isexpressed transiently indeveloping myotomes and enhances...

Vol. 9, 1-11, Janualy 1998 Cell Growth & Differentiation I

5 S. D. Zabludoff and B. J. Wold, unpublished data.

p27K� Is Expressed Transiently in Developing Myotomes andEnhances Myogenesi&

Sonya D. Zabludoff,2 Marie Csete, Roger Wagner,Xin Vu, and Barbara J. Wold3Division of Biology, 156-29 California Institute of Technology,

Pasadena. California 91125

Abstract

Vertebrate skeletal muscle development ischaracterized by tight coupling of muscledifferentiation with cell cycle arrest in G1IG�. Keyregulators of G1 progression are the C1 cyclin-dependent kinases, their positive regulators, the GIcyclins, and their negative regulators, the cyclin-dependent kinase inhibitors (CDIs). Here we show thatp27� protein, a GI CDI, is expressed in a prominentbut transient wave in the developing myotomes of themouse embryo. We relate fts expression to expressionof MyoD and myogenin proteins, which aredetermination and differentiation class myogenicregulatory factors, respectively. Functional assaysshowed that ectopic p27 expression can powerfullyenhance the efficiency of MyoD-initlated muscledifferentiation in cell culture. When consideredtogether with the myotomal expression patterns of p18,p21, and p57, these results suggest a model In which

p27 acts as a “trigger” CDI while myoblasts are exitingthe cell cycle and initiating differentiation. At latertimes, when p27 protein has been down-regulated, It Isproposed that accumulation of p18, p21, and p57maintain the differentiated myocytes in a postmltoticstate.

Introduction

Vertebrate skeletal muscle development is characterized byirreversible withdrawal of differentiating myoblast precursors

into a G1/G0 arrest (1 , 2). Environmental signals that promotecell cycle progression through G1 effectively suppress dif-ferentiation in myogenic cell culture models (3-5). Much ev-idence now supports the idea that G1 CDKs,4 together with

Received 9/2/97; revised 10/20/97; accepted 11/26/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was supported by NIH Grants AR40708 and AR42671 (toB. J. W.). S. D. Z. was supported by a postdoctoral research fellowshipaward from the National Cancer Institute.2 Current address: Novartis Pharmaceuticals. Oncology Research, EastHanover, NJ 07936.3 To whom requests for reprints should be addressed.4 The abbreviations used are: CDK, cyclmn-dependent kinase; CDI, CDKinhibftor MRF, muscle regulatory facton MCK, muscle creatine kinase;CAT, chloramphenicol acetyltransferase; MHC, myosin heavy chain; Br-dUrd, 5-bromodeoxyundmne; E9.0-E1 1.5, embryonic days 9.0-11.5,CMV, cytomegalovirus.

their positive-acting coregulators, the Gi cyclins, directlygovern progression through the G1 phase of the cell cycle(6-12). Because cyclin D/CDK4 and cyclin E/CDK2 regulatecell cycle progression during G1 in several vertebrate celltypes (1 3, 14), they are attractive candidates for a central rolein integrating cell cycle status with muscle differentiation.Thus, the G1 cyclins are downstream targets of growth factorpathways, and enforced expression of cyclin Dl (1 5, 16) andcyclin E4 suppress muscle differentiation in cell culture mod-els. In addition to positive regulation by the cyclins, the CDKsare also subject to negative regulation by phosphorylationand by interaction with CDls, some of which also act astumor suppressors (1 7). CDIs described thus far are from twofamilies: the p16/INK4/MTS (including p15, p16, p18, andp19) and p21/CIP1 (including p21, p27, and p57) families.CDls from both families inhibit cyclin/CDK activity in vitro,

inhibit colony formation in transfected cells, and cause cellsto accumulate in G1 (1 8). In this manner, the CDls can act aspositive regulators of G1 arrest.

Skeletal muscle determination and differentiation path-

ways depend on the action of the MRF family of basic helix-loop-helix transcription factors (1 9, 20). MyoD and myf5 to-gether play an essential role in muscle fate determination.Expression of MyoD and/or myf5 can precede cell cyclearrest and differentiation (21 , 22). Myogenin, in contrast, isessential for differentiation of most skeletal muscle in vivo,

and its expression is typically up-regulated at the onset ofdifferentiation (23-26). In cell lines, myogenin expression isoften initiated in response to low mitogen medium, followedby cell cycle arrest and then terminal differentiation (27).Evidence is accumulating for a significant functional interac-tion between the G1 CDIs and regulators of muscle differen-tiation. p1 6 and p21 have been shown to enhance myogenicdifferentiation in transfection assays by 1 .5-3-fold (16),whereas in this work, we show that p27 is shown to enhance

myogenesis in a range from 5- to several hundred-fold. Also,in the C2C1 2 skeletal muscle cell line, withdrawal of seruminduces expression of p21 and p57 mRNA and stronglyup-regulates p27 protein levels (27-29).� Further evidence

that the G1 CDKs are important for muscle differentiationcomes from the observation that wild-type retinoblastomaprotein (pRb, a major downstream effector of CDI-sensitiveGi cyclin/CDK signaling) is needed to mediate a subset ofMyoD-initiated muscle differentiation functions in cell cul-

ture, although a second group of relatively early markers ofmuscle differentiation seem relatively unaffected by Rb func-tion (30).

