Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix. Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including SMalphaactin, SM22alpha, calponin, SMgammaactin or SM-myosin heavy chain detectable by RT-PCR or by immunostaining. To identify factors required for coronary smooth muscle differentiation, we excised proepicardial cells from Hamburger-Hamilton stage-17 quail embryos and examined them ex vivo. Proepicardial cells initially formed an epithelial colony that was uniformly positive for cytokeratin, an epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers were undetectable, indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours, cytokeratin-positive cells became SMalphaactin-positive. Moreover, serum response factor, undetectable in freshly isolated proepicardial cells, became strongly expressed in virtually all epicardial cells. By 72 hours, a subset of epicardial cells exhibited a rearrangement of cytoskeletal actin, focal adhesion formation and acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22alpha and SMgammaactin became expressed. By 5–10 days, SM-myosin heavy chain mRNA was found, by which time nearly all cells had become mesenchymal. RT-PCR showed that large increases in serum response factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative serum response factor constructs prevented the appearance of calponin-, SM22alpha- and SMgammaactin-positive cells. By contrast, dominant-negative serum response factor did not block mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires transcriptionally active serum response factor.

Reference

Arsenian
S.
,
Weinhold
B.
,
Oelgeschlager
M.
,
Ruther
U.
,
Nordheim
A.
(
1998
)
Serum response factor is essential for mesoderm formation during mouse embryogenesis.
EMBO J
17
,
6289
6299
Belaguli
N.
,
Schildmeyer
L.
,
Schwartz
R.
(
1997
)
Organization and myogenic restricted expression of the murine serum response factor gene: a role for autoregulation.
J. Biol. Chem
272
,
18222
18231
Blank
R.
,
McQuinn
T.
,
Yin
K.
,
Thompson
M.
,
Takeyasu
K.
,
Schwartz
R.
,
Owens
G.
(
1992
).
Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle.Evidence for cell type-specific regulation.
J. Biol. Chem
267
,
984
989
Bogers
A.
,
Gittenberger-de Groot
A.
,
Poelmann
R.
,
Peault
B.
,
Huysmans
H.
(
1989
)
Development of the origin of the coronary arteries, a matter of ingrowth or outgrowth?.
Anat. Embryol
180
,
437
441
Browning
C.
,
Culberson
D.
,
Aragon
I.
,
Fillmore
R.
,
Croissant
J.
,
Schwartz
R.
,
Zimmer
W.
(
1998
)
The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes.
Dev. Biol
194
,
18
37
Carroll
S.
,
Bergsma
D.
,
Schwartz
R.
(
1986
)
Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs.
J. Biol. Chem
261
,
8965
8976
Chen
C.
,
Schwartz
R.
(
1996
).
Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.
Mol. Cell. Biol
16
,
6372
6384
Chomczynski
P.
,
Sacchi
N.
(
1987
)
Single-step method of RNA isolation by acid quanidinium thiocyanate-phenol-cholroform extraction.
Anal. Biochem
162
,
156
159
Croissant
J.
,
Kim
J.
,
Eichele
G.
,
Goering
L.
,
Lough
J.
,
Prywes
R.
,
Schwartz
R.
(
1996
)
Avian serum response factor expression restricted primarily to muscle cell lineages is required for alpha-actin gene transcription.
Dev. Biol
177
,
250
264
Dettman
R.
,
Denetclaw
W.
,
Ordahl
C.
,
Bristow
J.
(
1998
)
Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts and intermyocardial fibroblasts in the avian heart.
Dev. Biol
193
,
169
181
Duband
J.
,
Gimona
M.
,
Scatena
M.
,
Sartore
S.
,
Small
J.
(
1993
)
Calponin and SM 22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development.
Differentiation
55
,
1
11
Gittenberger-de Groot
A.
,
Vrancken Peeters
M.
,
Mentink
M.
,
Gourdie
R.
,
Poelmann
R.
(
1998
)
Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.
Circ. Res
82
,
1043
1052
Grueneberg
D.
,
Natesan
S.
