A Sonic Hedgehog Signaling Domain in the Arterial Adventitia Supports Resident Sca1؉ Smooth Muscle Progenitor Cells

A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1؉ smooth muscle progenitor cells

Jenna N. Passman*†, Xiu Rong Dong*‡, San-Pin Wu*§, Colin T. Maguire*‡, Kelly A. Hogan¶, Victoria L. Bautch*†¶, and Mark W. Majesky*†‡ʈ**

*Carolina Cardiovascular Biology Center, Departments of ‡Medicine, ʈGenetics, and ¶Biology, and †Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 22, 2008 (received for review December 6, 2007) We characterize a sonic hedgehog (Shh) signaling domain re- interstitial fibroblasts (13, 14), and promotes endothelial cell stricted to the adventitial layer of artery wall that supports resi- chemotaxis and tube formation (17, 18). Therefore, Shh plays dent Sca1-positive vascular progenitor cells (AdvSca1). Using important roles in cell–cell communication during vascular patched-1 (Ptc1lacZ) and patched-2 (Ptc2lacZ) reporter mice, adven- development and adult wound repair. titial Shh signaling activity was first detected at embryonic day (E) Hedgehog (Hh) proteins signal by binding to patched-1 (Ptc1) 15.5, reached the highest levels between postnatal day 1 (P1) and or patched-2 (Ptc2) receptors and cdo/boc accessory proteins at P10, was diminished in adult vessels, and colocalized with a the cell surface, resulting in derepression of smoothened (smo) circumferential ring of Shh protein deposited between the media (19). Ptc proteins are 12-transmembrane domain receptors that ؊/؊ and adventitia. In Shh mice, AdvSca1 cells normally found at the are structurally related to the resistance-nodulation-division aortic root were either absent or greatly diminished in number. (RND) family of bacterial membrane permeases (20) and the Using a Wnt1-cre lineage marker that identifies cells of neural crest Niemann-Pick C1-like1 cholesterol transporter (21). Ptc proteins origin, we found that neither the adventitia nor AdvSca1 cells were contain a conserved sterol-sensing domain, and recent studies labeled in arteries composed of neural crest-derived smooth muscle cells (SMCs). Although AdvSca1 cells do not express SMC marker suggest that Ptc1 represses smo by transporting activating sterols proteins in vivo, they do express transcription factors thought to be out of the cell (22). Hh binding to Ptc1 inhibits transporter required for SMC differentiation, including serum response factor activity thereby allowing activating sterols to accumulate (19, (SRF) and myocardin family members, and readily differentiate to 22). Activated smo trafficks into the primary cilium where Hh SMC-like cells in vitro. However, AdvSca1 cells also express potent signal mediators are concentrated (23, 24) and triggers phos- repressors of SRF-dependent transcription, including Klf4, Msx1, phorylation of gli factors that then move to the nucleus and and FoxO4, which may be critical for maintenance of the SMC stimulate gene transcription (25). Among Hh target genes are progenitor phenotype of AdvSca1 cells in vivo. We conclude that a Ptc1 and Ptc2; therefore, Ptc1lacZ and Ptc2lacZ activities are used restricted domain of Shh signaling is localized to the arterial as sensitive reporters of Hh signaling in transgenic mice (26, 27). adventitia and may play important roles in maintenance of resi- The origins and functions of the adventitia are poorly under- dent vascular SMC progenitor cells in the artery wall. stood. Hu et al. (6) reported that stem cell antigen-1 (Sca1)- positive cells with a potential to differentiate into smooth muscle artery ͉ differentiation ͉ Klf4 ͉ self-renewal ͉ serum response factor cells (SMCs) were found in the aortic adventitia of adult ApoEϪ/Ϫ mice (AdvSca1 cells). When transferred to the adven- n adventitia surrounds most blood vessels where