In this work, we monitor p27 expression dunng mouseembryo myogenesis at the protein level because, as in sev-eral other tissues, it appears to be regulated at the posttran-

H D

B E

2 p27�1 and Myogenesis

p27

Myogenin

DAPI

scriptional level. We focus on the early skeletal myogenesis

in vivo that occurs in the somites of the mouse embryo. An

immune reagent directed against p27 demonstrated a sub-

stantial but transient wave of p27 expression that is corre-

lated with the timing of expansion of the MyoD-initiatedmyotomal subdomain (31). Comparison of p27 protein ex-pression with other CDIs expressed in myotomes suggests a

short-term role for p27 as cells are withdrawing from thecycle, whereas persistent expression of p21 and p27 in post-mitotic myocytes suggests a long-term rule for these CDls.Tests for the functional consequences of p27 expression

were performed and showed that p27 can greatly enhance

MyoD-initiated muscle differentiation.

Fig. 1. Immunocytochemicai localization ofp27 in the myotome at low magnification. Topis rostral; bottom is caudal; left is ventral; right

is dorsal. Parasagittal sections from El 0.5C57BL6 mouse embryos were coimmuno-stained with p27 (green) and myogenin (ted)and visualized by conventional fluorescent mi-croscopy. Nuclei were stained with 4;6-dia-midino-2-phenylindole (blue). A-C, x200;0-F, x400.

Results

p27 in the Developing Myotome. In several nonmuscle celltypes, it has been shown that p27 is regulated by posttran-

scriptional mechanisms (32-34). We therefore monitored thedistribution of p27 antigen to learn whether and where it is

expressed in the developing mouse muscle. The immune

reagent used was an affinity-purified rabbit polyclonal anti-

body raised against recombinant mouse p27 (see “Materialsand Methods”; Ref. 35.). This antibody reacted with only a

few cells of forelimb level myotomes at E9.5 (data not shown)

and with many more myotomal cells at E10.5 (Figs. 1 , 2A and

B, and 3). At these times, no other cells within the paraxial

mesoderm showed significant reactivity with this antibody. In

II MyoD p27 MyoD/p27

B a-RCTININ p27 cx A CT I N I N I p27

I,

ti.,,

,

‘5’

Cell Growth & Differentiation 3

Fig. 2. Immunocytochemical local-ization of p27 in the myotome byconfocal microscopy. Bars, 50 �m.Top is ventral; bottom is dorsal. In Aforelimb level transverse sectionsfrom El 0.5 C57BL6 mouse embryoswere coimmunostained for MyoD(red) and p27 (green) and visualizedwith a confocal microscope. In B,forelimb level transverse sectionsfrom El0.5 C57BL6 mouse embryoswere coimmunostained for a-actinin(red) and p27 (green). In C, forelimblevel transverse sections from El 0.5129 heterozygotic (+1-) and ho-mozygotic (-I-) p27 mouse em-bryos were coimmunostained formyogenin (red) and p27 (green).

C Myogenin/p27+1-

contrast to p21 and p57 RNAs, which are expressed in thedeveloping myotomes and also later in muscles derived from

them, virtually all myotomal reactivity for p27 disappeared by

El 1 .5 (Fig. 3). At El 1 .5, strong p27 reactivity in the nervous

system, including dorsal root ganglia adjacent to myotome-

derived muscle, was observed (Fig. 3). Preincubation of the

antibody with purified p27 protein eliminated immuno-

staining, as expected. Moreover, the myotomal staining can

be attributed specifically to p27 protein because in embryoshomozygous for a deletion of p27 (36), immunoreactivity was

absent (Fig. 2C).

-I--

We next asked how p27 expression is related to the onset

of MRF expression and to the execution of muscle differen-

tiation as the myotome develops. p27 immunostaining was

measured relative to the expression of MyoD (a determina-tion class MRF), myogenin (the major differentiation class

MRF), and a-actinin (a representative terminal differentiationgene). At El0.5, all p27-positive cells in the paraxial meso-

derm were also positive for the expression of MyoD. Be-

cause essentially no somitic cells were found at this time thatwere positive for p27 alone, we conclude that p27 protein

does not accumulate significantly until progenitor cells com-

Myotome

4 p27�’� and Myogenesis

El 05

El 1.5

Mgogenin p27 Mgogenin/p27

Fig. 3. Developmental expression pattem ofp27 in the myotome and dorsal root ganglia(DRG). Bats, 50 �m. Top is dorsal; bottom isventral. Forelimb level transverse sectionsfrom El0.5 and Ell.5 C57BL6 mouse em-bryos were coimmunostained for myogenin(red) and p27 (green) and visualized with aconfocal microscope.

mit to the myogenic pathway by expressing MyoD. This

result also argues that within paraxial mesoderm at this

stage, p27 expression is highly specific for myogenic cells,because only myogenic cells express MyoD. However someMyoD positive cells did not contain detectable p27 at thisstage (Fig. 2, A and B). In contrast to MyoD, only a subset ofp27-positive cells expressed myogenin (Fig. 3). And, as wastrue for MyoD, some myogenin-positive cells expressed no

detectable p27. The region of a-actinin expression in cyto-

plasmic domains defines the maturing myotome and showed

overlap with p27 expression, as expected from myogenincostaining (Fig. 2B). However, it is not possible in these

images to assign a specific nucleus to its cr-actinin-positive

or -negative cytoplasm; therefore, it is uncertain whether

individual cells coexpress a-actinin and p27, but the obser-

vation that p27 expression is myotomal is unequivocal.

