Transcription factor families: muscling in on the

myogenic program
DAVID C. LUDOLPH AND STEPHEN F. KONIECZNY’
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA

ABSTRACT Embryonic skeletal muscle develop- ultimately culminate with intracellular signal cascades to
ment has become a paradigm for understanding the generate changes in gene expression patterns. It is this
molecular basis of how cell lineages are established signaling complexity that allows the embryo to respond to
and how cells differentiate into specialized struc- changing environmental conditions or to changes in cell-
tures. Most vertebrate muscles are derived from cell interactions. As with many developmental events,
individual somites that produce two distinct muscle transcriptional control is both precise and yet surprisingly
populations: the myotomal muscles that generate the plastic in nature. Despite this inherent plasticity, altering
axial and trunk musculature and a second migratory the level or pattern of expression of many genes often
cell population that colonizes regions of the develop- produces severe developmental abtiormalities. However,
ing limbs. In both instances, muscle differentiation is when the system is fully operational, the result is the for-
accompanied by cell cycle arrest, fusion of individual mation of a complex multitissue organism that is intricate
myoblasts into niultinucleate myotubes, and the tran- in 1)0th its design and function.
scriptional activation of muscle-specific genes. Re- Of the many systems that have l)een experimentally ex-
cent experimental progress has led to greater 1)loited to model developmental control, skeletal muscle
understanding of the molecular mechanisms that may represent the single best system. Because myogene-
control myogenesis in the embryo. Most of the ad- sis involves morphological as well as cellular and mo-
vances have come from the identification and isola- lecular changes that occur during the stages of terminal
tion of regulatory genes that are involved in differentiation, embryonic skeletal mttscle development
controlling specific transcriptional events. In par- has become a pai’adigm for understanding the tuolecular
ticular, the muscle regulatory factor (MRF) and inyo- basis of how cell lineages are established and how cells
cyte enhancer factor 2 (MEF2) families have been differentiate into specialized structures. Most vertebrate
implicated in establishing the myogenic lineage as muscles are derived from individual somites that produce
well as controlling terminal differentiation. Two ad- two distinct muscle populations (t’eviewed in refs 1, 2).
ditional transcription factors, Pax-3 and MLP, also The myotomal muscles cotisist of individual mnononu-
appear to play a role in the production of a mature cleate myocytes that initially form axial muscle structures
muscle cell. This review focuses on these four verte- as well as the trunk musculature. A second migratory cell
brate transcription factor families and discusses the population colonizes regions of the embt’yo, producing
experimental evidence that these factors play impor- founder populations that generate the muscles of individ-
tant, non-overlapping roles in regulating skeletal ual structures, such as those of the developing limbs. In
niuscle development.-Ludolph, D. C., Komeczny, both instances, myogenic differentiation is accompanied
S. F. Transcription factor families: muscling in on the by cell cycle arrest, fusion of individual myoblasts into
myogenic program. FASEB J. 9, 1595-1604 niultinucleate myotubes, and the transcriptional activa-
tion of muscle-specific genes encoding structural and
Key Words: MyoD . 6HLH . MEF2 . myogenesis ‘ regulalory contractile proteins such as desinin, myosin, actin, tro-
palhways ponin, and tropomyosin (reviewed in refs 3, 4).

A BASIC PREMiSE OF DEVELOPMENT is that sets of genes

need to be precisely controlled at the transcriptional level To whom correspondence and reprint reeldmests should lit’ uciehresse’cl.
in order to establish the correct specification and differ-
at: Department of Biological Sciences. PlmrdluteUmiive’rsity, 1392 Lilly hail
entiation of tissue and organ types. Transcriptional regu- of Life Sciences, W. Lafayette. IN 47907-1392. USA.
lation per se is not merely the ability to turn genes on or 2Abbres’iations: M RF, mnust’le regtthatetrv factor; liii LII. iwesim Imelix-
off, but rather requires genes to be ti’anscriptionally ac- hoop-helix; TAD, transcript ion ac’tivat ion dutmnumi mi; FC F-2. fi bitulciast

tive in a correct positional, temporal, and quantitative growth factor 2; TGF-1. transfeirnuing growth factor 1; PKA. cyclic
AMP-dependent kinase: PKC, protein kinase C: Rb. retinohulastomna
fashion. The transcriptional control of regulatory amid
protein; MEF2. myocyte enhancer factor 2; MADS family. MCMI,
structural genes also is not solely dependent on simpli- agumnous. defmciens and serum response factor family; Pmmx
family. 1mired
fied ci.s- and Irans- regulatoty systems, but involves sig- box family; LIM famnihy, Lin-Il. Ish-! and Mec-3 family; MLP. mnuscle
naling pathways that itiitiate extracellulat’ly and Lt.1

