C 2014 Wiley Periodicals, Inc. V
genesis 52:809–816 (2014)
LETTER
Constitutive Notch Signaling in Adult Transgenic Mice Inhibits bFGF-Induced Angiogenesis and Blocks Ovarian Follicle Development Ju Liu,1,2,3* Urban Deutsch,4 James Jeong,1 and Corrinne G. Lobe1,2* 1
Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, Toronto, Ontario, M4N 3M5 Canada
2
Department of Medical Biophysics, University of Toronto, Toronto, Canada
3
Laboratory of Microvascular Medicine, Medical Research Center, Qianfoshan Hospital, Shandong University, Jinan, China
4
Theodor-Kocher-Institute, University of Berne, Berne, Switzerland
Received 20 February 2014; Revised 22 April 2014; Accepted 7 May 2014
Summary: Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock-out and transgenic mice have precluded studies of the role of Notch postnatally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre-conditional expression of the constitutively active intracellular domain of Notch1 (IC-Notch1). Double transgenic ICNotch1/Tie2-Cre embryos with endothelial specific ICNotch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline-inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC-Notch1 expression inhibited bFGF-induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF-induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angioC 2014 V genic therapies. genesis 52:809–816, 2014.
the menstrual cycle and wound healing (Risau, 1997). Re-initiation of angiogenesis in adults is associated with various pathological conditions such as tumor growth, retinopathies, and rheumatoid arthritis (Costa et al., 2004). Notch signaling pathway plays a crucial role in the regulation of angiogenesis (Gridley, 2010; Krebs et al., 2000; Shawber et al., 2003; Shawber and Kitajewski, 2004). Targeted disruption of the Notch1, Notch1/ 4, Jag1, Dll4, Hey1/2, and RBP-J genes all lead to vascular defects and embryonic lethality at embryonic day (E) 9.5 to 10.5 (Duarte et al., 2004; Fischer et al., 2004; Krebs et al., 2000, 2004; Xue et al., 1999). The primary vascular plexus forms in mutant embryos and yolk sacs but vessel remodeling does not occur. Surprisingly, a Notch gain-of-function experiment produced a similar phenotype to the Notch knockout phenotype: A constitutively active form of Notch4 expressed under the regulation of the endothelial cell-specific Flk-1 locus caused embryonic lethality at E9.5 with restricted
Wiley Periodicals, Inc.
* Correspondence to: Corrinne G. Lobe, Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5 Canada E-mail: [email protected] and Ju Liu, Laboratory of Microvascular Medicine, Medical Research Center, Qianfoshan Hospital, Shandong University, Jinan, China E-mail: [email protected] Contract grant sponsors: Heart and Stroke Foundation of Canada, Shandong Taishan Scholarship Published online 10 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22790
Key words: angiogenesis; Notch signaling; basic FGF; follicular development; transgenic mice
Angiogenesis is an active process in mammalian embryos and newborns, whereas in mature adult organisms angiogenesis is generally inactive except during
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vasculature growth and a disorganized vessel network (Uyttendaele et al., 2001). Studies of Notch signaling using transgenic and knockout approaches have been limited by early embryonic lethality. To circumvent this problem, we established ZEG-IC-Notch1 transgenic mice to take advantage of tissue-specific Cre recombinase-expressing mouse lines to tailor gene expression to particular tissues (Liu and Lobe, 2007). ZEG-IC-Notch1 utilizes a loxP-flanked STOP sequence between a CMV enhancerchicken b-actin promoter and the coding sequence for intracellular portion of the Notch1 protein (IC-Notch1) with an internal ribosomal entry site (IRES) linked enhanced green fluorescence protein (EGFP) (Fig. 1a). The ZEG-IC-Notch1 mice are available to the research community through the Jackson Laboratory (JAX Stock Number 019074). We crossed ZEG-IC-Notch1 mice with Tie2-Cre mice to activate IC-Notch1 expression in endothelial cells (ECs) (Kisanuki et al., 2001). Double transgenic Tie2Cre/ZEG-IC-Notch1 embryos died at E9.5 to 10.5 and exhibited pale yolk sacs with fewer blood vessels than the littermates (Fig. 1b,d). These embryos were developmentally retarded and displayed hemorrhaging around the vessels (Fig. 1c,e). Immunohistochemical staining of CD31 of the sections of the embryos demonstrated that major blood vessels such as the dorsal aorta, cardinal veins, and intersomitic arteries were present in Tie2-Cre/ZEG-IC-Notch1 double transgenic embryos, however the vessels were restricted and disorganized. Large caliber vessels such as dorsal aorta and cardinal veins were collapsed (Fig. 1h,i). These vascular remodeling defects are similar to the observations from other mouse models with conditional activation of the ICNotch1 in ECs (Cortegano et al., 2014; Krebs et al., 2010; Venkatesh et al., 2008). We also used a tetracycline-regulated system to activate Cre activity in ECs (Gossen and Bujard, 1992). ZEG-IC-Notch1 mice were crossed with Tie2-tTA/tet-OCre mice to obtain triple transgenic Tie2-tTA/tet-O-Cre/ ZEG-IC-Notch1 embryos. The Tie2-tTA transgene provides tetracycline transactivator (tTA) expression in ECs. In the absence of the tetracycline, tTA binds the tet-O-Cre transgene and activates expression of Cre recombinase, which should provide the same Cre activity as Tie2-Cre. Indeed, we found that the triple transgenic embryos exhibited a phenotype resembling the double positive Tie2-Cre/ZEG-IC-Notch1 mice and died around E10.5 (Fig. 1f,g), demonstrating Tie2-tTA/tet-OCre reiterates the tissue-specific Cre activity equivalent to Tie2-Cre. To overcome the embryonic lethality caused by ICNotch1 activation, we bred ZEG-IC-Notch1 mice with Tie2-tTA/tet-O-Cre mice as described above but maintained the pregnant female mice on tetracycline analog doxycycline throughout pregnancy (Fig. 2a). In this