These spatiotemporal patterns of coexpression and individ-ual expression do not have lineage tracing information. How-

ever, the data are consistent with a simple temporal progres-

sion in which MyoD expression precedes p27 accumulationwhich, in turn, precedes myogenin expression, followed bydifferentiation and p27 down-regulation (see “Discussion”).

p27 Enhances MyoD-initiated Myogenesis by MultipleCriteria. We next examined the functional effects of p27 on

myogenesis in C3H1OT1/2 cells, an embryonic fibroblast line,

by using transient transfection assays. Transient transfection

with MyoD or other MRF family regulators renders C3H1 01#{189}

cells myogenic, as measured by the activity of cotransfected

muscle differentiation reporter genes such as MCK-CAT orby the expression of endogenous muscle differentiation

genes such as MHC. C3H1 01#{189}cells were transfected with

a MyoD expression vector (pECE-MyoD), a myogenic re-porter composed of the promoter and enhancer regions of

MCK driving CAT gene (E1-117MCK-CA7) and varyingdoses of a pECE-p27 expression vector. In this expressionconstruct, p27 sequences began with the initiator AIG andincluded the entire protein coding sequence of murine p27(1 1). As shown in Fig. 4A, a very substantial enhancement(-200-fold) of MCK-CAT activity was observed under differ-

entiation conditions (culture for 72 h in 2% horse serum). Inother similar experiments, the plateau level of enhancementvaried from 5-fold to 800-fold, but within an experiment, thevariation between duplicate or triplicate transfection sampleswas very small. Myogenic enhancement was not limited tothe cotransfected reporter gene, because immunostaining oftransfected cells with antibodies specific for muscle MHCrevealed an increase in the number of MHC-positive cells in

the presence of added p27 (see below and Fig. 8). Thep27-mediated increase in myogenesis was not the result of asimple global increase of transfection efficiency by p27, norwas it due to an effect of p27 on the SV4O-driven MyoDexpression vector. Thus, the addition of p27 did not system-atically increase transfection efficiency of C3H1 01#{189}cells, asmeasured by expression from a CMV-CAT reporter, nor didit increase expression from a pECE-CAT vector. Similar p27-

mediated enhancement of myogenesis was observed whena cytomegalovirus-dnven MyoD expression vector wasused. Finally, p27 did not activate MCK-CAT in the absenceof added MyoD (Fig. 4B).

p27 Enhancement of MyoD-initiated Myogenesis IsSuppressed by Cyclin Dl . Because cyclin Dl/CDK4 andcyclin E/CDK2 complexes are inactivated by p27 in bio-chemical assays, we examined whether overexpression of

cyclin Dl was capable of suppressing the p27 enhancementof MyoD-initiated myogenesis. Transfection assays were

performed as described above with no added p27 and at aconcentration of p27 known to consistently enhance MyoD-initiated myogenesis. Superimposed on this second point,

transfections were performed with added pECE-cyclin Dlexpression vector. As shown in Fig. 5A, cyclin Dl negated

the effect of p27 at a dose demonstrated to suppress myo-

genesis (Fig. SB). Cyclin vectors or expression vector alone

did not activate MCK-CAT in the absence of added MyoD.p27 Mediates a Reduction in Cell Cycling. The assay

conditions for the experiment shown in Fig. 4 used a rela-tively mitogen-poor differentiation medium coupled with high

cell density. These conditions are widely used to enhance the

pace and extent of muscle differentiation. This raised the

300

200

100

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B.

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Fig. 4. p27-induced enhancement of myogenesis. In A, C3H1OT#{189}cellswere transfected as described in “Materials and Methods” with 0, 0.5, 1,2, or 4 �g of pECE-p27 expression plasmid, 1 �g of pECE-MyoD, and 1.7�.tg of MCK-CAT reporter. In B, C3H1OT#{189} cells were transfected asdescribed in “Materials and Methods” with 2 �g of pECE-p27 expressionplasmid or 1 � of pECE-MyoD and 1 .7 �g of MCK-CAT reporter. mA andB pECE expression vector lacking an insert was used to normalize for totalDNA precipitate in the transfection mixtures. Following transfection, cellswere allowed to recover for 1 2 h in growth media and then placed indifferentiation media for 72 h. Results were normalized to samples lackingpECE-p27 expression plasmid. Columns, the mean of a total of six 6-cmplates, two precipitates each divided into three plates; bars, SD.

question of how the growth conditions affect the response toadded p27. Mitogen-rich and -poor culture conditions were,therefore, compared directly at low cell densities. As shownin Fig. 6, surprisingly similar levels of enhancement in MCK-CAT reporter activity were observed under all culture condi-tions as indicated by the MyoD+p27:MyoD ratio alone. Note,however, that the absolute level of myogenic differentiation issignificantly higher under differentiation conditions, and thatthis level could not be attained by adding even the highestamounts of p27 under high-serum, low cell density condi-tions. What remained uncertain was whether the p27 effectscorrelated directly with cell cycle status.