it functions titial side of experimental vein grafts, AdvSca1-derived SMCs Aas a dynamic compartment for cell trafficking into and out made up 30% of cells in the graft neointima after 4 weeks (6). of the artery wall. The adventitia is a site for formation and The adventitia responds to balloon catheter injury with rapid regression of microvessels that penetrate and nourish the medial increases in SM␣Actin and SM22␣ expression, particularly at the and intimal layers of vessel wall (1–3). The adventitia also media–adventitia interface (5). Endothelial progenitor cells are contains perivascular nerves and lymphatic vessels and is an reported to cluster between the media and adventitia in human important domain for NAD(P)H oxidase activity in the vessel arteries (7, 8). These cells formed capillary sprouts in ex vivo ring wall (4). Adventitial cells participate in vascular growth and culture assays and were recruited by tumor cells for capillary repair and are important determinants of lumen size via control vessel formation in vivo. Given the role of Shh signaling in the of inward or outward wall remodeling processes (2, 5). Recent maintenance of resident stem and progenitor cells in skin (28), studies report unexpected roles for the adventitia insofar as it nervous system (29), lymphoid tissue (30), and hematopoietic supports resident progenitor cells that can adopt both vascular cells (31), we sought to test for similar roles for Hh signaling in and nonvascular fates (6–8). Despite these important functional the adventitial tissue surrounding blood vessels. properties, the signals and environmental cues that confer such unique and dynamic properties to the adventitial layer remain largely unknown. Author contributions: J.N.P. and M.W.M. designed research; J.N.P., X.R.D., S.-P.W., C.T.M., Sonic hedgehog (Shh) is an essential morphogen and growth and K.A.H. performed research; V.L.B. contributed new reagents/analytic tools; J.N.P., factor for many developing tissues (9). In the vascular system, X.R.D., and M.W.M. analyzed data; and J.N.P. and M.W.M. wrote the paper. MEDICAL SCIENCES Shh signaling is important for specification of arterial-venous The authors declare no conflict of interest. identity of endothelial cells (10), remodeling of yolk sac blood This article is a PNAS Direct Submission. vessels (11), and recruitment of mural cells (12). In adult blood §Present address: Department of Molecular and Cellular Biology, Baylor College of Medi- vessels, Shh is angiogenic in ischemic hindlimb and corneal cine, BCM-130, One Baylor Plaza, Houston, TX 77030. micropocket assays (13, 14), promotes perineural neovascular- **To whom correspondence should be addressed. E-mail: [email protected]. ization in diabetic animals (15), and protects the myocardium This article contains supporting information online at www.pnas.org/cgi/content/full/ against chronic ischemia (16). Shh stimulates production of 0711382105/DCSupplemental. angiogenic factors, including VEGF-A and angiopoietin-1 by © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711382105 PNAS ͉ July 8, 2008 ͉ vol. 105 ͉ no. 27 ͉ 9349–9354 Downloaded by guest on October 1, 2021 Fig. 1. Hh signaling in artery wall. (A, B, and D–F) Tissues were isolated from PtclacZ mice at P2 and stained for ␤-gal activity. All major arteries examined, including coronary arteries, aorta, pulmonary trunk (A), intercostal (D), mesenteric (E), and femoral arteries (F) were positive for Ptc1lacZ or Ptc2lacZ activity as shown. (B) Adult hearts displayed low levels of ␤-gal activity in coronary arteries, aorta, and pulmonary artery. (C) A cross-section of the aorta at P2 reveals Ptc1lacZ-positive cells are restricted to the adventitia. Ica, intercostal artery; A, artery; V, vein; N, nerve. (Magniﬁcation: A and D–F, ϫ7; B, ϫ3; C, ϫ 40.)