way, we obtained a normal Mendelian ratio of live-born triple transgenic Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 pups. The triple transgenic mice continued to survive after removal of doxycycline and displayed no overt phenotype. Echocardiography revealed no significant differences in cardiac morphology and function between WT and the triple transgenic mice. To detect the expression of IC-Notch1 in these adult mice, organs were immunostained for the reporter EGFP. We found only a fraction of ECs expressed EGFP in the triple transgenic mice (Fig. 2c) compared to all ECs marked by CD31 (Fig. 2d). The mosaic expression could result from reduced activity of the Tie2 promoter in adult mice and/or incomplete Cre excision. Physiological anigiogenesis in adults occurs mainly in cyclic changes in the ovary and reproductive tract (Stouffer et al., 2001). To investigate the role of Notch in adult angiogenesis, we tested the reproductive capacity of IC-Notch1 expressing females compared to non-expressing females. We found that of seven females with IC-Notch1 expression, four were sterile and three produced small litters of less than three pups, whereas control female mice produced 12–16 pups per litter. In tissue sections, ovaries from wildtype females had developing follicles whereas ovaries from females expressing IC-Notch1 did not have apparent maturing follicles (Fig. 3a,b). The ovaries of sterile IC-Notch1 expressing females had only pre-antral and degenerative follicles but not antral follicles, as seen in the ovaries from wildtype mice (Fig. 3c–f). While implantation and placental function in female mice with endothelial ICNotch1 expression might be disturbed due to the possible effects on placental vasculature, infertility could result from a failure of ovarian follicle development to a stage that could produce mature oocytes. Maturation of ovarian follicles requires the development of a vascular bed. Preantral follicles consist of an ovum surrounded by a layer of follicular or granulosa cells and are each vascularized by a single surrounding capillary loop (Tamanini and De Ambrogi, 2004). Development of the secondary follicle is characterized by the appearance of the antrum (cavity) and formation of the thecal layer from the surrounding stroma. The theca acquires a vascular sheath, and movement of nutrients from the thecal blood vessels into the follicular fluid is essential for the development of the ovum and follicle (Plendl, 2000). In mice, Notch1, Notch4, and Jagged1 are expressed in a subset of ovarian vessels in mature ovarian vasculature and angiogenic neovessels (Vorontchikhina et al., 2005). Blocking Notch pathways inhibits follicular maturation and induces disruption of gonadotropin stimulated angiogenesis (Jovanovic et al., 2013). Our results suggest ectopic activation of Notch signaling in ECs also impairs the angiogenesis required for progression of follicles to the secondary stage.
FIG. 1. Endothelial-specific expression of IC-Notch1 causes defects in angiogenesis. (a) Strategy for Cre-conditional IC-Notch1 expression. A floxed bgeo coding sequence and three repeats of an SV40 polyadenylation signal were placed between a CMV-a actin promoter and the constitutively active IC-Notch1. The coding sequence for the EGFP reporter fused with an IRES sequence was placed downstream of the IC-Notch1 cDNA to allow co-expression of IC-Notch1 and EGFP from the same transcript. Prior to Cre excision, the bgeo is expressed. Upon Cre excision the floxed bgeo is removed, leading to expression of IC-Notch1 and EGFP. (b–i) Endothelial-specific expression of IC-Notch1 causes defects in angiogenesis. (b, c) E10.5 control single transgenic embryo with yolk sac (b) and without yolk sac (c). (d, e) E10.5 Tie2-Cre/ZEG-IC-Notch1 double transgenic embryo showed disorganized vasculature in the yolk sac (d) and hemorrhage in the embryo (e). (f, g) E10.5 Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 triple transgenic embryo with a similar phenotype to Tie2-Cre/ZEG-IC-Notch1 embryos but at a later stage. (h, i) CD31 immunostain of transverse sections of control single transgenic E10.5 embryo (h, 503) and Tie2Cre/ZEG-IC-Notch1 embryo (i, 503). DA, dorsal aorta; CV, cardinal vein.
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FIG. 2. IC-Notch1 transgene expression in adults. (a) Tetracycline-Cre inducible system: ZEG-IC-Notch1 mice were crossed with Tie2-tTA/tet-O-Cre mice. In the presence of tetracycline, the triple transgenic embryos (Tie2-tTA/tet-O-Cre/ZEG-ICNotch1) express tTA in ECs but the tetracycline-bound tTA cannot bind the tet operator. When tetracycline is withdrawn, the tTA binds the tet operator, resulting in activation of Cre and IC-Notch1 expression. Using this system, Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice survived to birth when the pregnant mothers were maintained on doxycycline, a tetracycline analog, until birth. (b, c) EGFP immunostain of adult liver from control single transgenic mice (b, 1003) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice (c, 1003). ZEG-ICNotch1 transgene showed mosaic expression in adult blood vessels. Thick arrow refers to ECs expressing EGFP; Dark chevron refers to ECs not expressing EGFP; Thin arrow refers to capillaries expressing EGFP. (d) By comparison, CD31 immunostain of liver (1003) from Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mouse stains all ECs.