Since studies in other experimental systems had shownthat either ectopic p27 or ectopic MyoD (21 , 37, 38) canarrest cells in G1 , we examined their effects on the cycling of10T#{189}cells used in a myogenicity experiment. Transfection

Cyclin Dl (ug DNA/precipitate)

Fig. 5. p27 enhancement of MyoD-initiated myogenesis is suppressedby cyclin Dl. In A, C3H1OT#{189} cells were transfected as described in“Materials and Methods” with 0, 0.5, or 2 j�g of pECE-cyclin Dl expres-sion plasmid, 1 �g of pECE-MyoD, 0 or 2 �g pECE-p27 expressionplasmid, and 1 .7 j�g of MCK-CAT reporter. In B, C3H1OT1/2 cells weretransfected as described in “Materials and Methods” with 0, 0.5, 1 , or 2 �gof pECE-cyclin Dl expression plasmid, 1 j�g of pECE-MyoD, and 1 .7 �gof MCK-CAT reporter. In A and B, pECE expression vector lacking aninsert was used to normalize for total DNA precipitate in the transfectionmixtures. Following transfection, cells were allowed to recover for 1 2 h ingrowth media and then placed in differentiation media for 72 h. Resultswere normalized to samples lacking pECE-p27 and pECE-cyclin Dl ex-pression plasmid. Columns, the mean of a total of six 6-cm plates: twoprecipitates each divided into three plates; bars, SD.

assays were performed as described above, and cells wereeither maintained in growth medium or switched to differen-tiation medium, which is expected to favor cell cycle arrest inG1/G0. Thirty-eight h after DNA precipitate was removed,cells were incubated for an additional 10 h with BrdUrd toassess their proliferative status. Transfected cells, irrespec-tive of cycling or differentiation status, were identified byimmunostaining for the membrane-bound CD8 cotrans-fected marker gene. CD8-positive cells were counted andassessed for BrdUrd incorporation to determine the S-phaselabeling index. As shown in Fig. 7A, MyoD alone only slightlyreduced the percentage of transfected BrdUrd-positive cells

40

30

20

10

DM DM GMhigh density low density low density

.- 0

Q�

0

0,�#{149} i�atio 5.5 8.9 7.9

Fig. 6. Effect of growth conditions on p27-induced enhancement ofmyogenesis. C3H1 0T1/2 cells were transfected as described in “Materialsand Methods” with 1 j�g of pECE-MyoD, 1 .7 �g MCK-CAT reporter, andeither 2 �g pECE-p27 expression plasmid or 2 �g pECE expression vectorlacking an insert. For examining effects of growth conditions, cells weretrypsinized and either replated on a 6-cm plate (high density) in thepresence of differentiation medium (DM) or diluted 1 :6 and plated on a10-cm plate (low density) in the presence of differentiation medium orgrowth medium (GM) and incubated for 72 h. Results were normalized tosamples lacking pECE-p27 expression plasmid in differentiation mediumplated at high density. The magnitude of enhancement in each growthcondition is stated as a MyoD+p27:MyoD ratio. Columns, the mean of atotal of six 6-cm plates: two precipitates each divided into three plates;bars, SD.

0

U differentiationmedium

�I growthmedium

U differentiationmedium

� growthmedium

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Fig. 7. p27-mediated cell cycle withdrawal. C3H1 0T1/2 cells were trans-fected as described in “Materials and Methods” with 1 .7 �g of pECE-CD8,1 .7 �g MCK-CAT reporter, and either 1 �g of pECE-MyoD, 2 �tg pECE-p27, or 1 �ig of pECE-MyoD plus 2 �tg pECE-p27, or 1 �g of pECE-MyoDplus 2 �g pECE-p27 or 3 �g pECE expression vector lacking an insert.pECE expression vector lacking an insert was used to normalize for totalDNA precipitate in the transfection mixtures. Two sets of plates weremaintained in growth medium, and two sets of plates were switched todifferentiation medium. Prior to harvest at 48 h after the removal of DNA,cells were incubated for 1 0 h in the presence of 1 0 �M BrdUrd. Cells wereeither fixed and processed for immunostaining as descnbed in “Materialsand Methods,” or cells were harvested for CAT assays. A, percentage ofcells incorporating Brdtird. Approximately 200 CD8-positive cells percondition were counted. a and b are statistical different as determined byFisher’s exact test. B, CAT activities, normalized to samples lackingpECE-p27 in differentiation media.