Results found in mesenteric and femoral arteries (Fig. 2 E and F). Close Hh Signaling in Postnatal Blood Vessels. We examined ␤-gal activity proximity was found between Shh protein and AdvSca1 cells in blood vessels of PtclacZ transgenic mice at postnatal day 2 (P2). (Fig. 2D). Extensive overlap was observed between ␤-gal- In whole-mount hearts, Ptc1lacZ and Ptc2lacZ activities were positive cells and AdvSca1 cells in the aortic adventitia of Ptc1lacZ detected in coronary arteries, but not in myocardium or epicar- mice (Fig. 2D Inset). These findings suggest that a zone of Shh dium (Fig. 1A). The ascending aorta and pulmonary trunk were signaling in the adventitia identifies a unique domain of artery strongly positive at this time (Fig. 1A). Both Ptc1lacZ and Ptc2lacZ wall within which resident Sca1ϩ vascular progenitor cells are activities declined with age and were detected at low levels in found in vivo. coronary arteries and transverse aorta from adult mice (Fig. 1B). In aortic cross-sections at P2, strong Ptc1lacZ activity was re- Analysis of Isolated AdvSca1 Cells. AdvSca1 cells were isolated by stricted to the adventitia, with only an occasional positive cell immunoselection and examined for progenitor cell markers. found in the media or intima (Fig. 1C). Adventitial Ptc1lacZ AdvSca1 cells exhibited a CD34ϩ/c-kitϪ/CD140bϩ marker pro- activity was also found in intercostal (Fig. 1D) and femoral (Fig. file (Fig. 3A) and were negative for CD45 and CD68 (data not 1F) arteries. In the mesenteric arcade, Ptc1lacZ staining of shown), similar to the profile reported by Hu et al. (6). Freshly arteries was more intense than that of veins (Fig. 1E), whereas isolated AdvSca1 cells consistently expressed markers of Shh lymphatic vessels were negative [supporting information (SI) signaling including ptc1, ptc2, smo, gli1, gli2, and gli3 (Fig. 3A). lacZ Fig. S1]. Ptc2 activity exhibited very similar patterns (Fig. 1 The distribution of Shh in the extracellular space is governed by D–F Right and Fig. S2). Thus, active Hh signaling was found in multiple binding and transport proteins including hedgehog large and medium-sized arteries and veins in the perinatal period interacting protein-1 (Hhip1), cdo, and boc (reviewed in ref. 19), and was localized to the adventitia of these vessels. all of which are expressed by AdvSca1 cells (Fig. 3A). In addition, AdvSca1 cells contain mRNA for Shh, with few, if any, tran- Shh in the Adventitia. Shh protein was localized to the interface scripts detectable for Dhh or Ihh (Fig. 3A). These results suggest between the media and adventitia in large and medium-sized that AdvSca1 cells both produce and respond to Shh, and that the arteries 2 days after birth (Fig. 2A). This pattern of Shh restricted distribution of Shh in the artery wall is, in part, locally distribution colocalized with both Ptc1lacZ (Figs. 1C and 2B Inset) controlled by factors produced by AdvSca1 cells themselves. and Ptc2lacZ activities. We could not detect Indian hedgehog (Ihh) in postnatal arteries and found only low levels of desert Hu et al. (6) reported that AdvSca1 cells differentiate into hedgehog (Dhh) in the endothelium (data not shown). These SMCs when incubated with PDGF-BB (6). We found that when results indicate that Ptc1lacZ and Ptc2lacZ activities in the artery cultured in DMEM containing 10% serum many AdvSca1 cells Ϸ wall correlate with localized deposition of Shh protein in a ( 30–50%) lost expression of Sca1, gained expression of ␣ ␣ circumferential ring in the extracellular matrix (ECM)-rich space SM Actin, SM22 , calponin, and SM-MHC, and adopted an between the media and adventitia (Fig. 