Download Expedited Publication Marks the Smooth Muscle Lineage

803
Expedited Publication
Smooth Muscle Myosin Heavy Chain Exclusively
Marks the Smooth Muscle Lineage
During Mouse Embryogenesis
Joseph M. Miano, Peter Cserjesi, Keith L. Ligon, Muthu Periasamy, Eric N. Olson
Abstract We cloned a portion of the mouse smooth muscle
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myosin heavy chain (SM-MHC) cDNA and analyzed its
mRNA expression in adult tissues, several cell lines, and
developing mouse embryos to determine the suitability of the
SM-MHC promoter as a tool for identifying smooth musclespecific transcription factors and to define the spatial and
temporal pattern of smooth muscle differentiation during
mouse development. RNase protection assays showed SMMHC mRNA in adult aorta, intestine, lung, stomach, and
uterus, with little or no signal in brain, heart, kidney, liver,
skeletal muscle, spleen, and testes. From an analysis of 14
different cell lines, including endothelial cells, fibroblasts, and
rhabdomyosarcomas, we failed to detect any SM-MHC
mRNA; all of the cell lines induced to differentiate also
showed no detectable SM-MHC. In situ hybridization of
staged mouse embryos first revealed SM-MHC transcripts in
the early developing aorta at 10.5 days post coitum (dpc). No
Studies in skeletal and cardiac muscle cells have
identified several transcription factors that play
a crucial role in the formation of these cell
types.12 Comparable factors have yet to be uncovered in
smooth muscle cells (SMCs), whose developmental molecular biology is poorly understood. The inability thus
far to clone SMC-specific transcription factors may
relate to several fundamental differences existing between this cell type and skeletal and cardiac muscle.
First, the mechanics of SMC contraction and its regulation are distinct from those of sarcomeric muscle.3
Second, SMCs display remarkable plasticity in phenotype, particularly in blood vessels, where such phenotypic modulation is considered a sine qua non for
vascular disease.45 Finally, unlike the skeletal and cardiac muscle lineages, whose embryonic origins have
been localized to progenitors in discrete regions of the
embryo, SMCs develop from multiple, rather ill-defined, areas throughout the embryo. For example, studies performed in chick-quail chimeras show a proportion of SMCs in the great vessels emanating from a
Received July 1, 1994; accepted August 10, 1994.
From the Department of Biochemistry and Molecular Biology
(J.M.M., P.C., K.L.L., E.N.O.), University of Texas M.D. Anderson Cancer Center, Houston, Tex, and Molecular Cardiology
Laboratory (M.P.), Division of Cardiology, University of Cincinnati (Ohio) College of Medicine.
Correspondence to Dr Eric N. Olson, Department of Biochemistry and Molecular Biology, Box 117, The University of Texas
M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston,
TX 77030.
©) 1994 American Heart Association, Inc.
hybridization signal was demonstrated beyond the aorta and
its arches until 12.5 to 13.5 dpc, when SM-MHC mRNA
appeared in smooth muscle cells (SMCs) of the developing gut
and lungs as well as peripheral blood vessels. By 17.5 dpc,
SM-MHC transcripts had accumulated in esophagus, bladder,
and ureters. Except for blood vessels, no SM-MHC transcripts
were ever observed in developing brain, heart, or skeletal
muscle. These results indicate that smooth muscle myogenesis
begins by 10.5 days of embryonic development in the mouse
and establish SM-MHC as a highly specific marker for the
SMC lineage. The SM-MHC promoter should therefore serve
as a useful model for defining the mechanisms that govern
SMC transcription during development and disease. (Circ Res.
1994;75:803-812.)
Key Words * myogenesis * smooth muscle * myosin
heavy chain . mouse * development
subpopulation of neural crest cells,6-8 whereas SMCs of
the coronary arteries appear to arise independent of the
neural crest.9 Moreover, SMCs of distal vessels and
viscera arise from local mesenchyme within developing
tissues, apparently through inductive processes.10 These
studies suggest that SMCs potentially constitute a number of distinct lineages whose differentiation is under
complex local cues that have yet to be defined. Thus, of
the three muscle types, SMCs clearly are unique in
terms of function, differentiation, and developmental
origin.
A central question in vascular biology relates to the
transcriptional control mechanisms underlying SMC
differentiation and diversity. Approaching this issue has
been hampered by the lack of SMC-specific genes,
whose promoters could be used to identify regulators of
SMC transcription. For example, a number of cytoskeletal and contractile genes have been used as markers for
SMCs during development and disease"; however,
many such markers, including extra-domain fibronectin
variants,'2 meta-vinculin,'3 a-tropomyosin,'4 and heavy
caldesmon,'15 represent alternatively spliced products
from single genes expressed in multiple cell types. Thus,
an analysis of the promoters of these genes may not
reveal transcription factors specific to SMCs. Other
SMC markers that are not the result of alternative
splicing have been found to be more widely expressed
than initially thought. The prototypic example is smooth
muscle a-actin, which is expressed in developing heart
and skeletal muscle,16"7 fibroblasts,18 endothelial cells,19
and some rhabdomyosarcomas.20 The smooth muscle
a-actin promoter has been cloned and initially charac-
804
Circulation Research Vol 75, No 5 November 1994
terized,21,22 but no SMC-specific transcription factors
have yet been isolated.
