ation potential

is not impaired

lack limb

u ill

II~~ISCICS as

of muscle jwogenitors

although

their

lllllscles

exist

and

In any

(33].

express

genetic fail

myogenic

diffcrenti-

[39,X)].

studies

to cxj>rcsT

function

of.s~/0t~/I/,j!1;/15 double iLlvo1)

in the mvotome

ni~~sclcs but not the muscles cells ;Iction

in the absence of .jfyliD.

t!-j>c :irgues for RlyoI)

that

in heiid muscles

can

only

in precursors

which

form

and lack all hod) arise

double

Pax.3 i\ ~iii essential

I>>

niicc which

in the head [.il”].

the .sp/i&/.M,$.j

cxprcsbion

way. ‘1%~ analysis

of LjQfl.T

revcalcd

mut;lnt

As m~iscle through

mutnnt

upstream

of body

the

phcnoregulator

muscle

but not

in a /‘/lo.?-independent

p:ith-

of sp/~f&/,j{yf.T

mutants

gene that

is

are also de\ oid of I)od) coregulators

in neural

tissiics

must

th:it

ne\er

.jf.y~I).

Cooperation of MDFs with other transcription factors for muscle-specific gene activation Other

thali

scription

the

IzlyoD

Lictors.

family

imjwrt;rnt generate

proteins

Rll:I‘1 +2 domains.

present

scqllcnc‘c

cell linoagcs in

the

myogenesis

tltc

XII’lT1

cell diffcr-

genes

conser\ul

JlIS:! control

csscntial

[X-.37]

hind

regions

of

most

gcnc

for differentiation

cardiac

3s

consen-

I)-I\IE:f7

in

of all

of the h1EFX:

niirtation

and

impairs

which

LlAIX

jwotcins

sing:lc

‘I’hc

mouse

3150 play 3n

muscle

to an A-‘1’ rich I)NA

in

gcncs.

tran-

cnh;inccr

fwtors

during

the highly

homo- or hcterodimers

hHI,H

ni!ogcnic

ha\e foilr

containing

3djaccnt

the

ntusclc

the

of transcrij)tion

1.341. \‘crtclwtcs

gene

of

role in gcnc acti\2tion

cnti:ition

anJ

of myogcnic

nienibcrs

factor 2 (\11<1:2) family

morphogenrsis

and

[3X].

It is beliebed vatc

&it

also argiics

for

\1111:2 transcription

target genes

b>. bindinK

to their

site Ijut also by protcin-protein t~HI,II/El7

heterodiniers

co~~sensl~s

jm)l,al,l!

j>roteins

miisclc-specific

gene

xti\

of

their motifs.

1.191. ‘l’he

:ihility

j3rotcins

on two conser\ ed amino acitj rcsidiics

Rll)Fs

-

the

Ixxausc

corresponding

domain ders

thrconinc

xubstitution two

of nonmyogcnic

the

mutated

in

myogcnic

the

bHI,Ii

h;lsic

of these

asjwragincs

bH1,F-I

in this \~a)- still

and j>roniotc

factors

bind DNA,

myogencsis -

ala-

domains

of

t\\o rcsiducs

\vith

jwvznt

factors.

for

of m!-o-

i\ dependent and

and

In this comcofwtors

gcnic I>I~L,H nine

to initiate

E-box

rcspccti\c

:ict as csscntial

ation

niyogcnic

to the

hll
through

and Ilcterodimerization

plcx. hlI
with

3rc bolind

interaction

occurs

hetcrodimers

factors not onI>- actiown l)i’XA recognition

intcractiotis

u.hich

‘I’his

secjllencc.

l~l~I,li

of Pr/;ci ~2s

are not

to note that mouse

additional

case.

is no

directl)

mesodcrm

3 I)HI,I1

ptu.c”si.s.

jwttcrning.

3s Z’U.L? is also expressed

l)NA-binding :\ rcmarkablc

in somitic

lacking

.I!$.5

date, there of ,j!yoD

mutants

in\.ol\,ed in mcsoderm

gene.

becomes

cells that

to form limb rn~~sclcs. Among other severe defects.