2A). elongated, mesenchymal cell shape (Fig. 3 D and E). To rule out the possibility that up-regulation of SMC markers resulted from Sca1-Positive Cells in the Adventitia. A previous report (6) de- expansion of a pool of medial SMCs carried over in the AdvSca1 scribed Sca1ϩ cells in the adventitia surrounding the aortic root cell isolation, we repeated our experiments in the presence of in adult ApoEϪ/Ϫ mice. One possible role of Shh may therefore inhibitors of cell proliferation (aphidicolin or hydroxyurea). be to maintain Sca1ϩ progenitor cells within this domain of After verifying growth arrest, we observed up-regulation of SMC artery wall. As shown in Fig. 2C, Sca1ϩ cells are restricted to the marker proteins over a similar time course and to a similar extent adventitia (AdvSca1 cells) and exhibit little or no overlap with as seen in the absence of cell cycle inhibitors (Fig. S3). We SM␣Actin-positive cells in the media. A similar distribution was conclude that AdvSca1 cells can directly differentiate into

9350 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711382105 Passman et al. Downloaded by guest on October 1, 2021 Fig. 3. Analysis of AdvSca1 cells. (A) RT-PCR analysis of AdvSca1 cells from adult arteries. (B) Freshly isolated AdvSca1 cells (red) contain SRF (green) Fig. 2. Distribution of Shh and Sca1ϩ cells in the adventitia. (A) Shh protein colocalized with DAPI-stained nuclei (blue) (arrows). (C) Freshly isolated is found outside the aortic external elastic lamina (marked by green autoﬂuo- AdvSca1 cells (red) contain Klf4 (green) colocalized with DAPI-stained nuclei rescence) at P2. (B) Shh protein (red) is found between the media (SM␣Actin, (blue) (arrows). (D) RT-PCR analysis of AdvSca1 cells cultured for 0, 12, or 28 green) and adventitia of descending aorta. (B Inset) Shh (red) is colocalized days. M is total RNA from aortic media used as a positive control for SMC with ␤-gal in aortic sections from Ptc1lacZ mice. (C) Sca1ϩ cells (red) reside markers. This analysis was performed three times with similar results. (E) outside the media (SM␣Actin, green) in P2 aorta. (D) Sca1ϩ cells (green) are AdvSca1 cells cultured in serum-containing medium for 9 days. Many cells found in close proximity to Shh (red) and are embedded within a ␤-gal-positive down-regulate ScaI (red) and up-regulate SM␣Actin (green). (Magniﬁcations: domain (Inset) in aortic adventitia from Ptc1lacZ mice. (E) Mesenteric vessels B and C, ϫ40; E, ϫ10.) stained for SM␣Actin (green) and Shh (red). (F) Femoral arteries stained for SM␣Actin (green) and Sca1 (red). Images are stacked Z-plane sections from confocal microscopy. Adventitial localization of Sca1ϩ cells correlates with the distribution of Shh protein and active Shh signaling in vivo. marrow-derived (6). To gain insight into the origins of AdvSca1 cells in vivo we examined mice from embryonic day 12.5 (E12.5) to 12 weeks after birth. AdvSca1 cells first appeared in the SMC-like cells, and that this differentiation does not require cell perivascular space between the ascending aorta and pulmonary proliferation. trunk between E15.5 and E18.5 (Fig. 4C, arrows), well after the AdvSca1 cells do not express SMC marker proteins in vivo tunica media had acquired SMCs and organized a full comple- (Figs. 2 and 4). However, they contain mRNAs for transcription ment of elastic lamellae. AdvSca1 cells were present in this factors involved in SMC differentiation, including serum re- position at all subsequent time points examined, and they sponse factor (SRF), myocardin family members, and cysteine- increased in number with postnatal growth of the artery wall (Fig. 4 