Myosin heavy chain (MHC) genes are expressed in all
three muscle types, where the protein product dimerizes and associates with two pairs of myosin light chains
to form a functional hexameric myosin polypeptide.23
Various cardiac and skeletal MHC proteins exist
through the expression of several related, yet independent, genes.23 Smooth muscle MHC (SM-MHC) isoforms, on the other hand, arise through the alternative
splicing of a single gene.24-27 Expression studies indicate
that SM-MHC transcripts are confined to adult tissues
containing SMCs28; however, several antibody studies
demonstrated SM-MHC protein in endothelial cells,
myoepithelial cells of the mammary gland, and, rarely,
rhabdomyosarcomas.29-31 Definitive proof for the exis-
Library Screening
Initially, a mouse genomic library (EMBL-3A, Stratagene)
was screened under low-stringency conditions (35% formamide, 5x standard saline citrate [SSCI, 5x Denhardt's solution, and 0.5% sodium dodecyl sulfate [SDS] at 40°C) with a
probe encoding exon II of the rabbit SM-MHC cDNA.28 An
18-kb clone was isolated and found to include exon II of the
mouse SM-MHC gene. A genomic probe encompassing exon
II was subsequently used to screen the random-primed mouse
uterine cDNA library at high stringency. From a total of 105
recombinants screened, five overlapping cDNAs were plaquepurified, subcloned into Bluescript II, and analyzed by restriction mapping. The longest clone (1.2 kb) was sequenced on
both strands with Sequenase 2.0 (USB) and analyzed by FASTA
with the genetic computer group software package (Department of Biomathematics, University of Texas M.D. Anderson
Cancer Center).
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tence of SM-MHC in these and other cell types requires
Isolation of Tissue and Cell Culture RNA
a more
Total RNA from pooled adult mouse tissues was isolated
by the acid phenol method.32 Briefly, tissues were rinsed in
cold PBS, flash-frozen in liquid nitrogen, and homogenized
in 4 mol/L guanidinium isothiocyanate with 2% f-mercaptoethanol. Particulate matter was removed by spinning in an
SS-34 rotor at 10 000 rpm for 10 minutes. The supernatant
was then sheared, and total RNA was extracted as described.32 Samples of human aorta (gift from Dan Johnson,
Texas Heart Institute) and human colon (gift from Marsha
Frazier, M.D. Anderson Cancer Center) were similarly processed for total RNA isolation.
The following cell lines were used to generate RNA by the
same procedure described for tissue RNA isolation: 1OT1/2
cells grown in DMEM with 10% fetal bovine serum (FBS),
undifferentiated C2C12 myoblasts grown in DMEM plus 10%
FBS and differentiated C2C12 myotubes (differentiation was
for 7 days in DMEM plus 2% horse serum), undifferentiated
BC3H1 cells grown in DMEM plus 20% FBS and BC3H1
induced to differentiate by serum withdrawal for 48 hours,
undifferentiated P-19 cells grown in 20% FBS in MEM and
P-19 cells induced to differentiate by 10-day treatment with
10-7 mol/L all-trans retinoic acid (Sigma Chemical Co), and
undifferentiated F9 cells grown in DMEM plus 15% FBS at
10% CO2 and F9 cells induced to differentiate with 10-7 mol/L
retinoic acid plus 1 mmol/L dibutyryl cAMP. A human
myometrial SMC line (gift from James McDougall, Fred
Hutchinson Cancer Center, Seattle, Wash) was maintained in
DMEM plus 10% Hyclone FBS. The human rhabdomyosarcoma cell lines RD, RH-18, and RH-30 (gifts from Peter
Houghton, St Jude Children's Research Hospital, Memphis,
Tenn) were grown in DMEM containing 10% FBS to 75%
confluency and then switched to DMEM with 2% horse serum
for 48 hours. HepG2, HeLa, BALB/c3T3, and NIH 3T3 cell
lines were grown according to the manufacturer's specifications (American Type Culture Collection). Human umbilical
vein endothelial cell RNA was generously supplied by Dr
Kirkwood Pritchard, Medical College of Wisconsin.
stringent assay than immunodetection. Morestudies are needed to ascertain whether, like
smooth muscle a-actin, SM-MHC is expressed in developing heart and skeletal muscle.