,
Alexandre
C.
,
Gilman
M.
(
1992
)
Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor.
Science
257
,
1089
1095
Hamburger
V.
,
Hamilton
H.
(
1951
)
A series of normal stages in the development of the chick embryo.
J. Morphol
88
,
49
92
Herring
B.
,
Smith
A.
(
1996
)
Telokin expression is mediated by a smooth muscle cell-specific promoter.
Am. J. Physiol
270
,
1656
1665
Hill
C.
,
Wynne
J.
,
Treisman
R.
(
1994
)
Serum-regulated transcription by serum response factor (SRF): a novel role for the DNA binding domain.
EMBO J
13
,
5421
5432
Himura
T.
,
Hirakow
R.
(
1989
)
Epicardial formation in embryonic chick heart.
Am. J. Anat
184
,
129
138
Hood
L.
,
Rosenquist
T.
(
1992
)
Coronary artery development in the chick: Origin and deployment of smooth muscle cells and the effects of neural crest ablation.
Anat. Rec
234
,
291
300
Hungerford
J.
,
Owens
G.
,
Aargraves
W.
,
Little
C.
(
1996
)
Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular markers.
Dev. Biol
178
,
375
392
Johansen
F.
,
Prywes
R.
(
1993
)
Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.
Mol. Cell. Biol
13
,
4640
4647
Johansen
F.
,
Prywes
R.
(
1995
)
Serum response factor: transcriptional regulation of genes induced by growth factors and differentiation.
Biochim. Biophys. Acta
1242
,
1
10
Kalman
F.
,
Viragh
S.
,
Modis
L.
(
1995
)
Cell surface glycoconjugates and the extracellular matrix of the developing mouse embryo epicardium.
Anat. Embryol. (Berl.)
191
,
451
464
Kim
S.
,
Ip
H.
,
Lu
M.
,
Clendenin
C.
,
Parmacek
M.
(
1997
)
A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages.
Mol. Cell. Biol
17
,
2266
2278
Kwee
L.
,
Baldwin
H.
,
Shen
H.
,
Stewart
C.
,
Buck
C.
,
Buck
C.
,
Labow
M.
(
1995
)
Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.
Development
121
,
489
503
LeLievre
C.
,
Le Douarin
N.
(
1975
)
Mesenchymal derivatives of the neural crest: Analysis of chimeric quail and chick embryos.
J. Embryol. Exp. Morphol
34
,
125
154
Li
L.
,
Liu
Z.
,
Mercer
B.
,
Overbeek
P.
,
Olson
E.
(
1997
)
Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal and cardiac muscle cells.
Dev. Biol
187
,
311
321
Lilly
B.
,
Zhao
B.
,
Ranganayakulu
G.
,
Paterson
B.
,
Schulz
R.
,
Olson
E.
(
1995
)
Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila.
Science
267
,
688
693
Madsen
C.
,
Regan
C.
,
Owens
G.
(
1997
)
Interaction of CArG elements and a GC-repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle.
J. Biol. Chem
272
,
29842
29851
Manasek
F.
(
1971
)
The ultrastructure of embryonic myocardial blood vessels.
Dev. Biol
26
,
42
54
Manner
J.
(
1992
)
The development of pericardial villi in the chick embryo.
Anat. Embryol
186
,
379
385
McNamara
C.
,
Thompson
M.
,
Vernon
S.
,
Shimizu
R.
,
Blank
R.
,
Owens
G.
(
1995
)
Nuclear proteins bind a cis-acting element in the smooth muscle alpha-actin promoter.
Am. J. Physiol
268
,
1259
1266
Mikawa
T.
,
Gourdie
R.
(
1996
)
Pericardial mesoderm generates apopulation of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ.
Dev. Biol
174
,
221
232
Moessler
H.
,
Mericskay
M.
,
Li
Z.
,
Nagl
S.
,
Paulin
D.
,
Small
J.
(
1996
)
The SM-22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice.
Development
122
,
2415
2425
Obata
H.
,
Hayashi
K.
,
Nishida
W.
