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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 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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). Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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 TCLNEASVLHNLRERYFSGLIYTYSGLFCVVVNPYKYLPIYSEKIVDMYKGKKRHEMPPH --------------Y--------------I- Q-N------E---------------F-H-PA--Y--K---AAWM----------T-----W--V-NAEV-AA-R----S-A---F-H-PA--Y--K---A-WM----------T-----W--V-NAQV-AA-R----S-A---H---PA--Y--KD--T-WM----------T-----W--V-TPEV-DG-R----Q-A---F-H-PA--Y--K---A-WM---------TI ----W--V-NAEV-AA-R----S-AIYAI -FS-FS-FS-FS- Mouse SMMHC Rabbit SMMHC ELEKQL Human NMMHC-A ---R- .AWASSHKGKKDSSITG: ______-------T----: --Y ------S ---QG:::: -SI-AIGDRS-KENPNAN 7-I-AIGDRS-KDQTP-: -TI-ATGDLA-KKDSKM: ------ Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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 0 .0 0 CL)co (I) CL 0 I.0 < m 10 I 0 a)c y -o > C:;~21 _i 1 0i ( CO_ E-O 0 2 Q z c r m c) 0 CD (U E (C0 XT C> SM-MHC C2 U D BC H1 U D F9 Pig D V) U D UD----I0 _ ir '(SM-MHC Vl 9 4 18S I 0 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 c) D0 W > CO 1,1 CL I C: I '(18S KB 0 Co or± er c (U CL 0) I: c 0 0 >, 0 i < 0 -_ SM-MHC 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 A '4, C, Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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. -.M-- -0 ._ v .. i.' 1 i15 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 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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 Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 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. 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J M Miano, P Cserjesi, K L Ligon, M Periasamy and E N Olson Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017 Circ Res. 1994;75:803-812 doi: 10.1161/01.RES.75.5.803 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1994 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/75/5/803 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. 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