Recent studies suggested that basic fibroblast growth factor (bFGF) plays an essential role in follicle development (Garor et al., 2009; Matos et al., 2007). Immunohistological studies have located bFGF and its receptors to preantral ovarian follicles in the rat (Nilsson et al., 2001) and humans (Ben-Haroush et al., 2005). bFGF promotes the growth and survival of primordial follicles (Matos et al., 2007), and stimulated a high primordial to primary and secondary transition (Kezele et al., 2002). To measure the angiogenic response to bFGF in ICNotch1 expressing mice, we injected matrigel supplemented with bFGF into the mice subcutaneously, and compared the number of ECs growing into the matrigel. Mice expressing IC-Notch1 had 30% less infiltration of ECs in bFGF matrigel plugs than wildtype mice and although capillaries formed, larger vessels did not (Fig. 4a,b,e). When VEGF was used as growth factor in the matrigel, no significant difference was observed between wildtype and IC-Notch1 expressing mice in the number of ECs (Fig. 4c,d,f). Therefore, IC-Notch1 inhibited bFGF-induced but not VEGF-induced angiogenesis
in the matrigel plug assay. The matrigel plug from the Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice was digested and the single cell suspensions were analyzed by flow cytometry. Green fluorescence protein (GFP) positive cells presented at a minimal level (0.59%) in the matrigel containing bFGF, and a higher level (25.8%) in the matrigel containing VEGF (Fig. 4g,h). Therefore, exogenous IC-Notch1 expression prevents ECs from responding to bFGF. This result supports the idea that ovarian folliculogenesis does not proceed because constitutive Notch signaling inhibits bFGF-induced angiogenesis. In summary, with the combination of tissue-specific and tetracycline-inducible Cre transgenic mice, this conditional expression system allows us to bypass the embryonic lethal phenotype and examine roles for Notch signaling in adult angiogenesis. This unique conditional model permitted us with a spatiotemporal control of IC-Notch1 activation, and provided insights into the role of Notch signaling during both embryonic development and adulthood. METHODS Transgenic Mice ZEG-IC-Notch1 mice were previously generated in our laboratory and the genotyping was determined by tail-clip lacZ assay (Liu and Lobe, 2007). Tie2-Cre mice (provided by Dr. Masashi Yanagisawa, University of Texas Southwestern Medical Center) express Cre recombinase under the direction of the Tie2 promoter/ enhancer. Tie2-tTA mice (provided by Dr. Urban Deutsch, Max Planck Institute) express the tTA (tet repressor fused to a VP16 activator) under the regulation of 2.1b Tie2 promoter. Tet-O-Cre mice (provided by Dr. Andras Nagy, Samuel Lunenfeld Research Institute) carry the Cre coding sequence downstream of a minimal CMV promoter and tetracycline operator. CRE and tTA genotyping was performed by PCR. All mouse strains were maintained on mixed backgrounds. Experiments complied with ethical standards of the Sunnybrook Health Sciences Center Research Institute Animal Care Committee. Tetracycline-Inducible System ZEG-IC-Notch transgenic mice were bred with Tie2tTA mice and double transgenic offspring were crossed with tet-O-Cre mice. The breeding pairs were maintained with 0.1 mg/mL doxycycline (tetracycline analogue, Sigma-Aldrich, Oakville, Canada) in drinking water with 5% sucrose (Sarao and Dumont, 1998). To maintain the efficiency of doxycycline, the drinking water was protected from light and replaced every 24 h. Doxycycline was withdrawn the day pups were born and the pups were genotyped to identify triple transgenic offspring (Tie2-tTA/tet-O-Cre/ZEG-IC-Notch).
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FIG. 3. IC-Notch1 expression in ECs suppresses ovarian follicle development. (a, b) CD31 immunostained sections of ovaries from wildtype mice (a, 1003) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice (b, 1003). (c, d) AP stained sections of ovaries from control mice (c, 2003) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice (d, 2003). (e, f) Larger magnifications of AP stained ovary sections from wildtype and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice, showing primary and degenerative follicles. Arrows: mature secondary follicles; p: primordial follicle; pr: primary follicle; s: secondary follicle; gr: graafian follicle; d: degenerative follicle.
Immunohistochemsitry and Alkaline Phosphatase Staining Tissues preparation and staining of frozen sections for immunohistochemsitry (IHC) and alkaline phosphatase (AP) activity were essential as previously described (Liu and Lobe, 2007). For IHC, primary antibodies included anti-CD31 monoclonal antibody (BD Pharmingen, San Jose, CA) and anti-GFP monoclonal (3E6) anti-
body (Molecular Probes, Eugene, Oregon). Secondary antibodies are biotinylated rabbit anti-rat and goat antirabbit antibodies from Vector Labs (Burlingame, CA). Slides were counterstained with hematoxylin (Surgipath, Winnipeg, Canada). The staining solution for AP activity includes 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 337 mg/mL NBT (nitroblue tetrazolium
FIG. 4. Matrigel plug assay on IC-Notch1 expressing mice. (a–d) CD31 immunostained sections of matrigel injected with bFGF in wildtype mice (a) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1mice (b) and matrigel injected with VEGF in wildtype mice (c) and Tie2-tTA/tet-O-Cre/ZEGIC-Notch1 mice (d). (e, f) Number of ECs in sections of matrigel injected with bFGF (e) or VEGF (f). Bars represent standard deviation. (g, h) FACS analysis to measure EGFP expression in the cell from matrigel injected with bFGF (g) and VEGF (h) in Tie2-tTA/tet-O-Cre/ZEG-ICNotch1 mice.
