Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling

Abstract

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1⁺CD31^dimvascular endothelial (VE)-cadherin⁻CD45⁻ precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1⁺ precursor cell, but not the VE-cadherin⁺ endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1⁺ precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1⁺ local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Vascular smooth muscle cells (VSMC) play an important role in vascular homeostasis in the adult and the embryo. Identification of VSMC progenitors in the embryo may define the source of new VSMC seen during arteriogenesis and atherosclerotic plaque formation in the adult. However, the developmental origin of VSMC is still under investigation. From lineage-tracing experiments using either a genetic reporter or cross-species transplants, embryonic tissues such as the neural crest, the proepicardium, and the somites have been shown to be sources of VSMC during development (1, 2). Although numerous cells from the somites migrate to the site of dorsal aorta formation during avian development, only a small percentage of these migrating cells actually integrate into the dorsal aorta (3), suggesting that further differentiation is required to specify a VSMC fate.

A few studies have been performed to identify immediate VSMC precursors localized to nascent vessels. Mesoangioblasts are progenitor cells isolated from dorsal aorta of embryonic day (E)9.5 mouse embryos and can be expanded in culture as clones expressing both endothelial and mesenchymal markers (4). Mesoangioblasts have been shown to differentiate into VSMC in vitro and in mouse–chick chimeras (4). Endothelial cells (EC) also have been suggested to be a possible source of VSMC through the process of endothelial–mesenchymal transdifferentiation in an avian developmental model and in pathological arterial neointimal thickening in pulmonary hypertension (5, 6). However, no studies have examined whether the endothelium is a source of VSMC precursor cells during mammalian development.

The Notch signaling pathway has been shown to induce endothelial-to-mesenchymal transdifferentiation during cardiac cushion formation and in mature EC in vitro (7, 8). Disruption of Notch signaling during embryonic development leads to cardiac malformations as well as to vascular defects (9). A decrease in aortic VSMC coverage has been observed in some Notch mutants; however, the cellular mechanism of this disruption has not been characterized (10). Blockade of Notch signaling in specifically mature mouse VSMC does not result in embryonic vascular defects, suggesting that mature VSMC do not require Notch during embryogenesis (11). However, Notch activation in murine Pax3⁺ neural crest-derived cells has been shown to be required for VSMC development of the branchial arch arteries and vessels derived from the outflow tract (12), and EC expression of the ligand Jagged1 has been suggested to provide the Notch-activation signal (13). The requirement of Notch signaling from sources other than the neural crest for VSMC differentiation in mammalian systems has not been investigated, and where VSMC Notch activity is required in the path from neural crest to branchial arch also is not clear.

To determine whether endothelial–mesenchymal transdifferentiation is involved in the development of VSMC in a mammalian system, we used a binary tetracycline-inducible transgenic system to follow EC during murine vascular development. Two endothelial promoters were used: one that is endothelial specific, vascular endothelial (VE)-cadherin, and another that is more promiscuous, Tie1. We found that mature endothelium is not a source of VSMC during murine development. We also observed that the Tie1 promoter is active in a VSMC precursor that gives rise to VSMC following localized Notch activation in vivo. Our results suggest that Notch signaling is essential for the differentiation of VSMC from local Tie1⁺CD31^dimVE-cadherin⁻CD45⁻ precursor cells.

Results

VSMC Are Derived from a Tie1⁺CD31^dimVE-Cadherin⁻CD45⁻ Precursor Cell.

To determine whether EC can transdifferentiate into VSMC during mammalian development, we used a tetracycline-inducible (Tet-off) binary transgenic system to mark EC-derived cells. Mice expressing the tetracycline transcription activator (tTA) under an EC-specific promoter were crossed with responder mice expressing a nuclear-localized β-gal reporter under the tTA-activated tetracycline-responsive promoter tetOS in the absence of tetracycline (Fig. S1A) (14). Both EC promoter driver lines, Tie1-tTA and VE-cadherin-tTA (VECtTA) were active in the embryonic vasculature during early stages of VSMC development (Fig. S1 B–E) (14, 15). LacZ⁺ cells from both driver lines were capable of contributing to EC-derived mesenchymal cells in the cardiac cushion mesenchyme at E10.5 (Fig. S1 F–G), demonstrating that these models can be used to follow endothelial-to-mesenchymal transition.