0892-6638/95/0009-1 595/$O1 .50 © FASEB 1595

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REVIEW

Although the embryological origin of muscle cells is heritable conversion of tile cells to the myogenic lineage.
fairly well established, tile molecular pathways that con- Fibroblasts expressing MRF4, lot’ instance, can be in-
trol rnyogenic precul-sor cell popmlitioiis and terminal dif- duced to withdraw from the cc11 cycle, fuse into multinu-
ferentiation events are just beginning to be defined. cleate cells, and express muscle-specific gene products
Tremetidous progress has been mnade recently toward utI- sttcii as myosin anti actin (Fig. 1). By these criteria, the
derstanding tile molecular cotiti-ol of myogenesis in the four muscle regttlatoty factors (MRFs) function as deter-
embryo. Most of the advances have conte from the identi- mination-specific geties (see below).
fication and isolation of regulatoty geties involved in comi- The remarkable ability of the MRFs to convert non-
trolling specific transcriptional events. This review muscle cells into myoblasts suggests that these factors
focuses on four diffet-ent vertebrate transcription factor may have a similar function in vivo. As predicted, the
families Ihat likely have itlll)ortant, and non-overlapping, four MRF genes are expressed early in development,
roles in controlling skeletal muscle development. when the first Inyogenic lineage decisions are being es-
tablished in somites as well as in the limbs (reviewed in
ref 2). Close examination of MRF expression, however,
THE MUSCLE REGULATORY FACTOR FAMILY
has revealed that the four MRF genes exhibit important
l’roni the earliest studies of myogenesis it became clear spatial and temporal expression differences in tile etni)ryo
that myogenic stem cells, referred to as myoblasts, can be as well as in myogenic cell lines. For muscle cells in cul-
maintained as a proliferating, self-renewing population ture, MyoD and/or Mf-5 are expressed in the proliferat-
that “reniemtibt’t-s” its developmetital history (5-7). The itlg, undiffetentiated mnyoblast population whereas
stability of tile myogenic phenotype suggests that perma- Inyogenin transcripts increase significantly as cells com-
nent changes. e.g., changes in chromatin structure or mit to differentiate. After differentiation, MRF4 tran-
gene expression patterns, likely are responsible for main- scripts become detected, suggesting that each MRF has a
taming cells in a committed, determined state. Sttb- distinct role iti regulating myogenic events. Although
sequent studies using the deniethylatitig agent MyoD amid Myf-5 typically are expressed in myoblasts,
5-azacyticline and the C3II1OT1/2 (1OTI/2) embryonic the somitically derived migratory cell population that
cell line cotlfirmed that stable, heritable changes in cells eventually populates the limb regions remains MRF tran-
can produce a Inyogenie stem cell population (8, 9). script-negative (2). Thus, these cells do not receive their
From these studies, four related but distinct vertebrate migratory cues from the MRF gene family, but likely are
transcription factors were identified, referred to as MyoD, instructed via different environmental and transcriptional
myogenin, Myf-5, atd MRF4 (reviewed in refs 10, 11). A regulatoty pathways (see below).
single homologue also exists in sevet-al invertebrates in-
eluding C. elegans. Drosophila, sea urchins, and ascidi-
Ti-ansgenic approaches to MRF functions
ans (12).
These factors appear to play a major role in
cotitrollitig myogenic events because ectopic expression Although tile MRF genes at-c expressed early in develop-
of any one factor in nontnuscle cells results in a stable, ment and their expm-ession is restricted to skeletal muscle

Figure 1. Indumutitirm of mnyogenesis by’ cc-topic expms-

sion of a single mrnmscleregulatory factor. Tm’ansfection of
nonmusele fibiohiasts (1OTI/2) with an expression vec-
tor containing the MRF4 c’DNA (or Mi’oD, Mjf-5, myo-
gcnilm. or MEF2 c-DNAs) heacis to the conversion of
Imbrolilasts to committed my’oblasts. Under low serum
conditions. myoblasts differentiate, fumsing into mnultinu-
cleate myotubes and expressing mnuscle-specifIc gene
products such as mnyosin. In the presence of growth
factors, clifferentiat ion is blocked. In both panels, the
cells am-cstained with an antibody that recognizes sar-
comeric myosin heavy chains. Only myotubes express
contractile protein genes (see text for details).

1596 Vol. 9 December 1995 The FASEB hournal LUDOLPH AND KONIECZNY
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 REVIEW

lineages, the predicted role for the MRFs in skeletal from the myogenin (-I-) animals do tiot differentiate in
myogenesis itlitially came fiom “gain of function” assays vivo but readily differentiate when placed in culture (17).
in which ectopic expressioti of the MRFs in vitro leads to Thus, tile embiyonic envi t-onmetlt requires myogenin ex-
the establishment of the myogenic lineage. However, the pression for muscle formation itt vivo, whereas this re-
ability to ablate individual MRF genes iti mice through quirernent is bypassed in vitro. MRF4 (-I-) mite also
embryonic stem cell technology has recently provided di- have been generated and ate phenotypically mlotnlal with
rect evidence for the importance of these factors in myo- tile exception that they exhibit mild t’i b anonial ies and
genesis. Investigators first generated homozygous null display an approximate threefold increase itl snyogenin

mice for the MyoD (-I-) atid Myf-5 (-I-) genes (13, 14). expression (18), leaving open the possibility that myo-
Interestingly, myogenesis in these animals proceeds nor- genin compensates for the loss of MRF4 in these em-
mally, although severe developmental defects involving btyos. A second series of MRF4 (-I-) mice have also beeti
rib formation are found in the Mf-5 (-I-) mice (Fig. 2). produced in which both MRF4 amid Myf-5 expression is
MyoD (-I-) mice show an appt’oximate threefold increase absetit (19). These mice exhibit tile Myj-5 (-I-) phenotype
in Mf-5 transcril)t levels, suggestitig that Myf-5 expres- with fairly normal muscle, yet due to rib abnormalities
sion may compensate for the loss of the MyoD gene. they die shoitly after bit-th. Using these various 4IRF (-I-)
However, with the double hotnozygous MyoD (-I-);Mf-5 mice, however, it now is possible to examine directly the
(-I-) mice, muscle formatiomi does not occur (15). The ability of different MRF proteitis to compensate for tile
muscle-forming regions of these animals exhibit a cotil- loss of atiy one factor. For example, can tile MyoD pro-
plete absence of myogenic stem cells, revealing that tein, when placed utider the control of the rnyogentn pIn-
MyoD or Myf-5 is required to generate the myoblast moter, functionally substitute fot’ my-ogenin activity iti tue
population. myogenin (-I-) mice? The generation of these and addi-
In contrast, myogenin (-I-) mice produce normal tiollal animals should gt-eatly- enhance our understanding
myoblast populations, but terminal differentiatioti of these of tile 1-egulatoty iietwotks operating to cotltt-ol MRF gene
myoblasts (i.e., formation of functional muscle) is absent expression patterns and MRF pi-otein functions.
(16, 17) (Fig. 2). This result supports the idea that myo- iie importance of the distinct expression patterns as-
genin is requii-ed to initiate terminal differentiation sociated with each mammalian MRF getie has led to an
events associated with muscle developtnent. Myoblasts examination of the cis-actitig regulatoty elements that aie
involved in controlling theit sotnitic an(l muscle-specific
Transcription Factor expressioti. Transgenic mice carrying ilI)-oD and myo-
genin promoter-LacZ genes cxii ibit -galactosidase cx-

Gene Ablation Experiments pt’ession that recapitulates the tiormal expression pitttertl
of the MRFs (20, 21). Thus, the role of each MRF family
tllenlber in auto- and cross-m-egulating expression of the
Gene Knockout Phenotype MRF genes can now he exatiiineci, smtwe MRF (-I-) mice
can be crossed with MRF-LacZ mice to determine
whether the transgetle expression patterti is mnaintaitie(i in
#{149}Myf-5 No skeletal muscle defects
the absence of a specific MRF. Itideed, the myogenin-
LacZ gene is expressed appm’opriately in the presumptive
#{149}MyoD No skeletal muscle defects muscle forming regions of the myogenin (-I-) mice, even
though muscle differentiation is blocked (22), revealing
Myf-5 that expression of the Inyogenin gene is not dependent on
. Embryos devotd of myoblasts tile presence of a functional myogetlin protein. Similar
+ and differentiated skeletal muscle
MyoD crosses should provide additional information regarding
the regulatory pathways operative in controlling expres-
sion of the four mammalian iI’JRF genes.
#{149}
Myogenin Skeletal muscle differentiation defects
Structural properties of the MRFs