IC-NOTCH1 BLOCKS ADULT ANGIOGENESIS
salt; Roche Diagnostics, Basel, Switzerland), and 175 mg/mL BCIP (5-bromo-4-chloro-3-indolyl phosphate, toluidinium salt; Roche Diagnostics, Basel, Switzerland), and the sections were counterstained with Nuclear Fast Red (Sigma-Aldrich, Oakville, Canada). All slides were photographed using a Leica DFC300 camera with Leica FireCam 120 program. Matrigel Plug Assay Matrigel (Collaborative Biomedical Products, Bedford, MA) was thawed at 4 C overnight and mixed with 500 ng/mL bFGF (R&D Systems, Minneapolis, MN) or 1,000 ng/mL VEGF (Sigma-Aldrich, Oakville, Canada) (Klement et al., 2000). An aliquot of 0.5 mL of this mixture was injected subcutaneously into the shaved flanks of Tie2-tTA/tet-O-Cre/ZEG-IC-Notch mice and their single transgenic ZEG-IC-Notch littermates (5–7 week old, male). At day 10 all mice were anesthetized with 2.5% isoflurane and sacrificed. The matrigel plug was removed and snap-frozen in liquid nitrogen. The matrigel plugs were sectioned at 7 mm and immunostained for CD31. The microvascular density count was carried out as previously described, using a Leica FireCam 120 (Viloria-Petit et al., 2003). For each sample six fields of 0.1 3 0.16 mm were chosen randomly and counted by two different people blind to sample designation. The Analysis of Variance (ANOVA) was used to determine the significance of differences in relative density of ECs. ACKNOWLEDGMENT Authors thank Dr. Andras Nagy for helpful discussions and critical reading of the manuscript and Dr. Masashi Yanagisawa for generously providing the Tie2-Cre mice. LITERATURE CITED Ben-Haroush A, Abir R, Ao A, Jin S, Kessler-Icekson G, Feldberg D, Fisch B. 2005. Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses. Fertil Steril 84 (Suppl 2):1257–1268. Cortegano I, Melgar-Rojas P, Luna-Zurita L, SigueroAlvarez M, Marcos MA, Gaspar ML, de la Pompa JL. 2014. Notch1 regulates progenitor cell proliferation and differentiation during mouse yolk sac hematopoiesis. Cell Death Differ. Costa C, Soares R, Schmitt F. 2004. Angiogenesis: now and then. APMIS 112:402–412. Duarte A, Hirashima M, Benedito R, Trindade A, Diniz P, Bekman E, Costa L, Henrique D, Rossant J. 2004. Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev 18:2474–2478. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M. 2004. The Notch target genes Hey1 and Hey2
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are required for embryonic vascular development. Genes Dev 18:901–911. Garor R, Abir R, Erman A, Felz C, Nitke S, Fisch B. 2009. Effects of basic fibroblast growth factor on in vitro development of human ovarian primordial follicles. Fertil Steril 91:1967–1975. Gossen M, Bujard H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551. Gridley T. 2010. Notch signaling in the vasculature. Curr Top Dev Biol 92:277–309. Jovanovic VP, Sauer CM, Shawber CJ, Gomez R, Wang X, Sauer MV, Kitajewski J, Zimmermann RC. 2013. Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4). Reprod Biol Endocrinol 11:43. Kezele PR, Nilsson EE, Skinner MK. 2002. Insulin but not insulin-like growth factor-1 promotes the primordial to primary follicle transition. Mol Cell Endocrinol 192:37–43. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M. 2001. Tie2-Cre transgenic mice: A new model for endothelial celllineage analysis in vivo. Dev Biol 230:230–242. Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, Bohlen P, Kerbel RS. 2000. Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105:R15–R24. Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark KL, Gridley T. 2004. Haploinsufficient lethality and formation of arteriovenous malformations in Notch pathway mutants. Genes Dev 18:2469–2473. Krebs LT, Starling C, Chervonsky AV, Gridley T. 2010. Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants. Genesis 48:146–150. Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, Smith GH, Stark KL, Gridley T. 2000. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev 14:1343–1352. Liu J, Lobe CG. 2007. Cre-conditional expression of constitutively active Notch1 in transgenic mice. Genesis 45:259–265. Matos MH, Lima-Verde IB, Bruno JB, Lopes CA, Martins FS, Santos KD, Rocha RM, Silva JR, Bao SN, Figueiredo JR. 2007. Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro. Reprod Fertil Dev 19:677–684. Nilsson E, Parrott JA, Skinner MK. 2001. Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol 175:123–130.