To address these issues of specificity and to document
the spatial and temporal pattern of smooth muscle
differentiation during development, we cloned a fragment of the mouse SM-MHC cDNA and analyzed its
mRNA expression in adult and embryonic tissues. The
results demonstrate that in both adults and developing
embryos, SM-MHC transcripts appear only in those
tissues with a known SMC component. The onset of
SMC differentiation begins in the aorta at 10.5 days post
coitum (dpc). Subsequent staged embryos reveal SMMHC mRNA in the bronchial buds of the lung, muscular layers of the gut, peripheral vasculature, esophagus,
bladder, and ureters. Collectively, these results provide
the first detailed analysis of mouse smooth muscle
myogenesis and establish SM-MHC as the most specific
marker for the SMC lineage identified to date.
over,
Materials and Methods
Animals
Approximately 200 mice (Harlan Sprague Dawley, Indianapolis, Ind) were boarded in the animal facility at the M.D.
Anderson Cancer Center and handled in accordance with
institutional guidelines. For in situ hybridization studies,
staged mouse embryos were isolated beginning 7.5 dpc, which
was assessed visually by inspection for a vaginal plug. Pregnant
females were killed by cervical dislocation, and the uterine
horns containing embryos were rapidly excised and placed in
sterile phosphate-buffered saline (PBS). Embryos were rapidly
dissected from the uterine horn and immediately immersionfixed as described below.
Library Construction
Pooled mouse uteri were combined for poly(A) + RNA
isolation by using the FASTrack kit (Invitrogen). A polydT/
random-primed cDNA library was constructed by using 5 1£g
of poly(A) + RNA according to the manufacturer's instructions (SuperScript Choice System, BRL). Partially filled-in
Xho I linkers were ligated to the library, which was subsequently cloned into compatible ends of the lambda ACT
vector (gift from Stephen J. Elledge, Baylor College of Medicine, Houston, Tex). The library was packaged (Gigapack,
Stratagene), amplified once to a titer of 1010 plaque-forming
units (pfu)/mL, and stored at -80°C in 7% dimethyl sulfoxide.
RNase Protection Analysis
A 387-bp fragment of mouse SM-MHC encoding exons 1 (75
nt untranslated sequence) and most of exon II (17 nt of
untranslated sequence plus 295 bp of coding sequence) was
directionally subcloned into the Xho I and Sma I sites of the
Bluescript II vector, linearized with Xho I, and transcribed in
vitro by using T3 polymerase (Ambion) in the presence of
[a-32P]UTP (3000 Ci/mmol, Amersham). A 108-bp portion of
murine 18S cDNA (Ambion) was transcribed in vitro with T7
polymerase (Ambion) and cohybridized with SM-MHC to
verify equivalent amounts of RNA loaded in each lane. A
250-bp fragment (nucleotides 302 to 552) of the human
SM-MHC cDNA33 was amplified by polymerase chain reaction
and transcribed in vitro by using T7 polymerase (Ambion).
Miano et al Mouse Smooth Muscle Myogenesis
Mouse SMMHC
Rabbit SMMHC
Human NMMHC-A
Mouse axCardiac
Rat ICardiac
Rat fast skl
Pig slow skl
Mouse SMMHC
Rabbit SMMHC
Human NMMHC-A
Mouse aCardiac
Rat pCardiac
Rat fast skl
Pig slow skl
805
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Mouse aCardiac
KGTLED
Rat ICardiac
KGTLED
Rat fast skl
KGTLED
FIG 1. Amino acid alignment of selected myosin heavy chain (MHC) proteins across a portion of the Si globular head domain. Shown
are amino acids 94 to 218 of the mouse smooth muscle MHC (SMMHC) protein aligned against homologous regions of other MHC
proteins (their GenBank accession numbers are given in parentheses): rabbit SM-MHC35 (M77812), human nonmuscle MHC type A
(NMMHC-A)36 (M69180), mouse a-cardiac MHC37 (M76600), rat p-cardiac MHC38 (Xl 5939), rat fast skeletal (ski) MHC39 (X04267), and
pig slow ski MHC40 (Li 01 29). The shaded region corresponds to the Mg 2+-ATPase domain.
Riboprobes were digested with DNAse I and purified in a 5%
polyacrylamide-7 mol/L urea gel. Approximately 15 gg of
total RNA was combined with 1.5 x 105 cpm of SM-MHC and
4x 104 cpm of 18S riboprobes and assayed according to the
manufacturer's instructions (Ambion). Protected fragments
were resolved in a 4.5% polyacrylamide-7 mol/L urea gel.
Assays were repeated at least twice with independently isolated samples of RNA.