#{149}
MRF4 No skeletal muscle defects The MRF genes encode nuclear phosphoproteins that
contain a conserved central pt’otein motif referi-ed to as
the basic helix-loop-helix domaiti (bHLI-l) (t-eviewed in
#{149}
D-mef2 Somatic, cardiac and visceral refs 10, 11). Many studies have suggested that the bIILH
muscle differentiation defects
motif is responsible for protein dimnerization as well as for
DNA binding. The recent elucidation of the MyoD-DNA
Figure 2. Sumnmam-yof results obtained with mice homozygous null for
ctystal structure (23) indicates that the a-amphipathic
the Mjj-5. Ml’oD, myogenin. and MRF4 regulatory genes as well as for
D-mef2 null Drosophila embryos. Only the striated muscle )hen0type is helices, separated by a varial)le loop region, are responsi-
indicated. In some instances, such as with the l!f-5 (-I-) and MRF4 (-I-) ble for protein dimnerizatioti while tile basic region pro-
mice, additional defects in rib formation at-c observed. See text anti refs ‘ides the contact points with an appropriate DNA target.
13-19 and 50-51 for details. For the bHLH MRF proteins, the consetisus DNA se-

MYOGENIC TRANSCRIPTION FACTORS 1597

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quence -CANNTG-, referred to as an E-box, serves as the MyoD, the positions of these residues are Alahit, Thr115,
molecular target, althottgh additional flanking nucleotides amid Lys’24. Mutational analysis has shown that the amino
also have a role in DNA target specificity. Binding of the acids of these positions are crucial to the myogenic activ-
MRFs to E-box elements located within the regulatory re- ity of the MRFs. Remarkably, placing the cotTesponding
gions of most muscle-specific genes often is sufficient to Ala4, Thr115, and Lyst2” from MyoD into the MyoD
activate muscle gene expression. partner E12 produces a protein that functions as a myo-
In vitro the MRFs are capable of forming homodimers genic activator (28). Thus, altering three amino acids in
(24), although in vivo the MRFs likely futlction as het- the widely expressed E12 bHLH protein imparts muscle-
erodimers forming a complex with a second bHLH pro- specific activity. The MyoD-DNA crystal structure has re-
tein from the E-protein family, such as E12, E47, E2-5, vealed that Lys’24 is exposed on the surface of the
or HEB (25). In most instances, the E-protein dimer part- complex and thus potentially participates in protein:pro-
tier is expressed in a wide variety of cell types, whereas tein interactions with either the MyoD activation domain
MRF expression remains restricted to skeletal muscle. or with other elements of tile transcription apparatus (23).
The E-protein partner preferred by each MRF iti vivo However, Ala114 and Thr115 remain buried within tile
currently is utiktlown, but partner choice likely repre- DNA major groove, raisit’mg the question of how these resi-
sents an important mechanism to control MRF activity dues participate in muscle-specific transcriptional activa-
duritig myogenesis. Indeed, positive as well as negative tion when they are not exposed oti the surface. One
t’egulatory partners are known to i)e involved in modulat- possibility is that Ala’” atid Thr115 establish the overall
ing MRF activity (see below). conformation of the MyoD protein when bound to DNA.
Although tile four MRFs are capable of binding to an Thus, their role in myogenic specificity may be indirect,
E-box DNA sequence and convertitig fibroblasts to a dictating protein conformation so that MyoD binds cor-
myogenic lineage, their ability to activate specific con- rectly to a DNA target and potentially interacts with spe-
tractile reporter genes is not equivalent. MyoD, for exam- cific coactivators that function in a muscle-specific
ple, efficiently activates M-creatine kinase, troponin I, fasilion. This is an iniportant concept to address because
a-aczin, and a-acetylcholine receptor subunit reportem’ it cetlters around whethem’ the MRFs, when boutid to DNA
genes, whereas MRF4 activates only -actin and a-ace- as heterodimer complexes with E-proteins, require the in-
tyicholine expression (26). Thus, the MRFs exhibit intrin- teraction of other muscle-specific transcription factors to
sic differences with regard to transcriptional activity. elicit a correct transcriptional t-esponse amid the complete
These differences may reside in the different transcrip- activation of the myogenic program (see below).
tion activation domains (TAD) presetit withitl each MRF.
MyoD and MRF4 contain a sitigle amino termitius TAD, Regulation of MRF activity through intracellular
whereas Myf-5 and myogetliti contain TADs at both tile signal transduction pathways
amitlo and carboxyl potiions of the proteins. The MRF
TAD regions are utirelated at the primary anlino acid se- Although the MRFs are capable of activating the myo-
quence level, suggesting tllat the TADs may be responsi- genie program, they do so in vitro otily in 1ow serum con-
ble for transctiptional specificity. Cllimeric protein ditions (Fig. 1). Myoblasts expressing MyoD or Myf-5
constructs between MRF4 and MyoD or between MRF4 remain in an undifferetitiated state even though high
amid myogeniti have revealed that the transcriptional MRF levels are present. Identical results are obtained if
specificity of the factors is in part dtte to differences in 1OT1/2 fibroblasts transfected with MRFs are maintaitied
their respective TADs (26, 27). These differences also in the presence of fibroblast growth factor-2 (FGF-2) or
ate manifested when the factors are tested in heterolo- transforming growth factor-fl (TGF-1). Under these
gous systems such as in yeast (K. Mak anti S. Kotiieczny, c)tiditioti5, tile MRFs are expressed and translocate to
unpublished observations), confim’ming that the TADs im- the nucleus but no must-ic differentiation or activation of
part essetitial information regarding how the factors func- muscle reporter genes occurs. Thus, the MRFs are sub-
tion in viti-o. Whether the TADs exhibit distinct activities ject to negative control when cells are exposed to specific
in vivo remains unknown, but, as discussed above, it now growth factors. Several lines of evidence support the cx-
is possible to address these important issues by generat- istermce
of multiple regulatory pathways by which the
ing mice expressing individual MRF proteins itl different MRFs may be inhibited. One mechanism itivolves expres-
MRF (-I-) etlvironmetlts. sioti of Id, a helix-loop-helix (HLH) protein that lacks a
An interesting aspect of MRF biology is that a large basic domain (29). Id dimerizes with E-proteins, thereby
superfamily of bHLH factors exists, but only the MRFs sequestering potential MRF dlimer Partners and inhibiting
are capable of generating a myogetlic phenotype. Amino MRF activity. Id levels increase substantially when cells
acid alignment of known factors reveals that tile bHLH are supplied with serum and the overexpression of Id in
domain represents a protein motif that is conserved myoblasts inhibits muscle differentiation (29). However,
across the entire bHLH supet’fatnily. However, within the cells tnaintained in FGF-2 or in TGF-1 do not up-regu-
basic domain and hinge region, the MRFs contain three late Id expression, yet are blocked from differentiating,
conserved amino acid residues (alatiine, threonine, arid suggesting the existence of additional pathways nega-
lysine) that ate not present in any other bHLH factot’. For tively comitroilitig myogemlic events.