We next examined the descending aorta at E12.5 to determine whether EC are a source of VSMC. β-Gal activity was detected in the EC and EC-derived hematopoietic cells but not in the surrounding periendothelial cells in VECtTA:TetOSLacZ (VEC:LacZ) embryos (Fig. 1A). In contrast, the Tie1 promoter drove the expression of LacZ not only in EC but also in the surrounding VSMC, as verified by costaining of α-smooth muscle actin (SMA), the earliest marker of VSMC (5), and by β-gal activity (Fig. 1B). This phenomenon was not restricted to the descending aorta but was observed in all vascular beds examined (Fig. S1 H–S), even though VSMC from the different arteries arise from different tissues of origin (16). β-Gal⁺ VSMC also were present in the dorsal aorta of E10.5 embryos, and the proportion of β-gal⁺ VSMC decreased as the embryo developed (Fig. 1C). These findings suggest that Tie1 can be used to mark either a subset of VSMC or a VE-cadherin⁻ precursor cell that is capable of VSMC differentiation. Further, these data show that mature VE-cadherin⁺ EC are not an embryonic source of VSMC during mammalian development.

Fig. 1.

The Tie1 promoter, but not the VE-Cadherin promoter, is active in VSMC precursor cells. (A and B) β-Gal activity in the descending aorta of E12.5 embryos. (A) VEC:LacZ embryos showed EC (CD31 stained in green) and rare hematopoietic cells (arrow) expressed LacZ (blue). (B) In Tie1:LacZ embryos, β-gal activity (blue) overlapped with both CD31 (green) and SMA expression (red). (C) Quantification of β-gal⁺ cells in SMA⁺ VSMC in the Tie1:LacZ embryo dorsal/descending aorta. (D) Cells positive for LacZ expression were isolated by FACS from E10.5 Tie1:LacZ and VEC:LacZ embryos. (E) qRT-PCR analysis of EC and mesenchymal markers in sorted Tie1:lacZ⁺ and VEC:LacZ⁺ populations compared with whole embryos. (n = 3; *P < 0.05). For primer information, see Table S2. Error bars in C and E represent SEM.

To characterize better the Tie1⁺ vascular cells at the initiation of VSMC development, LacZ⁺ cells from E10.5 Tie1tTA:TetOSLacZ (Tie1:LacZ) or VEC:LacZ embryos were flow-sorted using the FITC-based fluorescent β-gal substrate fluorescein di[β-d-galactopyranoside] (FDG) (Fig. 1D). Quantitative RT-PCR (qRT-PCR) analysis of E10.5 Tie1⁺ and VE-cadherin⁺ embryonic cells from both transgenic models revealed an enrichment of EC markers (CD31, CD34, Flk-1, Tie1, Tie2, and VE-cadherin) compared with the whole embryo, as expected (Fig. 1E). Although a similar level of EC marker expression was seen in the Tie1⁺ and the VE-cadherin⁺ populations, suggesting the same level of enrichment for EC, the Tie1⁺ population showed increased expression of mesenchymal progenitor markers Msx2 and SMA, whereas expression of neural crest markers Pax3 and Pax7 was not detected (Fig. 1E).

Coexpression of EC (CD31) and mesenchymal markers (SMA) was confirmed in a subpopulation of flow-sorted cells from E10.5 Tie1:LacZ embryos by immunofluorescent staining (Fig. 2A), suggesting the presence of a Tie1⁺ vascular precursor cell. However, expression of CD31 and Tie1 has been described in hematopoietic cells in addition to EC. To characterize further the cell lineage of the CD31⁺ population that expresses mesenchymal markers, CD31⁺ cells from wild-type E10.5 embryos were flow-sorted based on VE-cadherin and CD45 expression, and qRT-PCR analysis was performed to examine marker expression in each population (Fig. 2B). The CD31⁺VE-cadherin⁻CD45⁻ population expressed the early VSMC lineage markers h1-Calponin (17), Msx2, and SMA at levels more than 10-fold higher than seen in the CD31⁺VE-cadherin⁻CD45⁺ (hematopoietic) population, demonstrating that the presumptive VSMC precursor does not express CD45 and suggesting that these cells may not be of hematopoietic origin (Fig. 2C). The CD31⁺VE-cadherin⁻CD45⁻ population expressed the EC markers Flk1 and VE-cadherin at a lower level than seen in the CD31⁺VE-cadherin⁺CD45⁻ (mature EC) population, as expected. However, the expression of Tie1 was comparable in the two populations, confirming the expression of Tie1 in the VSMC precursor population (Fig. 2D). The CD31⁺VE-cadherin⁻CD45⁻ population also lacked expression of Sca-1, which is expressed on embryonic EC and hematopoietic cells (18, 19). Finally, markers for mature smooth muscle cells, Myocardin, SM22-α, and Smooth muscle myosin heavy chain (SM-MHC) were not expressed at detectable levels in the CD31⁺VE-cadherin⁻CD45⁻ population, showing that this presumptive precursor population does not contain mature VSMC (Fig. 2E). Interestingly, the populations also differ in the surface expression level of CD31, with the EC population showing the highest level and the hematopoietic and the CD31⁺VE-cadherin⁻CD45⁻ populations showing dimmer CD31 expression (CD31^dim) (Fig. S2). Taken together, these findings define the existence of a Tie1⁺CD31^dimVE-cadherin⁻CD45⁻ VSMC precursor that is positive for SMA and Msx2 and suggest that the Tie1tTA driver line can be used to isolate these presumptive VSMC precursors.