6 p27�Pl and Myogenesis

#{149}MyoD

0 MyoD + p27

in both culture conditions. This difference was not statisti-

cally significant (Fisher’s exact test). In contrast, MyoD and

p27 together generated a large, statistically significant re-

duction in S-phase labeling. In this experiment, replicate

plates were assayed for myogenic enhancement by p27, and

the result was a 5-fold increase in MCK-CAT (Fig. 7B), ver-

ifying that the cell cycle measurements had been made in the

context of a “typical” p27 enhancement of myogenesis. Thecorrelation between added p27, reduced S-phase index, and

elevated MCK-CAT levels were evident in both high and low

serum conditions. This is consistent with a mechanism in

which much of the p27 effect on MCK-CAT is correlated

directly with inhibition of S-phase. The mitogen-rich charac-

ter of growth medium also suppressed MCK-CAT, but it did

so in a manner surprisingly independent of S-phase effects,

at least at this time point. This suggests that much of the

effect of high serum on MCK-CAT expression is likely to be

independent of S-phase status and may, therefore, be me-

diated through other pathways.

p27 Increases the Number of Differentiated Cells. We

next investigated the effect of elevated p27 on the number of

cells scoring positive for MHC, an endogenous marker of

muscle differentiation. When p27 was included with MyoD,

we observed a 3.5-fold increase in the number of strongly

MHC-positive cells coupled with a -1 0-fold increase in myo-

genic activity as measured by MCK-CAT (Fig. 8). On average,

individual cells transfected with both MyoD and p27 stained

more intensely for MHC than did cells transfected with MyoD

alone. It therefore appears that the overall increase in MCK-

CAT is mimicked by endogenous MHC, and that the increase

derives from more cells differentiating coupled with more

CD8 CD8 CDSMyoD MyoD

p27

�LCD8 CD8 CD8

MyoD MyoDp27

efficient accumulation of differentiation products. We also

observed that the immunostaining for MyoD, which detects

both endogenous and exogenous MyoD expression, was

more intense in cells cotransfected with p27 than in cells

transfected with MyoD alone. Although the intensity was not

quantitated, it is plausible that elevated p27 enhances myo-

genicity by increasing MyoD stability either directly or as a

secondary consequence of G1/G0 arrest.

DiscussionIn skeletal muscle development where the linkage of terminal

differentiation with permanent G1/G0 arrest is prominent, we

found that the p27 CDK inhibitor is expressed transiently in

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�D MyoD+p27 0

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0 �1�Fig. 8. p27-mediated increase in the number of myogenic cells.C3H1 0T#{189}cells were transfected as described in “Materials and Methods”with 0 or 2 p.9 of pECE-p27 expression plasmid, 1 �g of pECE-MyoD, and1 .7 �g of MCK-CAT reporter. pECE expression vector lacking an insertwas used to normalize for total DNA precipitate in the transfection mix-tures. Following transfection, cells were allowed to recover for 12 h ingrowth media and then placed in differentiation media for 72 h. Resultswere normalized to samples lacking pECE-p27 expression plasmid. Cellswere either fixed and processed for immunostaining as described in“Materials and Methods” (one plate/precipitate), or cells were harvestedfor CAT assays (two plates/precipitate). A: columns, the mean of a total offour 6-cm plates: two precipitates each divided into two plates; bare, SD.B: columns, the mean total of 15 low magnification fields (xlOO)from twoprecipitates.


6 X#{149}Yu and B. J. Wold, unpublished data.

the early developing axial musculature of the embryo. Theearly axial musculature of the vertebrate embryo differenti-ates in the myotomal compartment of the somites that ma-ture in a rostrocaudal gradient. The emerging myotome isdetectable around E9.0 in forelimb level somites as a post-mitotic domain by the criterion of S-phase DNA labeling. Thisdomain also expresses members of the MRF family of myo-genic transcription factors and the p27-related CDls, p21and p57 (29, 39).6 The myotomal compartment expandsgreatly in the following days of development by the additionof postmitotic MRF-positive cells (40). This general picture ofcell cycle withdrawal and CDI RNA expression in the myo-tome has lacked the resolution needed to clarify how expres-sion of CDls of the p21/p27/p57 family are integrated withexpression and action of the myogenic regulators in theembryo. Here we monitored the accumulation and disap-pearance of p27 antigen by double immunostaining to definemore precisely its expression pattern relative to myogeninand MyoD.

Gene disruption studies indicate that myogenin function isrequired for most myocytes to execute muscle differentiationin vivo, and myogenin expression is up-regulated before theonset of muscle differentiation. In contrast, MyoD (together

with myf5) appears to play a prominent role in specifyingmuscle cell fate, as deduced from expression pattern andgene disruption studies (19, 20). MyoD is characteristicallyexpressed in proliferating myoblasts in cell culture and is firstexpressed in a subdomain of the myotome, beginningaround E10.25 in forelimb level somites (40, 41).