In Situ Hybridization
The same mouse probe used for RNase protections was
used for in situ hybridizations. Staged mouse embryos (7.5 to
17.5 dpc) were fixed overnight in 4% paraformaldehyde-PBS,
cleared in xylene, and embedded in paraplast. The methods
used for in situ hybridization were as described, with slight
modifications.34 Sections (8 gim) were floated onto triplewashed silane-coated slides (Histology Control Systems),
dried overnight at 37°C, and stored at room temperature
before use. For prehybridization, sections were cleared in two
changes of xylene and hydrated through decreasing amounts
of ethanol. Sections were briefly rinsed in 0.85% saline and
PBS before fixation in 4% paraformaldehyde for 20 minutes.
After rinsing in PBS, sections were subjected to a 7.5 -minute
digestion in 50 gg/mL proteinase K (Boehringer Mannheim),
followed by 0.1 mol/L triethanolamine-acetic anhydride acetylation and dehydration.
For hybridization, riboprobes were heated for 2 minutes at
90°C in 50% formamide, lx Denhardt's solution, 0.3 mol/L
NaCI, 20 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EDTA, 10%
dextran, 0.01 mol/L Na2HPO4, and 0.5 mg/mL yeast RNA
(Boehringer Mannheim) and placed on ice. Approximately
1x 100 cpm of probe was applied to each slide (25 to 30 gL),
which was then coverslipped and hybridized in a humidified
chamber at 52°C for at least 16 hours. After hybridization,
sections were washed in 2x SSC and 10 mmol/L dithiothreitol
at 50°C for 40 minutes; 50% formamide, 2x SSC, and 10
mmol/L dithiothreitol at 65°C for 40 minutes; and 0.1 mol/L
Tris-HCl (pH, 7.5) and 50 mmol/L EDTA containing 20 gg/mL
RNase A (Boehringer Mannheim) at 37°C for 45 minutes,
followed by 2x SSC and 0.1x SSC washes at 37°C for 20
minutes each. Sections were dehydrated, dried, and dipped in
Kodak NTB2 emulsion (Eastman Kodak Co). Autoradiography
lasted 1 week in light-tight boxes with desiccant at 4°C. Slides
were developed in D-19 developer (Eastman Kodak Co), fixed
with rapid fixer (Eastman Kodak Co), and dehydrated through
xylene. Slides were coverslipped with low-viscosity cytoseal
(Stephens Scientific) and examined under bright- and dark-field
microscopy. Sections from at least three embryos at 7.5, 8.5, 9.5,
10.5, 11.5, 12.5, 13.5, 14.5, and 17.5 dpc were examined.
Results
Cloning Mouse SM-MHC
By use of a mouse SM-MHC genomic fragment, a
uterine cDNA library was screened to obtain several
overlapping clones (see "Materials and Methods"). One
clone (1.2 kb) was sequenced on both strands and found to
encode the first eight exons of SM-MHC. The corresponding nucleotide sequence has been deposited in GenBank
(accession No. L25860). This sequence is nearly identical
to the recently cloned rat SM-MHC cDNA.25 Fig 1 shows
an amino acid alignment of the mouse SM-MHC protein
with several other MHC proteins across a portion of the
highly conserved S1 globular head domain. This sequence
analysis shows the mouse SM-MHC to be 98% homologous to the rabbit sequence and 89% homologous to the
type A human nonmuscle MHC. In contrast, mouse
SM-MHC is only 54%, 58%, and 57% similar to mouse
a-cardiac, rat P-cardiac, and rat fast skeletal MHC, respectively. The sarcomeric MHCs are =84% homologous
to one another in this region of MHC. The greatest stretch
of conserved amino acids among all the MHCs analyzed
is, as expected, within the Mg 2+-ATPase domain (shaded
region in Fig 1).
SM-MHC mRNA Expression in Adult Tissues and
Cultured Cell Types
We analyzed adult mouse tissues and several cultured
cell types by RNase protection to document the specificity of SM-MHC mRNA expression (Fig 2). This
method of detection is more stringent than Northern or
immunodetection, both of which are susceptible to cross
hybridization/reactivity to other myosin isoforms. Our
results support the work of others28 by demonstrating
SM-MHC mRNA in mouse aorta, intestine, lung, stomach, and uterus (Fig 2, top left). We consistently observed greater SM-MHC mRNA expression in stomach
Circulation Research Vol 75, No 5 November 1994
806
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FIG 2. Smooth muscle myosin heavy chain (SM-MHC) mRNA expression in
adult mouse tissues and cell lines. Top left, Approximately 15 .gg of total RNA
from the indicated tissues was hybridized in solution to a riboprobe corresponding to SM-MHC and processed for RNase protection (see "Materials and
Methods"). The probe is 463 bp, and the protected fragment is 387 bp in length.
A 108-bp protected band corresponding to 18S RNA is shown to demonstrate
comparable RNA loading. Exposure time was for 8 hours. Sk indicates skeletal.
Top right, Total RNA (15 gg) from the indicated cell lines either undifferentiated
(U) or differentiated (D) with appropriate stimuli (see "Materials and Methods")
hybridized to the SM-MHC and 18S riboprobes. Exposure time was 7.5 hours.