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Plendl J. 2000. Angiogenesis and vascular regression in the ovary. Anat Histol Embryol 29:257–266. Risau W. 1997. Mechanisms of angiogenesis. Nature 386:671–674. Sarao R, Dumont DJ. 1998. Conditional transgene expression in endothelial cells. Transgenic Res 7: 421–427. Shawber CJ, Das I, Francisco E, Kitajewski J. 2003. Notch signaling in primary endothelial cells. Ann NY Acad Sci 995:162–170. Shawber CJ, Kitajewski J. 2004. Notch function in the vasculature: Insights from zebrafish, mouse and man. Bioessays 26:225–234. Stouffer RL, Martinez-Chequer JC, Molskness TA, Xu F, Hazzard TM. 2001. Regulation and action of angiogenic factors in the primate ovary. Arch Med Res 32: 567–575. Tamanini C, De Ambrogi M. 2004. Angiogenesis in developing follicle and corpus luteum. Reprod Domest Anim 39:206–216. Uyttendaele H, Ho J, Rossant J, Kitajewski J. 2001. Vascular patterning defects associated with expression
of activated Notch4 in embryonic endothelium. Proc Natl Acad Sci USA 98:5643–5648. Venkatesh DA, Park KS, Harrington A, Miceli-Libby L, Yoon JK, Liaw L. 2008. Cardiovascular and hematopoietic defects associated with Notch1 activation in embryonic Tie2-expressing populations. Circ Res 103:423–431. Viloria-Petit A, Miquerol L, Yu JL, Gertsenstein M, Sheehan C, May L, Henkin J, Lobe C, Nagy A, Kerbel RS, Rak J. 2003. Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors. EMBO J 22:4091–4102. Vorontchikhina MA, Zimmermann RC, Shawber CJ, Tang H, Kitajewski J. 2005. Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expr Patterns 5:701–709. Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, Gridley T. 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 8:723–730.
genesis 52:809–816 (2014)
LETTER
Constitutive Notch Signaling in Adult Transgenic Mice Inhibits bFGF-Induced Angiogenesis and Blocks Ovarian Follicle Development Ju Liu,1,2,3* Urban Deutsch,4 James Jeong,1 and Corrinne G. Lobe1,2* 1
Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, Toronto, Ontario, M4N 3M5 Canada
2
Department of Medical Biophysics, University of Toronto, Toronto, Canada
3
Laboratory of Microvascular Medicine, Medical Research Center, Qianfoshan Hospital, Shandong University, Jinan, China
4
Theodor-Kocher-Institute, University of Berne, Berne, Switzerland
Received 20 February 2014; Revised 22 April 2014; Accepted 7 May 2014
Summary: Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock-out and transgenic mice have precluded studies of the role of Notch postnatally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre-conditional expression of the constitutively active intracellular domain of Notch1 (IC-Notch1). Double transgenic ICNotch1/Tie2-Cre embryos with endothelial specific ICNotch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline-inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC-Notch1 expression inhibited bFGF-induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF-induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angioC 2014 V genic therapies. genesis 52:809–816, 2014.
the menstrual cycle and wound healing (Risau, 1997). Re-initiation of angiogenesis in adults is associated with various pathological conditions such as tumor growth, retinopathies, and rheumatoid arthritis (Costa et al., 2004). Notch signaling pathway plays a crucial role in the regulation of angiogenesis (Gridley, 2010; Krebs et al., 2000; Shawber et al., 2003; Shawber and Kitajewski, 2004). Targeted disruption of the Notch1, Notch1/ 4, Jag1, Dll4, Hey1/2, and RBP-J genes all lead to vascular defects and embryonic lethality at embryonic day (E) 9.5 to 10.5 (Duarte et al., 2004; Fischer et al., 2004; Krebs et al., 2000, 2004; Xue et al., 1999). The primary vascular plexus forms in mutant embryos and yolk sacs but vessel remodeling does not occur. Surprisingly, a Notch gain-of-function experiment produced a similar phenotype to the Notch knockout phenotype: A constitutively active form of Notch4 expressed under the regulation of the endothelial cell-specific Flk-1 locus caused embryonic lethality at E9.5 with restricted
Wiley Periodicals, Inc.