.sp/~t& mice carrving from

bctor

in the embryo

mcsodcrm,

rcstrictcd

has been that the paired-box

dwclopment

to

the

regulatory

‘Ii)

In this context, it is intcrcsring

/h.w/~hi/~~ i\ absolutely

in the

tube

underscore

myogcnic

remains

An exciting

gcncs

pre-

be induced

can

ohscr\,ations

Pax3 controls

muscle-sj)ccific

key

of

cxprcssion.

and neural

naturally

unlikely.

sus

homcodomain

explants

,j/yf’.S

the I-‘c/.\;i-cxj>ressing rctrovirus.

striking

Pax3 is an upstream regulator of MyoD

the

in

mcsoderm

of 1’/1.\:~as 3 dominant

its mode of action

cithcr

recent discovery

using a

cxpres-

Lvith 50 kb proximal

has not

distinct

[Z7].

cur)-ing

the prwisc

correct spatiotcmpor3l

the mechanisms

in

jjlatc

ectopic

tissues

In this stud?; it is shown

maintains

cells which

it by infection

by

chick embryo

expression

and

and more general

in

to activate the gene in

be achicwd

Inusclc-formin:

in response

expression

\zhcrcar correct somitic

clcmcnts

the

located >.50 kb upstream

17X]_ Although

that

In

prcrursors

(l’A(:s)

arc ncccssq

can

promoter

cxtcnt,

,j!y~D

mcsoderni

Although

of ,llRZ*‘4

hlyf-ii

muscle

\vzu, demonstrated

cells of the limbs.

sion in myotomes

dri\w

facial

chromosomes

of the ,M,lf1.5 promotcr, muscle

in

to some

yeaat artifical

of.lfyj1.5.

-8 kb downstrum

region

only

complemented

clcments

observations).

regions

nicely

vector 1.3Z”].

f’/I.~.i induces

somitic

j3otcntial Littlc

;lrc

of /‘c/s.i in cultured csj3rcssion

express

in the mytome

distal

,\rnold, unjwblishcd

findings

expression retrovirus

expression

of ,bfRF4

‘l’hese

revcal,

lines

drives the correct fetal expression

early esprcssion

[%I.

transcrip-

whereas

and part of the early mytomal

‘1%~ comj+tc

tran-

by a proximal

and hlEF2

‘liansgenic

at all decelopmcntal fragment

of .jfRb‘4

to which

this

located in the

in .WRfq’4 regulation

in tissue

[25].

sites

iV~lscle-specific

properly

bind

that

expression

binding [21-23].

3rc also inwlvcd

i\ activated

promoter tion

and hlEF2

j3romotcr

541

in

the

IlaG

such as El 1, rcn-

inactibc. ii model

.tLs hlI>Fs suggchts

thnt

542

Differentiation and gene regulation

alaninc and thrconinc arc rcquircd for the myogenic bHLH factors to recruit an essential myogenic cofactor probably through adoption of a compatible conformation.

A recent study [40’] now provides evidence that the myogenie residues alanine and threonine in the basic domain of R;lyoD are required for synergistic activation of musclespecific transcription by MyoD and hlEF2 hut not for the interaction ,OU se. Obviously the hlyoD/MEF2 complex must acquire transcriptional competence through a mechanism that is dependent on the basic domain of hIpoD and complex formation is not sufficient. ‘I’he requirement to transmit the transcriptional activation signal within the complex can he bypassed through suhstitution of the MyoD or MEFZ activation domain with the constitutive activation domain of the herpes virus protein VP16. As the actual nature of signal transmission is not known, an intriguing possibility would he that yet another cofactor must be recruited into the complex. Indeed, it has been shown that the transcriptional adaptors p300 and CBP coactivatc the myogcnic hHLH proteins by physical interaction [41]. Stimulation of hlyoD’s transact&Con potential by p300 requires two domains of p300 and the transactivation domain of MyoD. Interestingly, p300 also interacts with the MADS domain of MEFZ proteins and potcntiates its transcriptional activation capability. hluscle LIkl protein (RILP) IS another potential coregulator of skeletal myogenesis which promotes myogenic differentiation [42]. A recent study shows that hlLP enhances the activity of MyoD and interacts with MDFs through the bHLH domain and the first I,IILl motif of MLP 1431. This interaction is specific for myogenic bHI,H transcription factors and does not occur with nonmuscle hHLH proteins, such as El2 or E47. Although the precise mechanism by which MLP enhances terminal differentiation is unknown, it may act by increasing the binding of myogenic hHLH proteins to specific DNA regulatory elements, as hlLP on its own lacks a transcription activation domain.