1598 Vol. 9 December 1995 The FASEB Journal LUDOLPH AND KONIECZNY
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 REVIEW

A second level of MRF regulation involves posttransla- Because cell cycle progression amid chiffem-entiation are
tional modification mechanisms that are operative under mutually exclusive events, cell cycle regulators, including
different growth conditions. Cyclic AMP-dependent pro- the retinoblastoma (Rb) protein, cyclin/cdk complexes,
tein kinase (PKA) and protein kinase C (PKC) are known and cdk inhibitors, are also likely to play importatlt roles
to inhibit the activity of the MRFs wheti constitutively ex- in regulating the tm’ansition from proliferation to differen-
pressed in myogenic cells (30-32). PKA-dependent inhi- tiation (Fig. 3). Several lines of evidence support the
bition occurs via indirect pathways, as mutation of the tiotion that these molecules control essetltial myogenic
PKA sites in myogenin and MRF4 have no effect oti the events. For example, hypophosphorylation of Rb is re-
ability of PKA to inhibit muscle differentiation (30, 32). quired for myoblasts to withdraw from the cell cycle and
In contrast, PKC-dependent inhibition of myogenin activ- fuse into myotubes (34). The effect of RI) OFt the MRFs
ity involves the direct phosphorylation of a conserved appears to be direct since RI) and MyoD interact with one
threonine residue located within the basic domain of tile another in vitro as well as im vivo via the pocket amid
protein (31). Threonine phosphorylation inhibits myo- bHLH domains, respectively (34). inactivation of Rb via
genin from binding to DNA and this phosphorylation phosphorylation or by genetic alteratiomi abolishes MyoD
event can be imiduced through a FGF-2 signal pathway. interactions, leading to inhibitioti of myogenesis (34).
Whether all MRFs are subject to similar control is un- Overexpression of cychin Dl also preveilts activation of
clear. In the case of MRF4 and MyoD, FGF-2 treatment the myogenic progl’am by the MRFs (35), possibly by
does not produce a phosphothreonine protein (32). In ad- phospliorylating Rb and thus maintaining cell cycle pro-
dition, MyoD and MRF4 proteins isolated ft’om FGF-2- gression. Conversely, ectopic expression of MyoD acti-
treated cells exhibit normal DNA binding activity (Y. vates tile cdk inhibitor a protein implicated iti
Kong and S. Koniecztiy, unpublished observations), sug- promoting Rb hypophosphotylation, cell cycle arrest, atld
gesting that FGF-2 inhibition of myogenesis does not op- terminal differentiation (36). Ectopic expression of
erate via PKC and phosphorylation of the PKC site in the l21t also enhances the ability of MyoD to gemlemate a
basic domain. TGF-131 similarly inhibits MRF activity muscle phemiotype, eveti ill tile presetice of high serum
through a pathway that does hot alter tile dimerization or concentrations (36), again supporting tile hypothesis that
DNA bitiding properties of the MRFs (33). These regula- M)-oD expression induces p2l’1, which in turn inhibits
tory mechanisms still may involve phosphorylation of the cyclin D1/cdk4 conlplexes. These events gemlerate c-eli
MRFs or E-protein partners, however, since regulatory cycle arrest and terminal differetltiation (Fig. 3). Addi-
events of phosphorylation and dimet-ization may be cou- tional studies examining the role of specific growth fat-
pled in myotubes (S. Johnson and S. Konieczny, unpub- tom’s in controlhimlg both prol iferat ion and diffetentiat ion
lished observations). should provide the information tieeded to explaiti how

Myogenic Regulatory Pathway

Mesodennal
Progenitor Myoblast Early Myotube Mature Myotube

MyoD
Myf’S Growth Myogenin MRF4
Pax’3 Id Factors MEF2 MLP

#{174}LL
#{174}#{174} (q (q
(V L , _______

cycliui
DI Rb-P04 p21#{176}” Rb “Inner,,ation”

Mesodermal Myogenic Early Late

Determination Genes? Differentiation Events Differentiation Events

Figure 3. Summary ofa nlyogenic regulatory cascade model showing mIte relative linear positions of known transcription factors. Muscle development
entails the specification of the myogenic lineage tltat likely involves Pax-3 as well astheMRFgenesMvoD and lhf-5. Myoldasts remain in a prolifei’ativi’.
undifferentiated state when maintained in the l)i’v’ of serum growth factors dmie to the cell cycle regulated gene pi’odticts cv-lin Dl and RI5. lit low
serum, Rb becomes hypophosphorylated with the aid of p2lri, inducing cell cycle an-est and the transcriptional activation of tite msogeoili anti 1JEI”2
genes. Early differentiation events are signified by the fusion of mnyoblasts into multintu-leate myotubes and the expression of tntis(-k’-slwcifii- gene
products such as myosin and ar-tin. Latet- differentiation events involve expression of the .41RF4 and MLP genes and, in thi emhno. inner atiiin of
pI-imnnry muscle fibers. This model repi-esents a compilation of data from both einlu)onic as well as tissue e-mtlttire model systems and is not iiittntletl to
i-eflect all possible regulamoiy networks, but rather to serve as a working hypothesis for how mite myogenic phenotj is estal l islied am I ma i nla i ned.