Bottom left, Human tissues and cell lines similarly analyzed by RNase protection. Exposure time was 8.5 hours. HUVEC indicates human umbilical vein
endothelial cells; RH-18, RD, and RH-30, three different human rhabdomyosarcoma cell lines; and LTR, a human myometrial smooth muscle cell line.45
and uterus than in other tissues, which is likely to reflect
the higher content of SMC in these tissues. On overexposure, tissues such as heart, skeletal muscle, spleen,
and testes showed a protected band corresponding to
SM-MHC (data not shown). Expression in heart and
skeletal muscle is attributable to the presence of blood
vessels (see below), whereas spleen and testes are
known to contain a nonvascular SMC component.41
To further test its specificity of expression, we assayed
for SM-MHC mRNA in a variety of cell types. No
detectable SM-MHC mRNA expression was ever observed in 10T1/2, C2C12, P-19, or F9 cell lines whether
undifferentiated or differentiated with appropriate stimuli
(Fig 2, top right). Moreover, HeLa cells as well as BALB/c
3T3 and NIH 3T3 fibroblasts failed to express SM-MHC
(data not shown). Because there has been a controversy
concerning the identity of BCQH1 cells as smooth versus
skeletal muscle,42'43 we assayed for SM-MHC mRNA
expression in both proliferating and differentiated BC3H1
cells. No SM-MHC expression was observed in this cell
line, consistent with the conclusion that these cells more
closely resemble the skeletal muscle cell type.44
To address the issue of SM-MHC expression in endothelial cells and rhabdomyosarcomas,293' we analyzed
several human cell lines. Fig 2, bottom left, shows that
although a human SM-MHC riboprobe easily detects
human aortic and colonic SM-MHC transcripts, no signal
was observed in RNA samples from human umbilical vein
endothelial cells, three rhabdomyosarcoma cell lines,
HepG2 cells, and a myometrial SMC line immortalized
with an amphotropic retrovirus containing the E6 and E7
open reading frames of human papillomavirus type 16.45
Expression analyses therefore show SM-MHC mRNA to
be highly enriched in adult tissues containing SMC and
absent in 14 different non-smooth muscle cell types.
In Situ Hybridization of SM-MHC in Adult
Mouse Tissues
To localize the faint expression of SM-MHC observed in RNase protections of adult heart and skeletal
muscle and to determine whether the riboprobe crosshybridized with other MHC mRNAs, sections of adult
heart, skeletal muscle, and uterus were processed for in
situ hybridization. Fig 3 shows representative sections of
three adult tissues hybridized with the antisense SMMHC riboprobe corresponding to exon I and most of
exon II (see "Materials and Methods"). No specific
hybridization signal was observed in myocardial or
skeletal muscle cells, although small arterioles in these
tissues were clearly labeled with the probe (Fig 3A
through 3D). Adult uterus was strongly labeled within
the myometrial layer, with little or no signal detected in
the glandular endometrial layer (Fig 3E and 3F). Sense
SM-MHC probe did not specifically hybridize to any
tissues (data not shown). Thus, despite the presence of
predominantly coding sequence, the SM-MHC probe
only hybridized to adult tissues containing SMCs.
Developmental Expression of SM-MHC in
Postimplantation Mouse Embryos
No studies to date have examined the mRNA expression of restricted SMC markers during mouse embryogenesis. Therefore, we examined SM-MHC mRNA
expression in developing mouse embryos to ascertain its
specificity of expression and to determine when and
where smooth muscle myogenesis is initiated during
Miano et al Mouse Smooth Muscle Myogenesis
807
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FIG 3. In situ hybridization of adult mouse
tissues for smooth muscle myosin heavy
chain (SM-MHC) expression. Bright-field
(A, C, and E) and dark-field (B, D, and F)
micrographs of SM-MHC mRNA expression in heart (A and B), skeletal muscle (C
and D), and uterus (E and F). Sections
(8-jm) were processed for in situ hybridization as described in "Materials and
Methods." m indicates myometrium; e,
endometrium. Arrowheads denote blood
vessels.
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embryogenesis. No SM-MHC mRNA was seen in embryos from 7.5 to 9.5 dpc. At 10.5 dpc, SM-MHC
transcripts were first observed in the developing dorsal
aorta (Fig 4A). The outflow tract, however, was notably
devoid of SM-MHC transcripts at 10.5 dpc (data not
shown). The signal intensity in the dorsal aorta was
weak at 10.5 dpc but consistently greater than background. No obvious expression gradient was apparent,
indicating that the initiation of SMC differentiation was
synchronized throughout the aorta (data not shown).
Significantly, no embryonic regions other than the dorsal aorta displayed SM-MHC expression at 10.5 days,
indicating that aortic SMC differentiation is dependent
on local environmental cues.