In this study, we found that in the E9.5 myotome, somecells coexpress myogenin and p27, while other cells arepositive for either antigen alone. We do not know whether allcells expressing p27 at this early time also express myf5protein, which is the earliest determination class MRF be-cause myf5 antibodies available to us were not entirely myf5specific. However, the p27-positive cells were located within

the postmitotic early myotomal domain, which is positive formyf5 RNA by in situ hybridization. We also know from genetic

arguments that myf5 function is essential for the myogenicspecification of these earliest muscle cells. We thereforethink it is very likely that all p27-positive cells in the emergingmyotome are also myf5 positive. By day El 0.5 when the

MyoD dependent compartment of the myotome has becomeprominent, we found that virtually all cells expressing detect-able p27 also expressed MyoD. Cells that were p27�/myo-genin, together with p2T’imyogenin� cells, were againobserved, just as they had been at day E9.5. Either of two

scenarios can explain the observed expression patterns: (a)p27 may be expressed in some specialized subset of myo-tomal cells that is not correlated with the temporal progres-sion from myoblast to differentiating myocyte as reflected in

MRF expression patterns; or (b) the distinctions in costainingpatterns might reflect simply the state of maturation of mdi-vidual cells within the expanding myotome. In the latter sce-nano, p27 expression during the myoblast to myocyte tran-sition is transient for an individual cell; within the MyoD-initiated somitic domain (20, 31 , 41), p27 protein expression

would begin after the onset of MyoD and before myogenin,continue through early myogenin expression, and then bedown-regulated after cell cycle withdrawal is complete andterminal differentiation has begun. We presently favor thelatter model, which is shown in Fig. 9. It would permit p27 toserve cell cycle arrest and/or differentiation permissivelyfunctions in vivo that are consistent with the effects observedin cell culture experiments. In this view, cells coexpressingp27 and myogenin would be among the newest myogenin-expressing cells in the compartment and would subse-quently convert to p27-negative status. Cells in the myo-tomal domain express p21 and p57 robustly, by the criterionof in situ hybridization (29, 39)5 and it seems likely that theyare also robustly expressed at the protein level, by analogywith their expression in cell culture (42). In any model, thelong-term down-regulation of p27 by day 1 1 .5 suggests thatp27 is not involved in maintaining the postmitotic state butcould play a role in establishing or coordinating it with theonset of differentiation.

The possible functional significance of p27 expressionobserved in vivo was examined in transfection studies incultured cells. Enforced p27 expression in C31 0T#{189}fibro-blasts did not, by itself, activate the expression of endoge-nous or cotransfected muscle differentiation genes. How-ever, added p27 vigorously enhanced expression of

c�9�Early Myoblast

MyoDMyogeninp18p21p27

Myocyte

8 p27(�P1 and Myogenesis

7 S. Wang and B. J. Wold, unpublished data.

MyoD

MyoD

p27

“Primed” Myoblast Early Myocyte

MyoDMyogeninp18p21

PS7

Fig. 9. A developmental model for p27 expression dunng axial myogenesis in vivo. Early myoblast expresses MyoD. Following an environmental signal,myoblast expresses both MyoD and p27. Expression of p27 initiates or “primes” the myoblast for cell cycle withdrawal. Early myocyte exits the cell cycleand expresses MyoD, myogenin, p27, and p21 . Myocytes continue to differentiate and express MRFs, downstream marker genes (a-actinin, MHC, andothers) and p21 , p57, and other CDI family members, while p27 is down-regulated.

differentiation reporter genes when cotransfected withMyoD. A previous study showed that p21 , alone and inconjunction with p1 6, increased myogenicity in C2C1 2 cellsmaintained in growth media by 0.5- and 4-fold, respectively(16). In our assay system, p21 enhancement ranged from 2-to 200-fold and showed a dose-response curve similar tothat for p27 (data not shown). These results, along with in situ

hybridization studies that showed p21 and p57 RNAS arestrongly up-regulated in the myotome (29, 39),6 suggest thatmultiple CDls are involved in skeletal myogenesis. Genedisruption studies in which either p1 6 (43),7 p27 (36, 44, 45),or p21 (46) have been knocked out in mice showed that noneof these CDls are individually essential for skeletal muscle

development, and this may reflect redundancy of function.Although it may be that one or more other CDls, such as p18,will be essential for skeletal myogenesis, a more likely pos-sibility is that there is substantial redundancy of CDI functionin myogenesis.

The data we have for p27 protein are consistent with atemporal cascade proposed in the model in Fig. 9 in whichinitial CDI action (postulated to be by p27), is governed byone set of environmental signals that target a specific subsetof CDKs, and that subsequent signals transfer CDK inhibition

to either single or multiple CDIs (p1 8, p21 , and/or p57) formaintenance following p27 down-regulation. If this simplemodel is correct, it predicts that any muscle phenotypes inp27 knock-outs would be subtle, involving a change in thetiming of cell cycle exit, in the onset of differentiation, or inthe level of MRF needed to initiate differentiation. And be-cause the myotome is a continuously growing domain, wewould also predict that at any given time, only a subset of allmyotomal cells would be affected because many will haveprogressed to p27-independent status. We also note thatalthough our data support a model in which many (perhapsall) early myotomal cells express p27 protein (before El 0 atthe forelimb level), we also found that at later times after dayEl 1 , p27 was no longer detected, but new myogenic pre-cursor cells continue to enter the myotomal muscle masses.For these later stages, other CDIs presumably initiate cell

cycle withdrawal in cells that do not express p27 or, alter-natively, the proposed early function for p27 is simply notneeded at later times in myogenesis. It remains possible thatp27 at these later times has simply changed its epitopepresentation, but this seems relatively unlikely because thispolyclonal reagent probably sees more than one epitope.