MYOGENIC TRANSCRIPTION FACTORS 1599

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cells respond to the many changing emlvironmentai condi- In--OSin heavy chain, desmin, and certain MRFs (39, 43
tiotis itl the embryo. and references withimi). Despite sharing similar DNA
binding activities amid amino acid comlservation within the
Future directions
MADS amid MEF2 domains, the four mammalian MEF2
For all that is known about MRF gene expression pat- geme products are diverged outside of these regions, sug-
terns, structural features of tile proteins, positive and gesting that each MEF2 factor plays a unique role in em-
negative di merization pam’tners, posttranslational modifica- bryotuc developmemit.
tions, and the comitrol of cell cycle evetits, it remaitis un- The ability of the MEF2 proteins to serve as regulators
clear how expression of a single MRF dramatically of muscle development is further supported by the ex-
produces a fully differentiated muscle phenotype. Several pression patterns of the MEF2 genes during eat’ly em-
imni)ortant issues remain unatiswered regarding our under- bryogenesis. MEF2 transcripts are detected in the
standing of how the MRFs function. First, it is essential presumptive cardiac niesoclerm, developing somites and
to determine how the MRFs control myogenesis in vivo. limb buds at 7.5, 8.5, amid 11.5 days p.c., respectively, as
Clearly, serum gm’owth factors have a role in regulatitig well as it) the adult organism (44). However, unlike the
MRF activity in vitm’o, but additional studies are required skeletal muscle-specific expression exhibited by the
to establish the mechanisms by which these factors are MRFs, the MEF2 genes are expressed in a broader m’ange
regulated imi the developitlg limb when tile myoblast of cell types, iticluding brain and neural crest cell deriva-
population is expatiding i)ut termitlal differentiatioti is tives as well as skeletal, cardiac, and visceral mitscle
blocked. Second, what are the true molecular targets for (44). This highly complex expression pattern is further
the MRFs? Tiiese regulatory factors activate muscle complicated by the use of alternative mRNA splicing to
stm’uctural genes such as M-creatine kinase, desmin, and yield tissue-specific MEF2 isoforms (39). In addition to
troponin I, i)ut is the activation of tllese structural genes alternative splicing, there also is compelling evidence
sufficient to gemlerate a complete muscle phetiotype? Are that posttranscriptional mechanisms may regulate the
othem’, as yet unknowmi, regulatory targets needed to acti- cell-specific expression and activity of different MEF2
vate the myogemlic program? Sinlilarly, are there MRF- i)roteim)s. For example, transcripts for MEF2A, MEF2B,
specific molecular targets in myoblasts that stabilize the and MEF2D are present in many cell types, yet MEF2
myogenic stem cell phetiotype? Finally, what activates DNA binding activity is largely restricted to skeletal and
tile earliest myogemlic factot’s. especially MyoD and Myf- cardiac muscles (ref 39 amid referemices within). There-
5, it) tile developing embmyo to initiate the myogenic cas- fom-e,it) nonmuscle tissues, MEF2 transcripts either fail to
cade? With the advancement of transgemiic and molecular be translated into their respective proteins or, alterna-
strategies, these questions should provide the ititellectual tively, the translated proteins are degraded selectively by
challenge for future studies. these cell types. Understamding the relationship between
MEF2 gene expression and activity will be essential to
umlderstanding how MEF2 proteins function in muscle
THE MEF2 FACTOR FAMILY
and itl nonmuscie cell types.
A secotid class of muscle transcription factors that control Similar to the four MRFs, MEF2 proteins also exhibit
myogemlic events is the myocyte enhancer factor-2 the ability to initiate the myogetiic program when overex-
(MEF2) fatnily, which belongs to the MADS superfamily pressed in nommuscle cells (Fig. 1) (45). Whether MEF2
(MCM1, gamous, deficiens, amid erum response factor) activates the myogenic program directly or itdirectly has
of DNA bimlding proteins (reviewed in ref 37). MEF2 was yet to be established. However, these results suggest that
identified originally in C2 myotube cultures by its ability the MEF2 proteins represent importamit regulatoty factors
to bind to a comiserved A/T-rich DNA sequence, - that are involved in the establishment of skeletal muscle
CTA[A/T]1TAG/A- (MEF2 site), that is present in the lineages, a trait previously attributed exclusively to the
regulatory regions of many muscle-specific genes (38). MRF protein family. How MEF2 fits into the myogenic
The manimalian MEF2 family is comprised of four dis- regulatory cascade, and whether the ability of MEF2 to
tinct genes, named MEP2A, MEF2B, MEF2C, and induce myogenesis demonstrates a potential relationship
MEF2D (reviewed in ref 39). Two homologues also exist between the MEF2 and MRF families, are addressed be-
in amphibians (SLJ and SL2) (40) as well as one in low.
Drosophila (D-meJ2,) (41, 42). Members of the MADS su-
perfamily share a common 56-amino acid motif, referred
Regulatory interactions between MEF2 and the
MRFs
to as tile MADS box, that is responsible for DNA binding
and protein dimerization. In addition, MEF2 factors con- Recent studies have provided evidence for a complex
tain a unique conserved 29-amino acid sequence (the regulatory network between members of the muscle
MEF2 domain), which also may dictate DNA target speci- bHLH and MEF2 family of proteins. MEF2 DNA binding
ficity. The MEF2 proteins interact with) MEF2 sites as sites, for example, often are positioned within close prox-
either homodimers or heterodimers with other members of imity to MRF binding sites (E-hoxes) in the regulatory re-
the MEF2 family to transcriptionally activate numerous gions of many muscle genes. Forced expression of
muscle-specific genes including M-creatine kinase, myogenin or MyoD in 1OT1/2 fibroblasts induces MEF2

1600 Vol. 9 December 1995 The FASEB Journal LUDOLPH AND KONIECZNY
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 REVIEW