* Correspondence to: Corrinne G. Lobe, Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5 Canada E-mail: [email protected] and Ju Liu, Laboratory of Microvascular Medicine, Medical Research Center, Qianfoshan Hospital, Shandong University, Jinan, China E-mail: [email protected] Contract grant sponsors: Heart and Stroke Foundation of Canada, Shandong Taishan Scholarship Published online 10 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22790
Key words: angiogenesis; Notch signaling; basic FGF; follicular development; transgenic mice
Angiogenesis is an active process in mammalian embryos and newborns, whereas in mature adult organisms angiogenesis is generally inactive except during
810
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vasculature growth and a disorganized vessel network (Uyttendaele et al., 2001). Studies of Notch signaling using transgenic and knockout approaches have been limited by early embryonic lethality. To circumvent this problem, we established ZEG-IC-Notch1 transgenic mice to take advantage of tissue-specific Cre recombinase-expressing mouse lines to tailor gene expression to particular tissues (Liu and Lobe, 2007). ZEG-IC-Notch1 utilizes a loxP-flanked STOP sequence between a CMV enhancerchicken b-actin promoter and the coding sequence for intracellular portion of the Notch1 protein (IC-Notch1) with an internal ribosomal entry site (IRES) linked enhanced green fluorescence protein (EGFP) (Fig. 1a). The ZEG-IC-Notch1 mice are available to the research community through the Jackson Laboratory (JAX Stock Number 019074). We crossed ZEG-IC-Notch1 mice with Tie2-Cre mice to activate IC-Notch1 expression in endothelial cells (ECs) (Kisanuki et al., 2001). Double transgenic Tie2Cre/ZEG-IC-Notch1 embryos died at E9.5 to 10.5 and exhibited pale yolk sacs with fewer blood vessels than the littermates (Fig. 1b,d). These embryos were developmentally retarded and displayed hemorrhaging around the vessels (Fig. 1c,e). Immunohistochemical staining of CD31 of the sections of the embryos demonstrated that major blood vessels such as the dorsal aorta, cardinal veins, and intersomitic arteries were present in Tie2-Cre/ZEG-IC-Notch1 double transgenic embryos, however the vessels were restricted and disorganized. Large caliber vessels such as dorsal aorta and cardinal veins were collapsed (Fig. 1h,i). These vascular remodeling defects are similar to the observations from other mouse models with conditional activation of the ICNotch1 in ECs (Cortegano et al., 2014; Krebs et al., 2010; Venkatesh et al., 2008). We also used a tetracycline-regulated system to activate Cre activity in ECs (Gossen and Bujard, 1992). ZEG-IC-Notch1 mice were crossed with Tie2-tTA/tet-OCre mice to obtain triple transgenic Tie2-tTA/tet-O-Cre/ ZEG-IC-Notch1 embryos. The Tie2-tTA transgene provides tetracycline transactivator (tTA) expression in ECs. In the absence of the tetracycline, tTA binds the tet-O-Cre transgene and activates expression of Cre recombinase, which should provide the same Cre activity as Tie2-Cre. Indeed, we found that the triple transgenic embryos exhibited a phenotype resembling the double positive Tie2-Cre/ZEG-IC-Notch1 mice and died around E10.5 (Fig. 1f,g), demonstrating Tie2-tTA/tet-OCre reiterates the tissue-specific Cre activity equivalent to Tie2-Cre. To overcome the embryonic lethality caused by ICNotch1 activation, we bred ZEG-IC-Notch1 mice with Tie2-tTA/tet-O-Cre mice as described above but maintained the pregnant female mice on tetracycline analog doxycycline throughout pregnancy (Fig. 2a). In this
way, we obtained a normal Mendelian ratio of live-born triple transgenic Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 pups. The triple transgenic mice continued to survive after removal of doxycycline and displayed no overt phenotype. Echocardiography revealed no significant differences in cardiac morphology and function between WT and the triple transgenic mice. To detect the expression of IC-Notch1 in these adult mice, organs were immunostained for the reporter EGFP. We found only a fraction of ECs expressed EGFP in the triple transgenic mice (Fig. 2c) compared to all ECs marked by CD31 (Fig. 2d). The mosaic expression could result from reduced activity of the Tie2 promoter in adult mice and/or incomplete Cre excision. Physiological anigiogenesis in adults occurs mainly in cyclic changes in the ovary and reproductive tract (Stouffer et al., 2001). To investigate the role of Notch in adult angiogenesis, we tested the reproductive capacity of IC-Notch1 expressing females compared to non-expressing females. We found that of seven females with IC-Notch1 expression, four were sterile and three produced small litters of less than three pups, whereas control female mice produced 12–16 pups per litter. In tissue sections, ovaries from wildtype females had developing follicles whereas ovaries from females expressing IC-Notch1 did not have apparent maturing follicles (Fig. 3a,b). The ovaries of sterile IC-Notch1 expressing females had only pre-antral and degenerative follicles but not antral follicles, as seen in the ovaries from wildtype mice (Fig. 3c–f). While implantation and placental function in female mice with endothelial ICNotch1 expression might be disturbed due to the possible effects on placental vasculature, infertility could result from a failure of ovarian follicle development to a stage that could produce mature oocytes. Maturation of ovarian follicles requires the development of a vascular bed. Preantral follicles consist of an ovum surrounded by a layer of follicular or granulosa cells and are each vascularized by a single surrounding capillary loop (Tamanini and De Ambrogi, 2004). Development of the secondary follicle is characterized by the appearance of the antrum (cavity) and formation of the thecal layer from the surrounding stroma. The theca acquires a vascular sheath, and movement of nutrients from the thecal blood vessels into the follicular fluid is essential for the development of the ovum and follicle (Plendl, 2000). In mice, Notch1, Notch4, and Jagged1 are expressed in a subset of ovarian vessels in mature ovarian vasculature and angiogenic neovessels (Vorontchikhina et al., 2005). Blocking Notch pathways inhibits follicular maturation and induces disruption of gonadotropin stimulated angiogenesis (Jovanovic et al., 2013). Our results suggest ectopic activation of Notch signaling in ECs also impairs the angiogenesis required for progression of follicles to the secondary stage.