D–F). We then used Wnt1-cre transgenic mice to label rich LIM proteins Csrp1 and Csrp2 (Fig. 3A). In addition, a ␤ subset of freshly isolated AdvSca1 cell nuclei were immunopo- cells of neural crest origin (34). Wnt1-cre-activated -gal reporter sitive for SRF protein (Fig. 3B). However, AdvSca1 cells also labeled neural crest-derived SMCs in proximal aorta, pulmonary trunk, common carotid arteries, and ductus arteriosus, but did express potent silencers of SRF-dependent transcription, includ- not label AdvSca1 cells in these vessels (Fig. 4 G and H). ing Msx1, Klf4, and FoxO4 (Fig. 3D and data not shown). Each of these factors can inhibit SRF and myocardin-dependent SMC Analysis of AdvSca1 Cells in Shh؊/؊ Embryos. To determine whether gene expression (32, 33), and they are down-regulated as Ad- Shh was required for the formation or maintenance of AdvSca1 vSca1 cells acquire SMC markers and increase SRF coactivator cells, we examined ShhϪ/Ϫ mice. Two anatomically defined sites expression (Fig. 3D). Therefore, a subset of AdvSca1 cells may were studied. The presence of AdvSca1 cells at the aortic root, be maintained as SMC progenitors by expression of silencers of where they are first detected during development (Fig. 4C) and SRF-dependent transcription. When AdvSca1 cells were incu- are most abundant in adult ApoEϪ/Ϫ mice (6), was confirmed in bated with VEGF-A (10 ng/ml, 9 days), PECAM1-positive WT mice (Fig. 5A). By contrast, we could not detect AdvSca1 endothelial cell clusters appeared in elongated cords that often cells at the aortic root in ShhϪ/Ϫ embryos (Fig. 5B). A small ␣ colocalized with SM Actin-positive cells (Fig. S4). When incu- number of AdvSca1 cells were found in the transverse portion of bated with BMP2 (50 ng/ml, 20 days), colonies formed that thoracic aorta (Fig. 5 E and F). An absence of septation of the MEDICAL SCIENCES stained with alizarin red or von Kossa’s stain consistent with common truncus arteriosus in Shh-deficient embryos has been differentiation of a subset of AdvSca1 cells to osteogenic cells described (35). AdvSca1 cells were present in the descending (Fig. S4). Under conditions described above, 30–50% of Ad- thoracic aorta of ShhϪ/Ϫ embryos, but always in reduced num- vSca1 cells differentiated to SMC-like cells, Ϸ10% produced bers (Fig. 5 C–F and Fig. S5). The presence of residual AdvSca1 endothelial cells, and 1–5% formed osteogenic colonies in vitro. cells suggests that factors other than Shh are involved in homing and/or maintenance of these cells. Because Dhh expression was Developmental Origins of AdvSca1 Cells. Bone marrow reconstitu- detected in the aorta, low levels of Dhh may allow some of these tion experiments previously showed that AdvSca1 cells are not cells to escape a requirement for Shh in the artery wall.

Passman et al. PNAS ͉ July 8, 2008 ͉ vol. 105 ͉ no. 27 ͉ 9351 Downloaded by guest on October 1, 2021 Fig. 5. AdvSca1 cells in ShhϪ/Ϫ arteries. (A–D) Aortic tissues from WT (A and C) and ShhϪ/Ϫ (B and D) embryos at E18.5. Cross-sections through aortic root (A and B) and descending aorta (C and D) were immunostained for Sca1 (red), SM␣Actin (green), and DAPI (blue). (E) Solid lines in indicate relative positions of sections in A–D.