Fig. 2.

Tie1⁺CD31^dimVE-cadherin⁻CD45⁻ cells express both endothelial and mesenchymal markers but not mature smooth muscle markers. (A) (Upper) Tie1:LacZ⁺ cells pooled from four E10.5 embryos were flow sorted by FDG (see Fig. 1D), cytospun, and immunostained for CD31 (green) and SMA (red) and with a DAPI nuclear counterstain (blue). (Lower) The proportions of CD31⁻SMA⁻, CD31⁺SMA⁻, CD31⁻SMA⁺, and CD31⁺SMA⁺ cells were quantified. (B) Single-cell suspensions from E10.5 wild-type embryos were analyzed for surface marker expression on the viable cells. The CD31⁺ population contained ∼20% CD45⁺ (hematopoietic), ∼30% VE-cadherin (VEC)⁺ (endothelial), and ∼50% VEC⁻CD45⁻ cells. (C) CD31⁺VEC⁻CD45⁺ hematopoietic cells express low or nondetectable levels of early VSMC markers compared with the CD31⁺VEC⁻CD45⁻ population. (D) The CD31⁺VEC⁻CD45⁻ population (presumptive VSMC precursors) expresses low levels of EC markers [Flk-1 and VE-cadherin (VEC)] compared with VEC⁺ mature EC but express comparable levels of Tie1. (E) The CD31⁺VEC⁻CD45⁻ population does not express the mature VSMC markers Myocardin, SM22-α, and SM-MHC. ND, not detected. *P < 0.05. Error bars in C and D represent SEM.

Blockade of Notch Signaling in Tie1⁺ Cells Leads to Hemorrhage Localized in Newly Forming Vasculature During Embryonic Development.

To determine whether Notch is required for vascular precursors to generate mature VSMC, the Tie1tTA driver line was used to express an N-terminal dominant-negative mutant of Mastermind-like 1 (dnMAML) fused to GFP in presumptive VSMC precursors. dnMAML inhibits Notch activity by binding Notch and another binding partner, CSL [CBF1/Su(H)/Lag2 protein] but not to coactivators required for transcription of target genes, as demonstrated by specific blockade of Notch target gene induction (Fig. S3A) (20). The VECtTA driver line also was used to express dnMAML to exclude effects caused by blockade of Notch signaling in mature EC.

Constitutive expression of dnMAML in EC under the control of either the VE-cadherin or Tie1 promoter led to embryonic lethality with developmental delay at E10.5 (Fig. S3 B and C). The cardiac defect-induced lethality is consistent with previous studies using Tie2Cre-mediated inactivation of Notch1 (21). To bypass early embryonic death, dnMAML-GFP expression was blocked by adding tetracycline to the drinking water. Withdrawal of tetracycline blocked expression of the Notch target, Hey2, in E10.5 EC and induced dnMAML-GFP expression within the first 24 h (Fig. S3 D and E) but had no effect on the expression or activity of other putative MAML binding partners (Fig S3D) (12, 22).