FIG. 1. Endothelial-specific expression of IC-Notch1 causes defects in angiogenesis. (a) Strategy for Cre-conditional IC-Notch1 expression. A floxed bgeo coding sequence and three repeats of an SV40 polyadenylation signal were placed between a CMV-a actin promoter and the constitutively active IC-Notch1. The coding sequence for the EGFP reporter fused with an IRES sequence was placed downstream of the IC-Notch1 cDNA to allow co-expression of IC-Notch1 and EGFP from the same transcript. Prior to Cre excision, the bgeo is expressed. Upon Cre excision the floxed bgeo is removed, leading to expression of IC-Notch1 and EGFP. (b–i) Endothelial-specific expression of IC-Notch1 causes defects in angiogenesis. (b, c) E10.5 control single transgenic embryo with yolk sac (b) and without yolk sac (c). (d, e) E10.5 Tie2-Cre/ZEG-IC-Notch1 double transgenic embryo showed disorganized vasculature in the yolk sac (d) and hemorrhage in the embryo (e). (f, g) E10.5 Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 triple transgenic embryo with a similar phenotype to Tie2-Cre/ZEG-IC-Notch1 embryos but at a later stage. (h, i) CD31 immunostain of transverse sections of control single transgenic E10.5 embryo (h, 503) and Tie2Cre/ZEG-IC-Notch1 embryo (i, 503). DA, dorsal aorta; CV, cardinal vein.
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FIG. 2. IC-Notch1 transgene expression in adults. (a) Tetracycline-Cre inducible system: ZEG-IC-Notch1 mice were crossed with Tie2-tTA/tet-O-Cre mice. In the presence of tetracycline, the triple transgenic embryos (Tie2-tTA/tet-O-Cre/ZEG-ICNotch1) express tTA in ECs but the tetracycline-bound tTA cannot bind the tet operator. When tetracycline is withdrawn, the tTA binds the tet operator, resulting in activation of Cre and IC-Notch1 expression. Using this system, Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice survived to birth when the pregnant mothers were maintained on doxycycline, a tetracycline analog, until birth. (b, c) EGFP immunostain of adult liver from control single transgenic mice (b, 1003) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice (c, 1003). ZEG-ICNotch1 transgene showed mosaic expression in adult blood vessels. Thick arrow refers to ECs expressing EGFP; Dark chevron refers to ECs not expressing EGFP; Thin arrow refers to capillaries expressing EGFP. (d) By comparison, CD31 immunostain of liver (1003) from Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mouse stains all ECs.
Recent studies suggested that basic fibroblast growth factor (bFGF) plays an essential role in follicle development (Garor et al., 2009; Matos et al., 2007). Immunohistological studies have located bFGF and its receptors to preantral ovarian follicles in the rat (Nilsson et al., 2001) and humans (Ben-Haroush et al., 2005). bFGF promotes the growth and survival of primordial follicles (Matos et al., 2007), and stimulated a high primordial to primary and secondary transition (Kezele et al., 2002). To measure the angiogenic response to bFGF in ICNotch1 expressing mice, we injected matrigel supplemented with bFGF into the mice subcutaneously, and compared the number of ECs growing into the matrigel. Mice expressing IC-Notch1 had 30% less infiltration of ECs in bFGF matrigel plugs than wildtype mice and although capillaries formed, larger vessels did not (Fig. 4a,b,e). When VEGF was used as growth factor in the matrigel, no significant difference was observed between wildtype and IC-Notch1 expressing mice in the number of ECs (Fig. 4c,d,f). Therefore, IC-Notch1 inhibited bFGF-induced but not VEGF-induced angiogenesis
in the matrigel plug assay. The matrigel plug from the Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice was digested and the single cell suspensions were analyzed by flow cytometry. Green fluorescence protein (GFP) positive cells presented at a minimal level (0.59%) in the matrigel containing bFGF, and a higher level (25.8%) in the matrigel containing VEGF (Fig. 4g,h). Therefore, exogenous IC-Notch1 expression prevents ECs from responding to bFGF. This result supports the idea that ovarian folliculogenesis does not proceed because constitutive Notch signaling inhibits bFGF-induced angiogenesis. In summary, with the combination of tissue-specific and tetracycline-inducible Cre transgenic mice, this conditional expression system allows us to bypass the embryonic lethal phenotype and examine roles for Notch signaling in adult angiogenesis. This unique conditional model permitted us with a spatiotemporal control of IC-Notch1 activation, and provided insights into the role of Notch signaling during both embryonic development and adulthood. METHODS Transgenic Mice ZEG-IC-Notch1 mice were previously generated in our laboratory and the genotyping was determined by tail-clip lacZ assay (Liu and Lobe, 2007). Tie2-Cre mice (provided by Dr. Masashi Yanagisawa, University of Texas Southwestern Medical Center) express Cre recombinase under the direction of the Tie2 promoter/ enhancer. Tie2-tTA mice (provided by Dr. Urban Deutsch, Max Planck Institute) express the tTA (tet repressor fused to a VP16 activator) under the regulation of 2.1b Tie2 promoter. Tet-O-Cre mice (provided by Dr. Andras Nagy, Samuel Lunenfeld Research Institute) carry the Cre coding sequence downstream of a minimal CMV promoter and tetracycline operator. CRE and tTA genotyping was performed by PCR. All mouse strains were maintained on mixed backgrounds. Experiments complied with ethical standards of the Sunnybrook Health Sciences Center Research Institute Animal Care Committee. Tetracycline-Inducible System ZEG-IC-Notch transgenic mice were bred with Tie2tTA mice and double transgenic offspring were crossed with tet-O-Cre mice. The breeding pairs were maintained with 0.1 mg/mL doxycycline (tetracycline analogue, Sigma-Aldrich, Oakville, Canada) in drinking water with 5% sucrose (Sarao and Dumont, 1998). To maintain the efficiency of doxycycline, the drinking water was protected from light and replaced every 24 h. Doxycycline was withdrawn the day pups were born and the pups were genotyped to identify triple transgenic offspring (Tie2-tTA/tet-O-Cre/ZEG-IC-Notch).