At 11.5 dpc, SM-MHC mRNA was observed in
branching arches of the ascending aorta (Fig 4B) and,
for the first time, the outflow tract (data not shown).
The signal intensity at 11.5 days was greater than at 10.5
days, suggesting an accumulating population of differentiated SMCs. At 12.5 dpc, SM-MHC mRNA could be
seen in developing gut (Fig 4C) and bronchi of the lung
(data not shown). No SM-MHC signal was ever observed in heart or somites at these early time points.
As development progressed, SM-MHC mRNA expression became widespread throughout the gut and lung (Fig
5). Both the inner circular and outer longitudinal layers of
the external muscularis of the gut could be delineated (Fig
5A). For the first time, distal blood vessels, such as those
of the head and intersomitic region, could be seen (Fig
5A). The signal intensity in visceral SMCs was consistently
greater than in the vasculature. Again, no SM-MHC signal
was observed in heart or somites.
By 17.5 dpc, SM-MHC mRNA was observed in every
tissue with an SMC component. Thus, in the thoracic
cavity, SM-MHC transcripts were seen in the large
blood vessels and bronchi of the lung and in vessels of
the heart and surrounding musculature (Fig 6A). Surprisingly, the hybridization intensity in the large bronchi
appeared somewhat less than that observed at earlier
time points (compare Fig 6A with Fig 5). The esophagus
showed intense expression of SM-MHC transcripts. At
this level of the thorax, the proportion of smooth muscle
within the muscularis of the esophagus is far greater
than skeletal muscle.41 As with embryos at earlier
stages, sense SM-MHC probe showed no specific hybridization (data not shown).
808
Circulation Research Vol 75, No 5 November 1994
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FIG 4. Smooth muscle myosin heavy chain (SM-MHC) transcripts in postimplantation mouse embryos at 10.5, 11.5, and 12.5 days post
coitum (dpc). Frontal (A and B) and sagittal (C) sections through mouse embryos at 10.5 (A), 11.5 (B), and 12.5 (C) dpc are shown. a
indicates aorta; Ib, limb bud; nt, neural tube; g, primitive gut; o, outflow tract; and b, brain. Arrowheads indicate branching arches of the
aorta.
SM-MHC transcripts were present throughout the
gut of 17.5 dpc mouse embryos but were confined to the
inner and outer layers of the external muscularis (Fig
6B); no signal was seen in the area of the muscularis
mucosae, probably because of the dearth of SMCs in
this region of the gut. The bladder showed intense
FIG 5. Smooth muscle myosin heavy chain (SM-MHC) mRNA in embryos at 13.5 and 14.5 days post coitum (dpc). Representative
sagittal sections from 13.5-dpc (A) and 14.5-dpc (B) embryos. Note the emergence of SM-MHC in developing lung buds (lu), gut (g),
and peripheral vessels (arrowheads). fi indicates liver; b, brain; i, intersomitic vessels; and h, heart.
Miano et al Mouse Smooth Muscle Myogenesis
809
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FIG 6. Smooth muscle myosin heavy chain (SM-MHC) transcripts in embryos at 17.5 days post coitum (dpc). Representative horizontal
sections through the thoracic region (A) and abdominal region (B) of 17.5-dpc embryos. Note that expression of SM-MHC in heart and
peripheral musculature is confined to blood vessels (arrowheads). The apparent signal around the periphery of the limbs is an edge
artifact that was present in sections hybridized with a sense SM-MHC riboprobe. a indicates aorta; e, esophagus; b, bladder; u, ureters;
g, gut; lu, lung; s, spinal cord; and h, heart.
hybridization to SM-MHC in all three of its muscle
layers with no signal in the epithelial layer. Paired
ureters were similarly labeled. The aorta and vena cava
were both positive; however, the intensity of the signal
was notably less than that in the bladder and ureters.
Neighboring blood vessels were variably labeled with
the SM-MHC probe (Fig 6B). No hybridization was ever
observed in the heart or skeletal muscle (Fig 6A and
6B). Thus, the results of our in situ hybridization studies
clearly demonstrate that SM-MHC mRNA is confined
to only those developing tissues with smooth muscle.
Discussion
SM-MHC: A Highly Specific Marker for the
SMC Lineage
Although the expression of several transcription factors has been documented in vascular SMCs,46-48 no
factors specific to this cell type have yet been identified.