Induction of dnMAML-GFP at E10.5 for 2 d led to localized hemorrhage at E12.5 in the forebrain and hindbrain of Tie1tTA:TetOSdnMAML (Tie1:dnMAML) double-transgenic embryos (Fig. 3A) but not in VECtTA:TetOSdnMAML (VEC:dnMAML) double-transgenic embryos (Fig. 3B). Tie1-driven induction of dnMAML at E11.5 for 2 d led to hemorrhage in the midbrain and the tip of the tail, in addition to the forebrain and the hindbrain, at E13.5 (Fig. 3C). In contrast, at E14.5 after 2-d induction of dnMAML, embryos displayed hemorrhage at the tip of the snout and the interdigital zones but not in the brain (Fig. 3E). Induction of dnMAML in Tie1⁺ cells at E14.5 for 2d led to hemorrhages in superficial vessels at E16.5 (Fig. 3G). The hemorrhagic regions correspond to areas undergoing active remodeling at the time of Notch blockade. However, after maturation of the vasculature in a particular region, Tie1 promoter-driven dnMAML expression no longer resulted in a phenotype in that region. The VEC:dnMAML embryos matured normally with 2-d induction of dnMAML (Fig. 3 B, D, F, and H and Table S1), suggesting that the hemorrhagic defect is caused by blockade of Notch signaling in a Tie1⁺VE-cadherin⁻ population and not by secondary effects of Notch blockade in mature VE-cadherin⁺ EC.

Fig. 3.

Notch blockade in Tie1-promoter active vascular cells leads to localized hemorrhage. (A–H) Whole-mount micrographs of embryos expressing dnMAML driven by the Tie1 promoter or the VE-cadherin promoter for 2 d before dissection. (A) At E12.5 Tie1:dnMAML embryos show hemorrhage localized to the forebrain and hindbrain. (C) At E13.5, Tie1:dnMAML embryos display widespread hemorrhage in the entire brain, as well as at the tip of the tail. (E) At E14.5, Tie1:dnMAML embryos display hemorrhage in the tip of the snout and the interdigital zones of the developing limbs. (G) At E16.5, Tie1dnMAML embryos show superficial hemorrhage in the torso and head. At all time points examined, no gross morphological defects are identified in the VEC:dnMAML embryos (B, D, F, and H). Red arrows indicate location of hemorrhage. Tg, transgenic. See also Table S1.

To rule out the possibility that different phenotypes were observed because the two promoters drive dnMAML expression to different levels, we examined dnMAML-GFP expression by flow cytometry in Tie1:dnMAML and VEC:dnMAML E12.5 embryos after 2 d of tetracycline withdrawal. In both strains the majority of the CD31⁺ cells coexpressed dnMAML-GFP. However, there was a significantly higher proportion of CD31⁺GFP⁻ cells in the VEC:dnMAML embryos (Fig. S4A), corresponding to the Tie1⁺CD31^dimVE-cadherin⁻ VSMC precursors described above. The mean GFP fluorescence intensities for the two strains were comparable (Fig. S4 B and C), indicating equal expression level of dnMAML-GFP with both promoters. Immunofluorescence analysis of the descending aorta showed colocalization of CD31 and GFP in the endothelium of both transgenic lines (Fig. S4 E and G). Interestingly, periendothelial CD31⁺ cells coexpressing dnMAML-GFP also were observed in the descending aorta of Tie1:dnMAML (Fig. S4E),but not VEC:dnMAML (Fig. S4G) embryos. These CD31⁺ periendothelial cells also coexpressed SMA, likely representing the Tie1⁺CD31^dim precursor cells that failed to differentiate into mature VSMC (Fig. 4A). The presence of GFP⁺ periendothelial cells coexpressing SMA also was confirmed and quantified (Fig. 4 C and D). The proportion of dnMAML-GFP⁺ periendothelial cells was decreased significantly as compared with the LacZ⁺ control, in which Notch activation was not perturbed, suggesting that dnMAML-expressing cells failed to differentiate into VSMC.

Fig. 4.

Periendothelial localization of Tie1⁺CD31^dimVE-cadherin⁻ cells that are blocked from undergoing VSMC maturation. (A and B) The descending aortas from E12.5 Tie1:dnMAML (A) and VEC:dnMAML (B) embryos were immunostained for SMA (red) and CD31 (green), and nuclei were stained with DAPI (blue). In Tie1:dnMAML embryos, periendothelial coexpression of CD31 and SMA was observed (arrows in A), which was absent in the VEC:dnMAML embryos (B). (C) The periendothelial GFP⁺ cells in Tie1:dnMAML embryos also costained for SMA (arrows). (D) Quantification of GFP⁺ periendothelial SMA⁺ cells in E12.5 Tie1:dnMAML descending aortas (n = 4). Error bars represent SEM.