Negative regulators of muscle gene transcription Given the dominant myogenic functions of MDFs, it seems important to regulate their activity in order to prevent premature differentiation of myohlasts and ectopic development of muscles. Peptide growth factors, such as ‘I’GF-p, BhlPs, and FGFs, arc known to inhibit myogenesis by blocking the expression and transcriptional activity of myogenic factors. Stern total. [44] have shown that TGFpl and FGF may also collaborate to induce myogenesis in somites. Myostatin, a TGF-P-related factor which acts in an autocrinc fashion, was recently identified as a potential inhibitor of skeletal myogenesis [45]. A number of nuclear factors have also been implicated in the inhibitory role, most notably the Id (inhibitor of DNA

binding) HI,H proteins which lack the basic DNA-binding domain and act to sequester hHLH proteins into nonbinding complexes. Differential interactions of the four known Id proteins with hHLH transcription factors may contribute to their specific biological functions [40]. ‘I’he mouse hHLH protein twist was also found to inhibit hlDF activity by blocking DNA binding, by titrating E-proteins, and by interference with the transactivation of the hlyoD cofactor ILIEFZ [47,48].

Mistl,

a novel

hHLH

protein

that

accumulates

genie stem cells and decreases as myohlasts to myotuhes, also functions as a negative

in myo-

differentiate regulator of

RlyoD activity [49]. hlistl-induced inhibition of diffcrcntiation involves formation of inactive hlyoD-hlistl heterodimers and occupancy of E-box target sites b); Mist1 homodimers. Mist1 possesses no transcription activation domain hut a repressor region capable of inhibiting heterologous acti\,ators. I-mfa protein may he another factor which prevents inappropriate myogenesis. This protein is highly cxprcsscd in the sclerotome and in oitro inhibits transactikation b) hlyoD family members [SO]. I-mfa associates with hlyoD and retains it in the cytoplasm but it can also interfere with DNA binding of hlDFs. ZEB (zinc-finger/E-box binding), a vertebrate homolog of the Dm.sophil~ zinc finger/homeodomain protein zfh-1, constitutes a completely different type of negative regulator of muscle differentiation [Sl]. ZEB hinds to a subset of E-boxes in muscle genes and actively represses transcription until it becomes replaced by high concentrations of myogenic hHLH factors. This may provide a mechanism to regulate temporal order of muscle gene expression depending on the relative concentrations of ZEB and m);ogcnic hHLH proteins as well as on their affinities to various E-box target sites.

Conclusions During the past decade, myogencsis has become a paradigm for cell lineage determination and differentiation most importantly through the discolzry of the four myogenie hHLH transcription factors. ‘I’hc genetic approach of targeted gene disruptions for each mcmbcr of the farnil) and even more powerful double mutations in diffcrcnt pairs of the myogcnic hHLH genes ha\,e revealed unique and overlapping functions of these genes and placed them in a network of regulators in the myogcnic pathway. ‘l‘he important issue of whether the different phcnotypcs that result from inactivations of these genes reflect their disexpression patterns or specialized functions tinct associated with the different myogenic hHLH proteins has now been approached. The collective evidence from many experiments suggests that both the le\cls and possibly timing of expression and unique functional properties are important to properly execute muscle lineage determination and differentiation in the embryo. As an open topic for

Muscle

the future,

it remains

unclear

how precisely

these

factors

discriminate between the distinct sets of target genes that they must activate to promote cells through the different stages

important

topic

in the field has been

genie lineage commitment, a parallel pathway with hlyf-5 upstream of MyoD must also exist. A third myogenic pathway

independent

of both

hIyf-5

and

Pax3

appears

to

opcratc for head muscle formation. It will be interesting to see which factors trigger myogenic commitment and My011 gcnc activation there. It is also important to kno\f how precisely Pm? muscles and whcthcr

activates JZyoD expression in body Pa.? plays a role in 22!,if1.5regulation.