The proposed temporal cascade of CDI action is alsoconsistent with data from tissue culture models. In the

C2C1 2 myogenic cell line, we observed a large and rapidinduction in p27 upon initiation of differentiation (in less than

12 h), followed by a slower further accumulation in p27 by

36 h.5 In contrast, p1 8 levels increase dramatically in C2C1 2cells but do so over a prolonged differentiation time course

starting at approximately 24 h, as does its association withCDK4 and CDK6 (42). This would seem most consistent witha role for p1 8 in maintaining the differentiated state.

A new although not entirely coherent set of clues concern-ing implied roles for G1-S CDKs and their CDIs is coming

from studies of pRb (2, 47-51). It has recently been reportedthat the inhibitory effects of some G1-S cyclins (E and A) onmuscle differentiation can be bypassed by a dominant-act-

ing nonphosphorylatable pRb mutation, but that the inhibi-tory effects of cyclin Dl could not be similarly bypassed (47).Because the dominant-acting Rb could not bypass the cyclinDl inhibition, the authors suggested that cyclin Dl functionsin an Rb-independent pathway (47). In contrast, anotherstudy demonstrated that cyclin Dl and A suppression can bebypassed by a hyperactive Rb, indicating that the CDKs

function to inhibit myogenesis through an Rb-dependent

pathway (52). In our study, p27-mediated enhanced myo-genesis was suppressed by cyclin Dl , which is consistentwith p27 functioning in the same pathway as cyclin Dl . Butbecause it is controversial whether cyclin Dl suppression ismediated through Rb, it remains to be demonstrated whether

p27 functions through an Rb-dependent and/or independent

pathway. Finally, a third study indicated that the negativeeffect of a Rb knock-out on myoblast differentiation is

strongest for cell cycle withdrawal and MHC accumulationand not for “early” events such as increases in myogenin andp21 (30). Because embryonic and C2C1 2 expression pat-terns of p27 suggest it as an early event, our observations


8 A. Wagner and B. J. Woid, unpublished data.

seem more consistent with p27 enhancement acting in theRb-independent pathway, as proposed by Lassar at a!. (2).

The biochemical mechanism for CDI action during muscledifferentiation is not yet clear. MRFs have been shown to bemultiply phosphorylated in vivo (53-55), and the presence ofmultiple possible CDK target sites in MRFs suggest that theymight be direct substrates for the CDKs. Although there is nodirect evidence for this, such phosphorylations could ac-count for CDK suppression of muscle differentiation. Thesephosphorylations may regulate MRF DNA binding and/or

transactivating activity and/or protein turnover rates. Our

observations of increased staining intensity of MyoD in the

presence of added p27, compared with no added p27, isconsistent with an increase in MyoD stability. Although notrigorously quantitative, this observation Is consistent with the

recent report that elevated cyclin Dl levels leads to an

ubiquitin-dependent reduction in MyoD half-life (56). Thus,p27 (and presumably other CDls) could promote the action ofMyoD or other MRFs by inhibiting their turnover through aCDK-dependent degradation pathway.

At present, it is at least equally possible that MRF coregu-lators such as the E proteins, MEF2, or CBP/p300 familymembers are targets of the CDKs because all are phospho-proteins. Interestingly, p300 is a known to be a target ofCDKs (57). Of course, these mechanisms and targets are notmutually exclusive, and the overall large effect of the CDK/CDI system on muscle differentiation may come from theaction of more than one biochemical pathway on more thanone target.

Materials and MethodsPlasmlds. The full-length p27 cDNA plasmld pET2la-p27 was kindlyprovided by J. Massagu#{233}.For expression studies, the pET2la-p27 plas-mid was used as atemplate In a PCR reaction to ampllfythe coding region

of p27 and was cloned Into Kpn and Xba sItes of the pECE eukaryotlcexpression vector, which contains the SV4O enhancer/promoter and 3’RNA processing sites (58). The MyoD cDNA and CD8 cDNA were clonedInto the pECE expressIon vector. The reporter plasmid used for expres-sion studies was El-i 17MCK-CAT (59).

lmmunohlstochomlst,y. C57BL6 mouse embryos were harvestedfrom timed pregnancies in sterile PBS, and semite counts were noted.Embryos were promptlyflxed in freshly prepared 4% paraformaldehyde inPBS for 4-12 h at 4#{176}C.FollowIng fixation, embryos were washed brieflywith PBS, immersed in 12.5 and 25% sucrose in PBS sequentially overseveral hours, and frozen at -70#{176}Cin OCT. SerIal i0-�&m sections werecut on a cryostat at -17#{176}C,collected ontoVectabond-coated slides, drIedat room temperature for 30 mm, and stored at -20#{176}C.