Notch Signaling Is Required for Differentiation of Tie1⁺ Precursors into VSMC.

To confirm that the hemorrhagic defect observed in Tie1:dnMAML embryos was secondary to defective VSMC maturation, we quantified arterial VSMC coverage by staining for SMA in the Tie1:dnMAML double-transgenic embryos at E12.5 following 2 d of dnMAML induction. In both the descending aorta and carotid arteries, we observed a reduction in the thickness of the VSMC layer in the double-transgenic embryos compared with single-transgenic littermates (Fig. 5 B, F, and J and Fig. S5 A and B). The vertebral artery in the Tie1:dnMAML mutants appeared to be collapsed, with no VSMC around the vessel (Fig. 5F); this observation correlates with the cranial hemorrhage seen at this time. The limbs from E14.5 embryos were necrotic; thus it was not possible to subject the vasculature to the same type of analysis. However, no difference was observed in smooth muscle coverage of the arteries in the VEC:dnMAML double-transgenic embryos, (Fig. 5 D, H, and L and Fig. S5 C and D), suggesting that the VSMC defect was caused solely by the blockade of Notch activity in the Tie1⁺VE-cadherin⁻ population and not by a paracrine effect or by heterotypic signaling from EC to VSMC. As an alternate measure of VSMC numbers, we also quantified the proportion of surface PDGFR-β⁺ cells by flow cytometry of single-cell suspensions from E12.5 embryos and found a significant decrease in the proportion of PDGFR-β⁺ cells in the Tie1:dnMAML double-transgenic embryos as compared with littermate controls (Fig. S5E).

Fig. 5.

Notch blockade in Tie1⁺ vascular progenitors impedes VSMC differentiation in vivo. (A–L) SMA expression in E12.5 embryonic arteries in Tie1:dnMAML and VEC:dnMAML embryos 2 d after dnMAML induction was detected by immunostaining. Decrease in SMA expression is seen in the Tie1:dnMAML descending aorta (A and B) (P = 0.009), vertebral arteries (E and F), and carotid arteries (I and J) (P < 0.0001), but not in VEC:dnMAML aorta and arteries (C and D, G and H, K and L). For quantification, see Fig. S5 A and B. (Scale bars, 50 μm.) Tg, transgenic.

Examination of the descending aorta in E14.5 Tie1:dnMAML embryos following 2-d induction of dnMAML did not reveal a difference in VSMC coverage (Fig. S6 A and B). At E12.5, when dnMAML was induced in these embryos, multiple layers of VSMC already invest the descending aorta (Fig. 5 A and C), suggesting that blocking Notch in Tie1⁺ precursor cells does not affect VSMC coverage of mature arteries because a mature VSMC layer already has been established. Consistent with this observation is the absence of periendothelial dnMAML-GFP expression in the E14.5 descending aorta (Fig. S6A), despite detection of dnMAML-GFP expression in the endothelium, suggesting a relative decrease in the Tie1⁺VE-cadherin⁻ precursor population in the mature vessel.

We examined VSMC coverage along the entire length of the carotid artery to confirm that Notch is required for maturation, rather than maintenance, of VSMC. Although both the proximal and distal halves of the carotid artery in Tie1:dnMAML embryos showed reduced VSMC coverage as compared with littermate controls (Fig. S6 C–G), the decrease in VSMC was more pronounced in the less mature distal half (Fig. S6H). This finding suggests that, as the embryo grows and as the distal region of the artery is actively invested with mature VSMC, blocking Notch signaling in Tie1⁺ precursor cells has a greater effect on mature VSMC proportions. Thus, our findings suggest that Tie1⁺CD31^dimVE-caherin⁻CD45⁻ cells represent local VSMC precursors and that VSMC from different segments of the same vessel develop independently, so that mature VSMC of one segment do not compensate for the lack of precursor differentiation in a less mature segment.