543

Patapoutian A, Yoon K, Miner H, Wang S, Stark K, Wold B: Disruption of the MRF4 gene identifies multiple waves of myogenesis in the myotome. Development 1995, 12:3347-3358.

10

Zhang W, Behringer RR, Olson EN: Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. Genes Dev 1995, 9:1388-l 399.

11

Rawls A, Morris JH, Rudmcki M, Braun T, Arnold HH, Klein WH, Olson EN: Myogenin’s functions do not overlap with those of MyoD or My+5 during mouse embryogenesis. Dev Bioll995, 172:37-50.

12

Wang Y, Schnegelsberg P, Dausman J, Jaenisch R: Functional redundancy of muscle-specific transcription factors Myf5 and myogenin. Nature 1996, 379:823-825.

understand-

ing what regulates the myogenic bHLH genes to initiate Compelling genetic evidence argues that myogcnesis. Pm.? is an csscntial upstream regulator for ,t[sflD expression in body muscle precursors. Although Pax3 is the first example of 3 non-cell type-specific factor involved in myo-

Arnold and Winter

9.

of myogenesis.

Another

differentiation

Wang Y, Jaenisch R: Myogenin can substitute for Myf5 in promoting myogenesis but less efficiently. Development 1997, 124:2510-2513. The first knock-In expenment replacing one myogenic bHLH factor gene by another addresses the issue of unique intrinsic properties of Myf-5 and myogenin versus functlonal differences as a result of their distinct expression patterns. The results reveal that myogenin expressed under the control of the Myf-5 gene can partially substitute for Myf-5 but is unable to compensate for loss of a second myogenic bHLH gene in double mutants also lacking either MyoD or myogenin. This suggests that myogemn only inefficiently compensates for Myf-5, probably owing to altered levels of gene expression and/or different transactivation properties. 13 .

14. .

The detailed genie bHLH insights into

analysis of important interactions of myofactors and MEFZ proteins has provided mechanisms of cell-type-specific transcrip-

tion. Kesearch over continue to identify

the next few years will undoubtedly novel factors lvhich will either syner-

gize with or repress thcsc principal and thus refine our view of muscle

players in myogencsis cell differentiation.

Acknowledgements

Gerber AN, Kleseri TR, Bergstrom DA, Tapscott SJ: Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis. Genes Dev 1997,11:436-450. This study provides evidence that MyoD and Myf-5 can remodel chromatin at binding sites In muscle gene enhancers and activate transcription at previously silent loci, whereas myogenin IS markedly less efficient at activating genes in transcriptionally silent chromatln. The ability of MyoD to activate endogenous genes IS associated with two regions in the protein that are distinct from its transactivation domaln and are conserved in the Myf-5 protein. TGF-P, basic FGF, and sodium butyrate - known inhibitors of myogenesls - block MyoD-mediated chromatin reorganization. These results may provide a molecular basts for the fact that MyoD and My+5 establish the myogenic lineage, whereas myogenin mediates terminal differentiation. 15.

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Patapoutian A, Miner H, Lyons GE, Wold B: Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice. Development 1993, 118:61-69.

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Zwelgerdt R, Braun T, Arnold HH: Faithful expression of the Myf-5 gene during mouse myogenesis requires distant control regions: a transgene approach using yeast artificial chromosomes. Dev Biol 1997, 191 :I 72-l 80.

29.

Daston G, Lamar E, Olivler M, Goulding M: Pax 3 is necessary for migration but not differentiation of limb muscle precursors in the mouse. Development 1996, 122:1017-l 027.

30.

Bober E, Franz T, Arnold HH, Gruss P, Trambley P: Pax 3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. Development 1994, 120:603-612.

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31. ..

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