(F) Dotted line boxes in A–D correspond to aortic segments shown here. AdvSca1 cells are absent in aortic root, greatly reduced in ascending and transverse aorta (termed ascending), and diminished in descend- Fig. 4. AdvSca1 cells in developing aorta. (A–F) Tissues were obtained at the ing aorta of ShhϪ/Ϫ embryos. time points indicated. Cross-sections through the aortic root were stained for Sca1 (red), SM␣Actin (green), and DAPI (blue). AdvSca1 cells first appear between E15.5 and E18.5 in the adventitial space between aorta and pulmo- increases in blood flow and blood pressure that are accom- nary trunk (white arrows in C) and persist in this location into adulthood. (G) Cross-section from Wnt1-cre/R26R (5 weeks old) aortic root stained for ␤-gal panied by corresponding increases in smooth muscle contrac- activity and nuclear fast red. SMCs (blue) but not adventitial cells originate tile protein mass (37). After 3 months of age, growth of the from neural crest. Dashed box is shown at higher magnification in H.(H) artery wall is diminished, cell proliferation rates are low, and Cross-section through aortic root shows no overlap between ␤-gal-positive wall structure has reached an adult state. We found PtclacZ ϩ and AdvSca1 cells. Ao, aorta; PT, pulmonary trunk. (Magnification scale bar activities and Shh protein levels were maintained at low levels ϫ in E applies to A–F; G, 10.) in adult mouse arteries, suggesting that adventitial Shh signaling activity correlates with rapid postnatal growth and Discussion remodeling of the artery wall. We report the characterization of a Shh signaling domain in the Distribution of Shh in the Adventitia. Ptc1lacZ and Ptc2lacZ activities adventitial layer of vessel wall. This domain is strongly positive for both Ptc1lacZ and Ptc2lacZ activities and colocalizes with a ring were colocalized with Shh protein deposited between the media of Shh protein deposited between the media and adventitia. and adventitia. Movement of Hh proteins in the extracellular Embedded within this domain is a population of Sca1-expressing space is limited by covalent lipid modifications at both amino- vascular progenitor cells, termed AdvSca1 cells, that can differ- and carboxyl-terminal domains (38, 39). Lipid-modified Hh entiate to SMC-like cells ex vivo. The number of AdvSca1 cells proteins are secreted as multimers that interact with heparan in the aortic root is greatly diminished in ShhϪ/Ϫ mice at E18.5. sulfate proteoglycans (HSPGs), which further restricts their These findings suggest that Shh signaling may play important movement (40, 41). Our data suggest that Shh protein is con- roles in development of the adventitia and maintenance of a centrated between the media and adventitia because of local resident population of Sca1ϩ vascular progenitor cells in the synthesis, secretion, and retention of Shh multimers by ECM artery wall. components, such as the HSPG perlecan (42), in the artery wall. PtclacZ-positive cells are located throughout the adventitia, Hh Signaling in Postnatal Artery Wall. Ptc1lacZ and Ptc2lacZ activi- whether directly adjacent to Shh protein at the media-adventitia ties in the adventitia of large and medium-sized arteries were interface or several cell layers removed. We do not yet know greatest between 1 and 10 days after birth. Although the basic whether this pattern of PtclacZ activity in vivo is the result of structure of major arteries is established at birth, substantial paracrine secretion of Shh by AdvSca1 cells themselves, ECM growth and remodeling occur in the perinatal period. For retention of Shh produced from another source, or some com- example, the number of SMCs in rat aorta increases 2.5-fold, bination of both. However, freshly isolated AdvSca1 contain wall thickness doubles, and collagen and elastin contents readily detectable amounts of Shh mRNA, suggesting that local increase Ͼ3-fold in the first month after birth (36). These synthesis and secretion of Shh is a normal function of the arterial changes in wall structure are adaptations to large perinatal adventitia.