The process of arteriogenesis also occurs in the adult during recovery from ischemia. Previous studies have shown that Notch activation is essential for postischemic arteriogenesis, although the cell population affected was not identified (23). Using a femoral artery ligation model, we examined the effect of blocking Notch in Tie1⁺ cells in the adult. Tie1tTA:dnMAML mice were maintained on tetracycline until induction of dnMAML 2 d after femoral artery ligation to examine the effect on VSMC remodeling rather than on the initial vasodilatory response to ischemia (24). Tie1:dnMAML mice showed impaired recovery of perfusion 10 d after ischemia compared with littermate controls, but the VEC:dnMAML mice demonstrated the same level of recovery as controls (Fig. 6A). Maintenance of perfusion through collateral arteries requires outward remodeling and increased VSMC coverage. We thus examined the effect of Notch blockade in Tie1⁺ precursor cells on VSMC coverage of the collateral arteries. We observed a significant decrease in arterial wall thickness in the Tie1:dnMAML mice as compared with controls (Fig. 6B), suggesting a role for Notch activation in Tie1⁺ cells in adult arteriogenesis. The VEC:dnMAML mice did not show a defect in arterial wall remodeling after induction of ischemia (Fig. 6B), demonstrating that blockade of Notch in mature EC does not affect arteriogenesis. Our results suggest that Notch activation in a Tie1⁺ VSMC precursor also is required in the formation of mature VSMC in the adult.

Fig. 6.

Blockade of Notch signaling in Tie1⁺ progenitor impedes adult arteriogenesis in an HLI model. (A) Tie1:dnMAML and VEC:dnMAML mice show normal perfusion before femoral artery ligation in the absence of Notch blockade. However, Tie1:dnMAML double-transgenic animals show reduced recovery of perfusion as compared with control mice with dnMAML induction. VEC:dnMAML mice show recovery similar to that of controls. (B) Tie1:dnMAML mice show less collateral artery VSMC coverage than control mice. Collateral artery remodeling is unaffected in VEC:dnMAML mice. Error bars represent SEM. Tg, transgenic.

Discussion

During development, VSMC are derived from different embryonic tissues (16). Previous studies on the developmental origins of VSMC have focused on derivation of VSMC from distant embryonic tissues, such as the neural crest and somites (1, 2, 25). Here, we show that during mammalian vascular development a local Tie1⁺ precursor population gives rise to VSMC in different arterial beds, including ones that previously were identified as neural crest- or somite-derived. This Tie1⁺ precursor expresses another EC marker, CD31, as well as SMA and Msx2, but not VE-cadherin or CD45. Further, our data suggest that this Tie1⁺CD31^dimVE-cadherin⁻CD45⁻ precursor requires Notch signaling for differentiation into VSMC in the embryo and adult (Fig. S7).

Two reports have suggested that murine ES cells differentiate into VSMC via an EC intermediate (26, 27). However, the ES cell-derived EC expressed EC markers at a much lower level and mesenchymal markers at a much higher level than seen in mature EC (27). The isolation of putative EC from differentiating ES cells with EC markers such as CD31 or Tie1 may result in the enrichment of immature EC or VSMC precursors, in addition to mature EC (26, 28). A recent study also suggested that pulmonary artery VSMC are derived from EC, based on studies using the Tie1 promoter (29). Studies using the Tie1 promoter for its EC-specific activity may need to consider the presence of this Tie1⁺ VSMC precursor population, because our findings suggest that VE-cadherin⁺ mature EC are not a source of VSMC in the embryonic or adult artery.

Many signaling pathways, including Notch, have been implicated in VSMC development (10, 30). Notch up-regulates transcription of the mesenchymal markers SMA and PDGF receptor β (PDGFR-β) and drives endothelial–mesenchymal transdifferentiation (8, 31, 32). The effect of Notch signaling on VSMC of neural crest and somitic origin has been studied using mammalian and avian models, respectively (3, 12, 33). Somitic cells expressing constitutively active Notch integrate preferentially into the avian dorsal aorta as EC (34). In contrast to these observations in chick development, we have seen that Notch signaling is required for the differentiation of precursor cells that can give rise to VSMC of different embryonic arteries in a mammalian system. The Tie1⁺ precursor appears to represent an immediate vascular precursor predisposed to a VSMC fate in the presence of Notch activation, regardless the embryonic tissue from which the cells originally migrated.