DNA bimldimig activity, suggestitlg that the MEF2 genes (50, 51). The cotllnlitted fl)yoi)lastS, hioweve,’, do riot cx-
are regulated by the MRFs in a linear, regulatoty pathway pl’ess muscle structural gemies and fail to fuse into mull-
(Fig. 3) (46). Reciprocally, MEF2 proteimls are capable of i nucleate myofibers even though they continue to expt’ess
transcriptionally activating expressioml of the myogenin, the Drosophila MyoD homologue nautilus. The D-nuj2
MRF4 and Xenopus MyoDa genes, raisitlg the possibility lrnitamlt phenotype can be rescued by pm-ovid i mg exogenous
that members of the MEF2 family regulate MRF gene ex- MEF2 to the somatic mesoderm, furlher suggesting that
pression, and vice versa (21, 47, 48). MEF2 is not necessamy for myogenic cell fate s1)e(’ifi(’a-
The relationship between the MEF2 and MRF factors tiomi, but instead1 acts i-elatively lalc in (‘omltm’oliimlg myo-
also is evident whemi MEF2 and MRF proteins are coex- gemlic differentiation and the expression of
pressed in cells. In this instance, MEF2 and MRF family mimscle-specific genes. It) this comltext, the D-meJ’2 mutant
metlibers coopet’ate to trans-activate expression of t’e- pilemlotype is similar to the phenotype of nyogenin (-I-)
porter genes containing both MEF2 and E-hox elements mice, suggesting that D-mef2, like Inyogenin, is required
in a synergistic fashion (43, 45, 47, 49). This synergistic for myoblasts to termllimlal hy differentiate. Despite these
response has been observed when myogenin and MEF2 pat’alleis, it is cleat’ that (lie m’egulatory pathways t’espom-
are coexpressed itl 1OT1/2 fibt’oblasts and when diffet’en- sible fom’ generating a normual tnust’ie phenotype in Droso-
tiated C2C12 cells are tramlsfected with m’eporter genes phila and itl vertebrates tnay have diverged. In
containing MEF2 and E-ljox comisensus sequences. The Drosophila, for example, expression of D-mef2 and nauti-
ability of myogemlitl and MEF2 to function together to lus are imidependent of ote another (51), as is MEF2 and
elicit high levels of gene expressiomi is due to the dit-ect Myf-5 in tnice (44). However, in vertebrates tIle other
association of tile MEF2 MADS domaiml witll the basic three MRF genes appear to be m’egulated ill pam’t by MEI”2
domain and helix I of tile MRF proteins (45). This asso- (21, 47, 48). Clearly, additional studies will be needled to
ciatioti is discm’imimiatory because the MADS donlaim) of identify MEF2 target geties that am’e d’onlmon to Droso-
MEF2 specifically recognizes MRF bHLH domains but phila and vertebrates amld to amlalyze MEF2 mutant mice
not nonmyogenic bHLH domaimis. Similarly, the bHLH to establish the role of the MEF2 mntmitigemlefamily in
domain of the MRFs distitiguishes between MEF2 and milamnlaiian cells.
the related, i)ut ubiqituously expressed, MADS protein
Future directions
SRF (45). These results confirmn that MRF and MEF2
protein interactions occur only in the context of muscle Umllike the MRFs, the MEF2 genes are expressed imi sev-
development. The precise mechanism by which these fac- eral nonmuscle tissue types, raising the possibility that
tors interact amid the role of specific DNA targets for MEF2 factors may also regulate many mlonmyogenic
MEF2 and MRF imlteractiomls will require further expem’i- evetits. The complexity of this multigene family is fum’ther
mentation. enhanced by the ability of tile MEF2 genes to produce al-
tertatively spliced mRNA products. Thus. umlderstanding
Functional activity of MEF2 in myogenesis
tile developmental timning amld tissrme disti-ibtmtion of the
The presence of MEF2 binding sites imi the tegulatomy re- MEF2 proteins likely will reveal subtle differences in
gions of many skeletal amid cardiac muscle genes, to- their biological activities. At this poimlt, however, the ma-
gether with the developmemtal expressiomi pattem’mls and jot’ function attributed to the MEF2 proteins is in i’egtmlat-
ability of MEF2 proteins to interact with the MRFs and ing various cardiac amid skeletal muscle developmental
activate muscle-specific gene transcription, strotlgly sug- evemits. MEF2 proteimls are imlvohved ml both E-box-de-
gests that the MEF2 proteins play a vital role in regulat- pendent and E-box-independemlt m’egttlatory cascades,
ing the development of skeletal amld cardiac cell limleages. whid’h suggests that MEF2 intel-acts with the MRFs in a
To establish) an in vivo m’oie for MEF2 durimig myogetlesis. skeletal muscle context whereas MEF2 functiomis in the
two recent independent “loss-of-function” studies have absence of the MRFs in cam’diac mnuscle. This sets up a
been described using Drosophila as a mnodel system (50, very interesting paradigm ml which MEF2 proteins are in-
51). Drosophila possess a single MEF2 gene (D-meJ2) en- volved in skeletal and cardiac developmemlt, but the fumlc-
coding a protein
with extensive homology to tile verte- tions of the MEF2 factors are distinct in the two cell
brate MEF2
MADS domain (41, 42). Dut-ing types. Thus, what distinguishes the role of MEF2 proteins
embryogenesis, D-meJ2 is expressed imi the presumptive in cardiac vs. skeletal muscle development? Are bULH
mesoderm aild becomes pI’ogressively restri(’ted to the so- factors required for MEF2 activity only in skeletal muscie
matic, cardiac, and visceral muscle limieages. Tile com- cells, or are cardiac bHLH factors also required for
mon structure, DNA binding activity, and localized MEF2 function in this tissue? A potential cardiac coregu-
expression of the D-mef2 gene suggest that D-mef2 plays latory factor for MEF2 is paraxis, which encodes a novel
a similar role in muscle developmemlt as its vertebrate bHLH factor that is expt’essed iml presumnptive cardiac
counterparts. amid skeletal muscle cells as well as in adult heai’t and
D-mej2 mutant embiyos exhibit an absence of differen- skeletal muscle tissues (52). Futum’e studies likely will fo-
tiated somatic, cardiac, and visceral muscle, although cus on the structural properties of the MEF2 proteins, the
these embryos retain muscle cell precursors that are cor- al)ility of MEF2 factors to interact with additional pro-
rectly specified amid positioned within tile tissues (Fig. 2) teins in different tissues, amid tile mole of the MRF and