Numerous cytoskeletal, contractile, and extracellular
matrix genes are expressed predominantly in vascular
SMCs and, as such, have been designated as markers for
the SMC lineage.1149 The promoters of these genes
could be useful in identifying novel transcription factors
that specify SMC identity. However, the specificity of
many SMC markers, and therefore the utility of their
promoters for identifying SMC-specific transcription
factors, has been called into question with their unexpected appearance in a variety of non-SMC types both
in vitro and in vivo.'6,18,1950-53 Thus, as an initial step
toward an investigation of the mechanisms that regulate
smooth muscle transcription, we have cloned a portion
of the mouse SM-MHC cDNA and carefully analyzed
its specificity of expression in adult and embryonic
mouse tissues. We show by RNase protection that
SM-MHC mRNA expression is, as previously documented in other species,24-26,28,54,55 restricted to adult
mouse aorta, intestine, lung, stomach, and uterus. We
further show by in situ hybridization that low-level
transcripts in adult heart and skeletal muscle are
solely attributable to the presence of blood vessels. It
should be noted that our data do not distinguish
different SM-MHC isoforms,24-27 because the probe
used contains a sequence common to all SM-MHC
transcripts. Recent studies, however, show SM-MHC
transcripts that are specific to visceral SMCs.25-27 Such
differential expression in SM-MHC mRNA isoforms
is postulated to account for the distinct functional
properties of contraction between vascular and visceral SMC populations.27
The specificity of SM-MHC mRNA was further assessed in cultured cells. From a survey of more than a
dozen cell types, including endothelial cells, fibroblasts,
and several rhabdomyosarcoma cell lines, we never
detected SM-MHC transcripts by RNase protection. In
fact, with the exception of primary rat aortic SMCs, we
have yet to detect SM-MHC mRNA in any cultured cell,
including a number of immortalized and multiply passaged SMC lines (Fig 2, bottom left, and data not
shown). The latter finding underscores the limited utility of some SMC lines that permanently lose phenotypic
markers.
Several antibody studies have examined SM-MHC
protein expression in a number of species at variable
intervals of time during development.49 These studies
have provided important information regarding isoform-specific SM-MHC expression but do not thoroughly address the specificity of SM-MHC during development or the precise time of onset for SMC
differentiation. Th&refore, we used in situ hybridization
of staged mouse embryos to clarify these issues. A
major objective was to ascertain whether SM-MHC
transcripts were expressed in developing cardiac and
810
Circulation Research Vol 75, No 5 November 1994
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skeletal muscle. Ruzicka and Schwartz16 first showed
smooth muscle a-actin mRNA in developing avian
hearts before the appearance of sarcomeric a-actins.
Subsequent studies documented smooth muscle a-actin
protein expression in developing rat heart and skeletal
muscle.17"56 Moreover, both smooth and sarcomeric
a-actin protein were found to coreside within developing sarcomeres of the heart and skeletal muscle.17'57 Our
in situ hybridization data clearly show that SM-MHC
transcripts are not present in either cardiac or skeletal
muscle at any stage of development. Indeed, with the
exception of blood vessels, SM-MHC mRNA was never
observed in tissues without an SMC component. Recently, we have observed the early expression of two
other SMC markers in the embryonic heart (J. Miano,
P. Cseresi, L. Li, and E. Olson, unpublished data,
1994). Thus, SM-MHC is the first smooth muscle
marker whose mRNA is expressed exclusively in SMCs
during mammalian development.
The only other SMC marker that has been analyzed
during mammalian development and shown to be restricted to the SMC lineage is r-enteric actin.17 Interestingly, the onset of -r-enteric actin protein expression
does not begin in the rat until day 15 of gestation,17
which corresponds approximately to 14 dpc in the
mouse. This would seem to suggest that SM-MHC and
i-enteric actin are regulated differently. It should be
pointed out however that the mRNA to i-enteric actin
was not measured, so it is possible that the 7-enteric
actin gene is activated earlier than 15 days of gestation.
Moreover, it is conceivable that i-enteric actin mRNA is
expressed in heart and skeletal muscle but is untranslated. This and other SMC markers should therefore be
analyzed at the mRNA level during development to rule
out gene activation without protein expression.
Smooth Muscle Myogenesis in Developing
Mouse Embryos
No studies have carefully examined the mRNA expression of SMC markers during mammalian development. Such an analysis is necessary not only to rigorously test the specificity of SMC markers during
embryogenesis but also to begin understanding the
spatial and temporal patterns of SMC differentiation
during development. Our in situ hybridization data
indicate that SMC differentiation (as defined by SMMHC mRNA expression) begins in the dorsal aorta at
-10.5 dpc. At this stage, we could not distinguish SMC
versus endothelial cell SM-MHC mRNA expression.
However, at least two lines of evidence support the
notion that SM-MHC mRNA expression at 10.5 days is
confined to SMCs. First, as reported in the present
study and others,3058 endothelial cells do not express
SM-MHC. Second, from studies performed in chickquail chimeras,59 endothelial cell differentiation occurs
with the initial formation of the aorta and other primitive blood vessels, which in the mouse corresponds to
days 7.5 to 8 of gestation; we did not observe SM-MHC
mRNA expression until day 10.5. Thus, it appears that
the reported expression of SM-MHC protein in bovine
endothelial cells29 is either species dependent or the
result of the antibody used cross-reacting with some
other potentially novel myosin family member.