,
Momiyama
T.
,
Uchida
A.
,
Ochi
T.
,
Sobue
K.
(
1997
)
Smooth muscle cell phenotype-dependent transcriptional regulation of the1-integrin gene.
J. Biol. Chem
272
,
26643
26651
Owens
G.
(
1995
)
Regulation of differentiation of vascular smooth muscle cells.
Physiol. Rev
75
,
487
517
Pardanaud
L.
,
Altmann
C.
,
Kitos
P.
,
Dieterlen-Lievre
F.
,
Buck
C.
(
1987
)
Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells.
Development
100
,
339
349
Pellegrini
L.
,
Tan
S.
,
Richmond
T.
(
1995
)
Structure of serum response factor core bound to DNA.
Nature
376
,
490
498
Poelman
R.
,
Gittenberger-de Groot
A.
,
Mentink
M.
,
Bokenkamp
R.
,
Hogers
B.
(
1993
)
Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.
Circ. Res
73
,
559
568
Schultheiss
T.
,
Xydas
S.
,
Lassar
A.
(
1995
)
Induction of avian cardiac myogenesis by anterior endoderm.
Development
121
,
4203
4214
Shimizu
R.
,
Blank
R.
,
Jervis
R.
,
Lawrenz-Smith
S.
,
Owens
G.
(
1995
)
The smooth muscle alpha-actin gene promoter is differentially regulated in smooth muscle versus non-smooth muscle cells.
J. Biol. Chem
270
,
7631
7643
Shore
P.
,
Sharrocks
A.
(
1994
)
The transcription factors Elk-1 and serum response factor interact by direct protein-protein contacts mediated by a short region of Elk-1.
Mol. Cell. Biol
14
,
3283
3291
Soulez
M.
,
Rouviere
C.
,
Chafey
P.
,
Hentzen
D.
,
Vandromme
M.
,
Lautredou
N.
,
Lamb
N.
,
Kahn
A.
,
Tuil
D.
(
1996
)
Growth and differentiation of C2 myogenic cells are dependent on serum response factor.
Mol. Cell. Biol
16
,
6065
6074
Szucsik
J.
,
Lessard
J.
(
1995
)
Cloning and sequence analysis of the mouse smooth muscle gamma-enteric actin gene.
Genomics
28
,
154
162
Triesman
R.
(
1994
)
Ternary complex factors: growth factor regulated transcriptional activators.
Curr. Opin. Genet. Dev
4
,
96
101
Vandromme
M.
,
Gauthier-Rouviere
C.
,
Carnac
G.
,
Lamb
N.
,
Fernandez
A.
(
1992
)
Serum response factor p67SRF is expressed and required during myogenic differentiation of both mouse C2 and rat L6 muscle cell lines.
J. Cell Biol
118
,
1489
500
Vrancken Peeters
M.
,
Gittenberger-de
G. A.
,
Mentink
M.
,
Hungerford
J.
,
Little
C.
,
Poelmann
R.
(
1997
)
The development of the coronary vessels and their differentiation into arteries and veins in the embryonic quail heart.
Dev. Dyn
208
,
338
348
Waldo
K.
,
Willner
W.
,
Kirby
M.
(
1990
)
Origin of the proximal coronary artery stems and a review of ventricular vascularization in the chick embryo.
Am. J. Anat
188
,
109
120
Yang
J.
,
Rayburn
H.
,
Hynes
R.
(
1995
)
Cell adhesion events mediated by4 integrins are essential in placental and cardiac development.
Development
121
,
549
560
Yano
H.
,
Hayashi
K.
,
Momiyama
T.
,
Saga
H.
,
Haruna
M.
,
Sobue
K.
(
1995
)
Transcriptional regulation of the chicken caldesmon gene: activation of gizzard-type caldesmon promoter requires a CArG box-like motif.
J. Biol. Chem
270
,
23661
23666
Zilberman
A.
,
Dave
V.
,
Miano
J.
,
Olson
E.
,
Periasamy
M.
(
1998
)
Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression.
Circ. Res
82
,
566
575
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