MYOGENIC TRANSCRIPTION FACTORS 1601

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REVIEW

MEF2 proteins iti cross-regulating expression of these early somitogenesis, when cells derived from the der-
gene families. mamyotome begin to differentiate and activate expression
of the MRF genes, whereas Pax-3 expressing cells of the
lateral dermamyotome, which migrate to populate the de-
PAX-3 PROTEINS
velopitig limbs, remllaiti negative for MRF expression until
Although the MRF and MEF2 transcription factor fami- the omset of differentiation. Epstein et al. (58) have
lies clearly play key regulatory roles mi controllimig vari- showti that Pax-3 expressiom) blocks myoblast differentia-
ous myogenic events, other transcription factors are also tion, again suggesting that Pax-3 may fumiction to inhibit
likely to be involved in regulatimig muscle developmemit. differentiation of nligratoly limb muscle precursor cells,
Tilis is pai’ticularly true because migratory somitic cells acting downstream of MyoD but upstream of myogenin.
that populate the early limb buds remain MRF- and The identification of cis-acting regulatoiy elements con-
MEF2-miegative until differentiation events are initiated trolling the developniental expression of the MRF and
(Fig. 3) (2, 44). Thus, identifying regulatory factors that Pax-3 genes should allow a direct test of whether they
control additional early myogenic decisions is cm’ucial to cross-regulate each other’s expression.
fttlly umiderstandimig how muscle cell lineages are estab- Although the expressiom) pattern of Pax-3 imi the devel-
lished amid maimitained. oping somites and limbs suggests an importamit i’ole in
One such class of factors tilat may regulate muscle de- myogenesis, a precise function for Pax-3 has yet to be de-
velopment is the paired box (‘Pax) gene family, which) cmi- ternlitled. Evidemice for a role of Pax-3 in somite and limub
codes transcription factors
contaimiing paired domaitis developmemit has been supported by investigatiomis of the
(m’eviewed in ref 53). One member of this family, Pax-3, splotch mouse, which carries a mllutant allele of tile Pax-3
has beeml implicated in controlling various regulatory gene that results in the production of trumlcated Pax-3
evemts associated with myogemiesis. The Pax-3 gene en- proteitis. The limbs of homozygous (-I-) splotch embryos
codes a 479-amino acid pm’otein containing two conserved are devoid of myogenic precursor cells whereas the axial,
DNA bindimig motifs: an amino terminus paired domain of facial, amid body wall musculatui’e appears normal (59,
128 amino acids amld a paired-type homeodomain of 60 60). The fact that only limb mnusculature is affected in
amimo acids (54). Although no itl vivo Pax-3 target gemie splotch mice supports the hypothesis that Pax-3 is essemi-
has been identified, in vitro evidence suggests that Pax-3 tial for the proliferation, specification, and migration of
binds to a regulatory sequemice found within the Droso- limb precursor cells and that Pax-3 plays a different
phila eveml-skipped promoter (54). In vitro studies have regulatoty role in distinct muscle precursor populations.
also revealed that Pax-3 possesses separate protemt do- Future studies should provide the framework to establish
nlaimls involved in tramiscriptional activation amid trail- a defimiitive role fot’ Pax-3 in muscle developmemit.
scril)tiOmlal repression that are active in a dose-depetidemit
fashioml (55). Thus, diffem’ent concentrations of Pax-3 may
LIM PROTEINS
dictate specific tramlscriptiomi functions during enlhryomlie
developnient. The LIM domaitl is a cystemne-t’ich motif that was identi-
Pax-3 expression is itiitially detected at 8 days p.c. fied itiitially in three developmentally impot-tamlt tran-
througllout the rostrum 1araxial mesoderm durimlg mouse scril)tion factors referred to as Lim-11, Isl-1, and Mec-3
embmyogenesis. As somites mature, Pax-3 expression be- (reviewed in ref 61). This domain consists of the se-
comes restricted to defimied comparttnemits of the somite. quence (CX2CX123HX2C)-X2-(CX2CX16..21CX2C/H/D
including the lateral dermomyotome (54, 56, 57). By 9.5 and is found in a growimig number of proteins that fall
days the Pax-3-positive
p.c., cells begin to migrate imlto into two major classes. One class contaimis two to three
the adjacemit limb regions and subsequently localize imlto LIM motifs ill comljunctioml witil homeoclomain motifs, and
diorsal amld ventral domains within tile limb mesemlchyme, a second class lacks a classical DNA bimiding domain.
markimlg the earliest limb premuscie masses. As these Tile LIM doniain defines a specific zimic binding structure
cells terminally differentiate, Pax-3 expression decreases that coom’dinates two atoms of zinc in a tetralledral fashioti
amid remains repressed in tile diffet’entiated muscles of via the comiserved cysteimie and histidine resi(iues itl the
tile embryo. LIM consensus. This domain has also been implicated in
The unique spatiotemporal expressiomi pattet’ns of tile the formatiomi of proteit) llomodmmers (62).
Pax-3 gene suggest tilat it is involved in myogenic speci- A mlovel LIM protein, referred to as MLP for muscle
fication tilroughout paraxial development, and in particu- LIM proteiti, has recently beet) identified amid foumid to
lar, Ifl tile specification of tile dermanlyotome precursor promote muscle differemltiation (63). MLP contains two
cells of tile limbs. As discussed earlier, Pax-3 expressioml adjacent LIM doniains that ate followed by glycine-rich
precedes MRF gene activation in sornites as well as itl regions. The MLP gene is expi’essed predominantly in
the developing limb buds, fum’thet’ suggesting that myo- skeletal and cardiac muscles. MLP expression is dt’amati-
genic specificatioti occurs before MRF expression (Fig. cally up-regulated during myoblast differetitiation, where
3). These observatiomls suggest that Pax-3 expression may the MLP proteiml accumulates in the nucleus of differenti-
be negatively coupled to the itiitiation of the MRF gene atimlg myotubes (Fig. 3). Drosophila MLP expressiot) is
cascade. Indeed, Pax-3 expressioml is repressed during associated with developing muscles of the viscei’al and

1602 Vol. 9 December 1995 The FASEB JoLirnal LUDOLPH AND KONIECZNY
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 REVIEW

somatic mesoderm subsequent to tIle formation of muscle how this elegant system is developmentally established
precursor cells, demonstrating that MLP expression in and maintained. Thus, we are left with the quandaty that
differentiating striated muscles has been conserved once the transcription factor families are fully charac-
throughout evolution (63). terized, what is the text step in the study of mnyogenesis?
Overexpression of MLP in C2 myoblasts produces cells Ai’e additional control poimits required to produce a func-
that differentiate with an increased efficiency compared tional muscle cell? These questions need to be, and will
to mock-transfected cells (63). The increase in differen- be, addressed only in future studies examining all facets
tiation is measured by an increase imi myotube frequency of development. Given the curm’emit status of the field,
and size as well as by an increase in the expression of ilowever, the future looks extremely promising for en-
muscle-specific gene products such as myosin and Ilancing our understanding of how cells commit to a par-
myomesin (63). C2 cells expressing amitisense MLP fail to ticular lineage.
form myotubes and express reduced levels of skeletal
muscle-specific genes. These results demonstrate that
We wish to ackmtowiedge our mutiny colleagues for (‘onhmnunmt’ating
MLP expression is essential to obtaitiing a differemltiated results plior to publication. In ad lit ion, we thank Sally Johnson anti
muscle phenotype. Elizabeth Taparowsky for helpful smiggesmions and Connie litilinook for