Although SM-MHC mRNA was present in the dorsal
aorta at 10.5 dpc, no detectable signal was seen in the
outflow tract until 11.5 dpc. The absence of SM-MHC
transcripts in this region of the vasculature at 10.5 dpc
could reflect a delay in smooth muscle differentiation.
Such a delay may be related to the relative number of
neural crest-derived versus mesoderm-derived cells migrating to the outflow tract. For example, studies in
chick-quail chimeras indicate that there is a population
of SMCs in the outflow tract that expresses smooth
muscle a-actin protein after SMCs in the aortic arches
(see Fig 6B in Reference 60). Our SM-MHC mRNA
data showing a similar temporal pattern of SM-MHC
mRNA expression suggest that at least one aspect of
smooth muscle myogenesis is conserved between
chicken and mouse.
No SM-MHC mRNA expression was observed beyond the outflow tract and aortic arches until 12.5 to
13.5 dpc, when early bronchial buds of the lung and
condensing mesenchyme in the gut showed an SMMHC signal. The expression of SM-MHC protein has
recently been documented in developing bronchi of the
rat lung.61 The presence of SM-MHC in the lung and
gut during fetal development is consistent with data
demonstrating fetal respiration and gastric motility in
early developing embryos.62'63 Such fetal activities likely
rely on functionally competent SMCs that are able to
contract.
The expression of SM-MHC mRNA remained confined to the SMC lineage as development progressed
with peripheral blood vessels of the head, musculature,
and intersomitic region initially displaying a positive
signal at 13.5 to 14.5 dpc and the esophagus, bladder,
and ureters showing intense labeling at 17.5 dpc. The
greater SM-MHC mRNA expression in visceral tissues
such as bladder, ureter, and gut is in agreement with a
report demonstrating a higher content of smooth muscle
a-actin mRNA in these tissues compared with vascular
tissue of the developing rat.M The latter findings may be
due to the relatively lower content of SMCs in the aorta
compared with visceral tissue. Alternatively, the lower
SM-MHC mRNA observed in vascular tissues such as
the aorta could be due to a population of SMCs that
have not fully differentiated. Indeed, there is evidence
for transitional SMC phenotypes in developing blood
vessels.65
Because adult vascular SMCs appear to recapitulate
their embryonic phenotype during disease,45 it may be
useful to understand the developmental context under
which SMCs differentiate. The formation of paired
aortas is coincident with heart formation, which begins
at the cardiogenic plate on day 8 of gestation in the
mouse.66 The expression of SM-MHC mRNA in the
embryonic aorta at 10.5 dpc is consistent with the
delayed onset of sarcomeric MHC expression in heart
and skeletal muscle34'67 and implies that such gene
expression occurs only after cells become committed
to each muscle lineage. Significantly, we did not
observe SM-MHC transcripts in regions of the embryo
where aortic SMCs are thought to arise (eg, the neural
crest), indicating that SMC differentiation occurs only
when such cells are in their proper environment. Local
cues (probably from adjacent endothelial cells) may
then stimulate the expression of SMC-specific transcription factors, which activate differentiated markers
such as SM-MHC. In this context, visceral SMC dif~
Miano et al Mouse Smooth Muscle Myogenesis
ferentiation has been shown to be dependent on
adjacent epithelium.10
Finally, the expression of SM-MHC mRNA in the
early aorta did not appear to display any discernable
expression gradient. This suggests that aortic SMC
differentiation occurs synchronously. Such a process is
compatible with a recent model for the formation of
coronary vessels whereby vessel growth proceeds in a
discontinuous rather than continuous manner.68 This
model of discontinuous vessel formation predicts that
SMCs along a segment of vessel are polyclonal.68 The
propensity for SMC phenotypic modulation and atherosclerotic disease within discrete regions of a vessel (eg,
abdominal versus thoracic aorta) may therefore be
intimately related to unique embryonic SMC phenotypes during vasculogenesis. Future work should be
directed toward defining these early developmental
phenotypes within vascular SMC lineages and the nature of the signals specifying them.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Acknowledgments
This study was supported by grants from the National
Institutes of Health, Muscular Dystrophy Association, and
The Robert A. Welch Foundation (Dr Olson). Dr Miano is a
recipient of a National Research Service Award. We thank Dr
John Schwarz for reading this manuscript. The editorial assistance of Kathy Tucker and the technical support of Mary Ellen
Perry, Michael Chase, Brian Mercer, and Alicia Tizenor is
appreciated. The computer software for analyzing SM-MHC
cDNA and protein sequences was offered through the Depart-
ment of Biomathematics, M.D. Anderson Cancer Center (NCI
CA-16672).
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J M Miano, P Cserjesi, K L Ligon, M Periasamy and E N Olson
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Circ Res. 1994;75:803-812
doi: 10.1161/01.RES.75.5.803
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