9352 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711382105 Passman et al. Downloaded by guest on October 1, 2021 Analysis of Isolated AdvSca1 Cells. Embedded within the PtclacZ- field (51). Lineage-specific cre mice are available for both of positive adventitial domain, we found a population of Sca1- these progenitors to test whether AdvSca1 cells arise from expressing cells that were negative for marker proteins of either of these sources. differentiated SMCs. However, when removed from the adventitia and placed in serum-containing medium, a significant Summary. We characterize a novel domain of Shh signaling in fraction (Ϸ30–50%) of these cells lost Sca1 from the cell the arterial adventitia and show that a population of Sca1ϩ surface, acquired a well spread mesenchymal cell shape, and vascular progenitor cells resides within this domain. Because expressed the SMC marker proteins SM␣Actin, SM22␣, cal- the adventitia lies between the vessel wall and surrounding ponin, and SM-MHC by 12–28 days. When AdvSca1 cells were tissues, progenitor cells within the adventitia could, in prin- exposed to BrdU to label replicating cells, a significant number ciple, contribute to remodeling, repair, or disease processes in of Sca1–BrdU double positive cells were found after 7 days, either the vessel wall or in neighboring nonvascular tissues. indicating that some AdvSca1 self-renew while, at the same Because essentially all tissues of the body contain blood time, others differentiate to SMC-like cells under the same vessels, the maintenance and fate of adventitial progenitor conditions (Fig. S3). These results suggest that AdvSca1 cells cells described here and by Hu et al. (6) may be relevant to a are a heterogeneous mixture of cells that can either self-renew wide variety of normal and pathogenic processes. Future work or respond to extracellular signals and differentiate to SMC- will examine the role of Hh signaling in responses of the like cells in vitro. adventitia to tissue injuries involving both vessels and surrounding nonvascular cell types. Maintenance of a Progenitor Phenotype by Transcriptional Corepres- sors. AdvSca1 cells do not express SMC marker proteins in vivo. Materials and Methods It was intriguing, therefore, to find that AdvSca1 cells con- Animals Used. All protocols were approved by the Institutional Animal Care tained transcripts for SRF and myocardin family members and and Use Committee at the University of North Carolina. Mice used include were immunopositive for SRF when examined immediately Ptc1lacZ (Ptctm1Mps) (26), Ptc2lacZ (Ptc2tm1Dgen; Jackson Laboratories; 005827), after isolation from the artery wall. These transcription factors Wnt1cre (Jackson Laboratories; 003829), Shhtm1Amc (Jackson Laboratories; are strong activators of SMC gene expression (37, 43–45) and 003318), Rosa26 (52), CD1 (Charles River), 129/SvEvJ and C57BL/6J (Jackson are necessary for vascular SMC differentiation in vivo. Yet Laboratories). Noon on the day of vaginal plug was designated E0.5. SRF also interacts with potent transcriptional corepressors, ␤ and it is the competition between coactivators and corepres- Whole-Mount -Gal Activity. Embryos or tissues were fixed in fresh 0.2% glutaraldehyde in 0.1 M sodium phosphate, 5 mM EGTA, 2 mM MgCl2, pH 7.3, sors that determines whether SRF-dependent target genes are for1honice, washed three times with rinse buffer (10 mM sodium phosphate, transcribed (44–48). Indeed, we found that the transcriptional 2 mM MgCl2, and 0.1% Triton X-100, pH 7.4) for 30 min each at room corepressors Msx1, Klf4, and FoxO4 were also expressed by temperature, and stained as described in SI Text. AdvSca1 cells. Msx1 forms a ternary complex with SRF and myocardin and inhibits binding of SRF-myocardin to CArG Immunofluorescence Staining. Tissues were fixed in 4% paraformaldehyde box motifs in SMC target genes (32). Klf4 is a zinc finger- (PFA), rinsed in PBS, saturated with 20% sucrose for cryoprotection, embed- containing protein that binds to a GC-rich element (TCE) and ded in agar, and frozen in OCT. Cryosections (12 ␮m) were fixed and stained inhibits SRF-dependent transcription of multiple SMC marker as described in SI