Prlorto antibody binding, slldeawereequilibrated to room temperature,and OCT was carefully peeled from around sections. Sections were en-circled with a hydrophobic barrIer, dried, and fixed to the slide in 4%paraformaldehyde/PBS for5 mm. Slides werethen washed twice for 1 mmin PBS, once for 5 mm in I % NP4O/PBS, and twice for 1 mln in PBS.

Sections were preblocked for 20 mm with a small amount of 10% goat

serum/3% BSA/PBS in a humid chamber. Preblock was drained, and asmall amount of primary antibody dIluted in 3% BSAJPBS was applied tothe section for 1 h at room temperature in a humid chamber. SlIdes werethen washed with PBS, 1% NP4O/PBS, and PBS as above, and secondaryantibody was applied for 30 mm at room temperature, followed by twoPBS washes of 2 mm each. Excess fluid was removed, and SlowFade(Molecular Probes, Inc.) mounting solution and a coverslip were applied.Fluorescence from labeled secondary reagents was then visualized byfluorescence or confocal laser scanning mIcroscopy.

Immune reagents were: (a) primary antibodies: p27 affinity-purifiedrabbit polyclonal antiserum raised against recombinant mouse p27, 1:50dilution, kindly provided by Dr. J. Roberts (35); F5D mouse lgG1 mono-

clonal against mouse myogenmn, 1 :5 dilution, kind gift of W. E. Wright;NCL-MyoDi mouse IgG1 against MyoDi , 1 :20 dIlution, Novocastra Labs,Ltd.; striated muscle specific a-actlnin mouse lgG1 monoclonal, 1:400dilution, Sigma Chemical Co.; and (b) secondary antibodies: goat anti-

mouse IgG1 FITC and TRITC conjugated, 1 :100 dilution, Southern Bio-technology Associates; goat anti-rabbit IgG FITC and TRITC conjugated,1 :160 dIlution, Sigma. It should be noted that we also investigated immu-nostaining using the anti-p27 antibody C19 from Santa Cruz and found

strong staining of myotomes that was not transient; this signal was corn-peted well by prelncubation with the immunizing peptide7 but remained

strong in p27 null embryos.8 The latter result probably reflects cross-reaction of the anti-C-19 reagent with p57 (p57 cross-reaction by thisantibody was also observed in an immunoblot experimental format5) orwith as yet unidentified cross-reacting antigens wIthin the myotome.

For BrdUrd incorporation experiments, cells were fixed In 4%parafornialdehydefor 10 mm, preincubated in 10% goat serum In PBS for30 mm, incubated with I :20 dilutIon anti-human CD8 mouse monoclonal(Sigma) for 2 h, 1 :500 dilution biotinylated anti-mouse (Vector Laborato-ries)for 1 h, and I :1000 dilution of avidln FITC (Vector Laboratories). Cellswere then refixed with 4% paraformaldehyde, acid treated with 2 M HC1in PBS, permeabilized with I % Triton-X for 10 mm, incubated with anti-ratBrdUrd (Accurate Chemical) for 2 h, and 1 :100 TRITC labeled anti-rat IgG

for 1 h (Jackson Laboratories). In between all steps, cells were washed

extensively with PBS. All antibodies were diluted in 3% BSAJPBS.Transfectlons. Transfections were performed as described by Neu-

hold and Wold (1993) with the following modifications. The day beforetransfection, approximately 1.5 x l0� C3H1OT#{189} cells were passagedonto a 6-cm plate. Calcium phosphate precipitates were generated con-taming equal total concentrations of DNA and incubated with the cells In

DMEM plus 15% fetal bovine serum (growth medium) with 30 �i�i chlo-roquinefor 8 h, followed by a 12-h incubation in fresh growth medium. ForCAT assays, cells were then washed with PBS and incubated for 72 h inDMEM plus 2% horse serum (dIfferentiation medium) or left in growthmedium for 72 h. Cells were harvested by trypsinization 96 h after trans-fection, and the pellet was suspended in 0.25 M Tris (pH 7.5). Afterfreeze-thawing, the supematant was assayed for protein content using

bicinchoninic acid detection reagent (Pierce, Rockford, Illinois) against analbumin standard curve. Fifteen �g of protein were then assayed for CATactivity byTLC (60). lfnecessary, the assay was repeated to achieve linearrange, i.e., unacetylated sample about 80% of total radiolabeled chlor-amphenicol. TLC plates were exposed and density analyzed on a Phos-phorimager, using ImageQuant software (Molecular Dynamics, Sunny-vale, CA). For BrdUrd Incorporation experiments, cells were rinsed withPBS and incubated for26 h in DMEM plus 2% horse serum (differentiationmedium)orleftin growth medium for26 h. Cells were then labeled for 10 hin the presence of 10 gsM BrdUrd.

AcknowledgmentsWe especially thank Dr. Jim Roberts for the p27 antibody.

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