The mechanism by which MLP induces myogenesis re- the l)iepai-ation of the inanttsd’ript. This work was su1)ported by iest’arch
giants to S.F. K. from the National Inst it ukw of Health, the A merit-an
mains unknown. Although LIM domains Ilave been impli- Heart Association, and the Muscular Dystrophy Association. DCL. was
cated in both DNA binding as well as in proteiti:proteimi supported by a Muscular Dystrophy Assitciation post(loctoral ft’lbosship.
im)teractions, precise LIM DNA bimiding domains ilave yet S.F.K. is an Establishedi Investigator of the Aint’rican Heart Association,
to be identified. Nonetheless, the nuclear localization of
MLP, together witil its ability to etihance the expression
REFERENCES
of muscle-specific genes, suggests that MLP has a direct
role imi regulatimig transcriptional processes. Potentially,
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2. Sassoon, 0. A. (1993) Myogenk- regulatory factors: tlissett lag their role and
the MRFs to induce cell cycle withdrawal and terminal regulation during vert-I,rate cml Iiyogenesis. Dee. Rio!. 156, 11-23
differentiation. Alternatively,
MLP may interact directly 3. But-kingham, M - E. (1994) Muscle: the regu at ion of myogeni’sis. Cssrr.Opus.
Genet. Dee. 4, 745-751
with the MRFs to function as a coregulatom’. Several stud- 4 Lassar, A B., Skapek, S. X,. antI Ntis itt-h, B. (191)1) Regulatory ineuhanisimis
ies have demonstrated that LIM proteimis can associate that coor(Ii nate skeletal mastIc tI iffercni latlon and tel I c tIe sit Iisl,assal_
Cure. Opin. Ce!!. Rio!, 6, 788-791
with bHLH factors to synergistically activate gene expres- 5. Bischt,ff, R., anti Holtzer, II. (1969) Mitosms and tIn’ l>05t55 (,fdllferentiatton
sion (64, 65). This potential partnership fot’ myogenesis is of iiiyogenic cells in stirs,. J. Ce!! Bit,!. 41. 188-200
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particularly intriguing simice high levels of tile MRFs and
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MLP and tile MRFs will provide additional itlformation IOTI/2 anti 3T3 cells treated is itli 3-azac titline. Ce!! 17. 771_77o)
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stable mesodermal stein cell Ii mit-ages tr,,ni I OT 1/2 cml Is: ci i,lt-nt.- for regu-
latory genes coittrolli ng deteniiinat ion. Ce!! 38, 791-800
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Des. 8, 1-8
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results in apparently nonisal iiius, It’ tlt’s i-lesi,nit’iit. Ce!! 71, 383-390
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II. Hussein.T., Rudisicki, II. A., anti Ainsst,I. 11.-lI. 11992) ‘rsiigeti’d inaelisaliss,i
From expressioti analyses to functional studies, the m’oies of tlte muscle regulatory gene- lItj.5 results iiiuI,nstriiial nh ties t’ltipisteist stilt1
of these regulators during developmetlt are beginning to PreIititiI death. Ce!! 71, 369-382
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Fig. 3. What is absent in this model, however, is the of skeletal inList-It’. Ce!! 75, 135 l_135t.t
16. hasty. P.. Bradley, A., Monk, J. Ii.. Ediiussniison, I). C.. Vi’nuti. J. II.. Olson,
likely ititet’related intet-actions that exist between factors. K. N., and KIt-sit, W. 11. I 1993) MustIm tie1 itim’iscy and ,ieoii.iial hailu iii islet-
For example, tile ability of the MRF proteimls to auto- and with a targeted inutatioti i a he my uigt-niii gt’sie. Vat ore (Ls,ndon) 364,
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anomalies. Genes Dee. 9, 1388-1399
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add additiomial levels to the alm’eady complex moleculat’ iii mice leads to alteiat ions in ski-lila I iistisulu- development. E 11130 J. 14.
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 sm-gillitmsl ity a IsighI’. etsissm’svcsI distal is esstsitl s It’isseuit . L)ese!tqtntt’ssi 121, ge’ise’ utls htusiiasis c’S1tie’ssitsss pststfile-
is tlsssiesg e’iiiltss sige-nesis. Pst,c. .(st!.
637-619 It-ad. Se,. (S.4 91.7320-7521
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list’ alssessce i,f positis e auii.me’guulatieess. I’ros. .\ut!.ieae!. Sts. I S-I 92. smueiLsse’essihii-yssgemie-sisDes-e!tspntens 120, 123 1-1263
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23. Ma, I’. C. II.. Rsssilsl. M Wi-isutrausi,. II.. antI Pals,,, (2. 0. (1991) Crystal Acti sat eta ssf Ise isis ttgsssssItsus’zigs- Its II EF2-\. a fsects,n I IssutI nuhisces ass,i
stieiitiiie’ si Mviii) lslll.Il iistsnasus-l)N.A stsmplt-x: tieisI)t’eiuses ttui l)N.A cssstpt’rssti-s siuthsM sst) .Sc,e,ss-e’ 266. 1236-12 II)
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ism’tu’mcesliisie’rs.J. Hiss!.Cheni. 268, 21115-21 t20 47. Naidu, P. S., Lseelolphs. D. C.. Tee. H. 9., ihisstcsheerger.T. J ..antl Kusi,ue’ezns
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I).,sie-s-izatisen sss’ei1iculy nI ieiyssgeiiit Iu.lix-lssu1t-he.Iix DNA-isineliisg factstis 11kb’.! puosnhster eluriisg asssuge’sse’sis.1/sd.Ce!!.1/is,!. 15. 2707-2718
elere’eue’sIlsy nttssconseivetl Iiydsoplsilie’ um’sislues. Genes Des. 7. 2456-2-171) I8. Wiessg, Il.-W.. Pisegsia, II., l.se. M.-F., l.i’ilslssess,, I).. auth Pu-ui). Il. 11991)
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sici0 atieeis elssssaessis rcsieiisu’eI its iiulue-c iisiis,lu-.stseeills geium’ expsessksn. h.uiessly.Des. Rio!. 166. 683-695
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1021
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1604 Vol. 9 December 1995 The FASEB Journal LUDOLPH AND KONIECZNY
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Transcription factor families: muscling in on the myogenic
program.
D C Ludolph and S F Konieczny

FASEB J 1995 9: 1595-1604

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