Text. Cultured cells were fixed in 4% PFA for 15 min at room genes (47). The forkhead transcription factor FoxO4 interacts temperature, then stained as described in SI Text. Primary antibodies and with myocardin and inhibits its coactivator function for SRF- imaging methods are described in SI Text. dependent SMC gene expression (49). These results are rem- Ϸ iniscent of a report by Matsuura et al. (50), showing that Sca1ϩ AdvSca1 Cell Isolation and Culture. The ascending aorta, aortic arch, and 1 cm of the descending aorta were dissected from adult mice and placed in cells from adult mouse hearts express Nkx2–5 and GATA4, PBS on ice. Endothelium was removed with a cotton swab. Adventitia was two transcription factors important for cardiac myocyte dif- dissected and rinsed in HBSS, digested with 14 mg/ml collagenase type 2 ferentiation, yet they do not express markers of differentiated (Worthington) and 0.75 mg/ml elastase (Roche) in HBSS for2hat37°C with ϩ myocardial cells. Heart-derived Sca1 cells differentiate to gentle rocking, and filtered (70 ␮m). Cells in the filtrate were pelleted at beating cardiomyocytes when exposed to oxytocin in vitro, thus 300 ϫ g then rinsed in PBS ϩ 0.5% BSA. Aortic Sca1ϩ cells were isolated by confirming their cardiogenic potential (50). These findings in using anti-Sca1 immunomagnetic MicroBeads and a MACS cell separation heart and artery wall suggest a model in which Sca1ϩ progen- system (Miltenyi). Cells were passed over two consecutive columns to ϩ itor cells in postnatal tissues are specified for certain cell fates increase purity of the isolation. A typical Sca1 fraction thus prepared is by expression of transcription factors that are required for 1–4% of total cells, with 80–95% of isolated cells positive for Sca1 antigen, and Ͻ1% of isolated cells positive for SM␣Actin by immunostaining. those fates. The maintenance of a SMC progenitor phenotype ϩ Isolated Sca1 cells were cultured in DMEM (Sigma) plus 10% FBS (HyClone) may therefore critically depend on expression of transcrip- and 1ϫ antibiotic/antimycotic solution (Gibco) at 37°C, 5% CO2. Cells were tional corepressors that block SRF-dependent transcription seeded in 48-well tissue culture plates at 8 ϫ 103 cells per well. Growth and recruit chromatin remodeling complexes to silence gene factors tested were VEGF-A (10 ng/ml; Clonetics) and BMP2 (50 ng/ml, expression. PeproTech).

Origins of AdvSca1 Cells. The origins of AdvSca1 cells are unclear. RT-PCR Analysis. Total cellular RNA was isolated by guanidinium isothiocya- Hu et al. (6) found no evidence that AdvSca1 cells arise from nate denaturation and phenol/chloroform extraction as described (53). Two- bone marrow. We investigated this question for aortic AdvSca1 step RT-PCR was carried out with the GeneAmp RNA PCR kit (Applied Biosys- cells by using Wnt1-cre transgenic mice in which ␤-gal is tems) according to the manufacturer’s instructions. Primer sequences and PCR permanently expressed in neural crest-derived cells (34). Ad- conditions are listed in Table S1. MEDICAL SCIENCES vSca1 cells directly adjacent to Wnt1-cre-positive aortic SMCs ACKNOWLEDGMENTS. We thank Da-Zhi Wang, Frank Conlon, and Jim did not express the neural crest marker. Moreover, our devel- Faber for helpful discussions. This work was carried out by J.N.P. in partial opmental time course studies showed that AdvSca1 cells did fulﬁllment of requirements for the PhD degree in the Curriculum in Ge- not appear in aortic arch arteries until between E15.5 and netics and Molecular Biology, University of North Carolina. This work was E18.5, much later than the migration of neural crest-derived supported by National Institutes of Health Grants HL-19242 (to M.W.M.), Ϸ HL-07816 (to C.M.), GM-00851 (to J.N.P.), and HL43174 and HL71993 (to SMC progenitors into the aortic arch complex E9.5. Two V.L.B.), National Research Service Award F32 HL68484 (to K.H.), American other lineages contribute SMCs to the aorta and other great Heart Association Grant 0715320U (to J.N.P. and M.W.M.), and the Carolina vessels, i.e., paraxial somitic mesoderm and secondary heart Cardiovascular Biology Center.

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