Failure of a local progenitor to differentiate into VSMC also provides insight into the process of embryonic VSMC development, because the pattern of defects defined by Notch inhibition corresponds to sites of active arteriogenesis. Our results suggest that the differentiation of VSMC in the thoracic dorsal aorta initiates before E10.5, because dnMAML expression in the Tie1⁺ precursor at E10.5 results in a mixture of mature VSMC and immature VSMC precursors, marked by the SMA⁺CD31⁺ phenotype, circumscribing the E12.5 aorta. In the carotid artery distal to the aortic arch, however, dnMAML expression at E10.5 severely reduces VSMC coverage of the vessel at E12.5, revealing that arteriogenesis occurs first in the thoracic region of the embryo and then progresses cranially and caudally toward the extremities. This progressive maturation also is demonstrated by the later onset of hemorrhage in the tail when Notch blockade is induced at E11.5. Cell-tracking studies during murine embryogenesis describe ventral migration of VSMC precursors of somitic origin toward the dorsal aorta along the dorsoventral axis but not along the rostral–caudal axis, suggesting that VSMC of different segments of the dorsal aorta differentiate independently and arise as patches from independent single precursor cells (35). These observations are in agreement with our analyses suggesting that activation of Notch in a local VSMC precursor is required to generate mature VSMC in a coordinated fashion during vascular development.

In summary, we demonstrate that a local Tie1⁺CD31^dimVE-cadherin⁻CD45⁻ precursor cell differentiates into VSMC in all vascular beds and requires Notch activation. Further, the Tie1:dnMAML transgenic system potentially can be used to map the process of embryonic arteriogenesis through development.

Materials and Methods

Mice.

Tie1-tTA and TetOSLacZ mice were maintained on a CD1 background. VECtTA mice were a gift from L. Benjamin (Harvard University, Cambridge, MA) and were maintained on the FVB/NJ background. Mice subsequently were backcrossed onto the C57BL/6J background for five generations. TetOS-dnMAML mice were generated by pronuclear injections of linearized DNA encoding the tetOS promoter followed by cDNA of the dnMAML-GFP fusion construct. dnMAML transgenic mice were identified with genotyping primers 5′-CAT GCC ATG GAT GGT GAG CAA GGG CGA G-3′ and 5′-CCA TCG ATT TAC TTG TAC AGC TCG TCC A-3′. All animal experiments were approved by and conform to guidelines of the Animal Care Committee of the University of British Columbia. For tetracycline treatment, timed-mated and plugged females were provided with drinking water containing 50 mg/L tetracycline. The treated water was replaced daily and removed at the times specified.

Flow Cytometry.

Mouse embryos at specified stages were dissected free of yolk sac tissue, minced, and digested in a solution containing 1% (wt/vol) BSA, 550 U/mL collagenase type II, 550 U/mL collagenase type IV, and 100 U/mL DNase I (Sigma-Aldrich) in PBS for 15 min in a 37 °C water bath. The digested embryos were treated with red blood cell lysis buffer, and single-cell suspensions were analyzed for GFP expression, stained with FDG, or immunostained with CD31 or PDGFRβ (BD Pharmingen) Ab followed by a secondary goat anti-rat Alexa Fluor 647 Ab (Invitrogen). Alternatively, phycoerythrin-conjugated CD31 Ab (BD Pharmingen), FITC-conjugated CD45 Ab (eBioscience), or allophycocyanin-conjugated VE-cadherin Ab (eBioscience) were used. FCS Express (De Novo) software was used to analyze flow cytometry data.

β-Gal Detection.

For whole-mount X-Gal staining, embryos were fixed, washed, and incubated with β-gal staining solution [1 mg/mL X-Gal (Invitrogen), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide in β-gal wash solution] at 37 °C for 2–4 h. The embryos were postfixed in 2% (wt/vol) paraformaldehyde and embedded in optimum cutting temperature compound for cryosectioning. Immunostaining was carried out as described (36). For flow cytometry analysis, single-cell suspensions from embryos were incubated with 0.5 mM FDG (Invitrogen) in DMEM at 37 °C for 2 min followed by 30 min at 4 °C.

Hindlimb Ischemia Model.

Tie1:dnMAML and VEC:dnMAML adults were generated by mating Tie1tTA and VECtTA mice with TetOSdnMAML mice. Mice were provided doxycycline or tetracycline (50 mg/L) in the drinking water until 2 d after hindlimb ischemia (HLI). HLI was performed as described when the mice were 8 wk old (23). Blood flow was monitored with a laser Doppler (PeriScan PIM II; Perimed) before ligation and on day 10 after HLI. The level of reperfusion was calculated by taking the ratio of blood flow in the ischemic leg over blood flow in the contralateral nonischemic leg using LDPI 2.6 (Perimed). Mice were killed, and hindlimbs were perfusion-fixed for cryosectioning and immunostaining.

Statistical Analysis.

Statistical analysis was performed with GraphPad Prism (GraphPad Software). P values were determined by Student’s t test.