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FIG. 3. IC-Notch1 expression in ECs suppresses ovarian follicle development. (a, b) CD31 immunostained sections of ovaries from wildtype mice (a, 1003) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice (b, 1003). (c, d) AP stained sections of ovaries from control mice (c, 2003) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice (d, 2003). (e, f) Larger magnifications of AP stained ovary sections from wildtype and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1 mice, showing primary and degenerative follicles. Arrows: mature secondary follicles; p: primordial follicle; pr: primary follicle; s: secondary follicle; gr: graafian follicle; d: degenerative follicle.
Immunohistochemsitry and Alkaline Phosphatase Staining Tissues preparation and staining of frozen sections for immunohistochemsitry (IHC) and alkaline phosphatase (AP) activity were essential as previously described (Liu and Lobe, 2007). For IHC, primary antibodies included anti-CD31 monoclonal antibody (BD Pharmingen, San Jose, CA) and anti-GFP monoclonal (3E6) anti-
body (Molecular Probes, Eugene, Oregon). Secondary antibodies are biotinylated rabbit anti-rat and goat antirabbit antibodies from Vector Labs (Burlingame, CA). Slides were counterstained with hematoxylin (Surgipath, Winnipeg, Canada). The staining solution for AP activity includes 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 337 mg/mL NBT (nitroblue tetrazolium
FIG. 4. Matrigel plug assay on IC-Notch1 expressing mice. (a–d) CD31 immunostained sections of matrigel injected with bFGF in wildtype mice (a) and Tie2-tTA/tet-O-Cre/ZEG-IC-Notch1mice (b) and matrigel injected with VEGF in wildtype mice (c) and Tie2-tTA/tet-O-Cre/ZEGIC-Notch1 mice (d). (e, f) Number of ECs in sections of matrigel injected with bFGF (e) or VEGF (f). Bars represent standard deviation. (g, h) FACS analysis to measure EGFP expression in the cell from matrigel injected with bFGF (g) and VEGF (h) in Tie2-tTA/tet-O-Cre/ZEG-ICNotch1 mice.
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salt; Roche Diagnostics, Basel, Switzerland), and 175 mg/mL BCIP (5-bromo-4-chloro-3-indolyl phosphate, toluidinium salt; Roche Diagnostics, Basel, Switzerland), and the sections were counterstained with Nuclear Fast Red (Sigma-Aldrich, Oakville, Canada). All slides were photographed using a Leica DFC300 camera with Leica FireCam 120 program. Matrigel Plug Assay Matrigel (Collaborative Biomedical Products, Bedford, MA) was thawed at 4 C overnight and mixed with 500 ng/mL bFGF (R&D Systems, Minneapolis, MN) or 1,000 ng/mL VEGF (Sigma-Aldrich, Oakville, Canada) (Klement et al., 2000). An aliquot of 0.5 mL of this mixture was injected subcutaneously into the shaved flanks of Tie2-tTA/tet-O-Cre/ZEG-IC-Notch mice and their single transgenic ZEG-IC-Notch littermates (5–7 week old, male). At day 10 all mice were anesthetized with 2.5% isoflurane and sacrificed. The matrigel plug was removed and snap-frozen in liquid nitrogen. The matrigel plugs were sectioned at 7 mm and immunostained for CD31. The microvascular density count was carried out as previously described, using a Leica FireCam 120 (Viloria-Petit et al., 2003). For each sample six fields of 0.1 3 0.16 mm were chosen randomly and counted by two different people blind to sample designation. The Analysis of Variance (ANOVA) was used to determine the significance of differences in relative density of ECs. ACKNOWLEDGMENT Authors thank Dr. Andras Nagy for helpful discussions and critical reading of the manuscript and Dr. Masashi Yanagisawa for generously providing the Tie2-Cre mice. LITERATURE CITED Ben-Haroush A, Abir R, Ao A, Jin S, Kessler-Icekson G, Feldberg D, Fisch B. 2005. Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses. Fertil Steril 84 (Suppl 2):1257–1268. Cortegano I, Melgar-Rojas P, Luna-Zurita L, SigueroAlvarez M, Marcos MA, Gaspar ML, de la Pompa JL. 2014. Notch1 regulates progenitor cell proliferation and differentiation during mouse yolk sac hematopoiesis. Cell Death Differ. Costa C, Soares R, Schmitt F. 2004. Angiogenesis: now and then. APMIS 112:402–412. Duarte A, Hirashima M, Benedito R, Trindade A, Diniz P, Bekman E, Costa L, Henrique D, Rossant J. 2004. Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev 18:2474–2478. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M. 2004. The Notch target genes Hey1 and Hey2
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are required for embryonic vascular development. Genes Dev 18:901–911. Garor R, Abir R, Erman A, Felz C, Nitke S, Fisch B. 2009. Effects of basic fibroblast growth factor on in vitro development of human ovarian primordial follicles. Fertil Steril 91:1967–1975. Gossen M, Bujard H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551. Gridley T. 2010. Notch signaling in the vasculature. Curr Top Dev Biol 92:277–309. Jovanovic VP, Sauer CM, Shawber CJ, Gomez R, Wang X, Sauer MV, Kitajewski J, Zimmermann RC. 2013. Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4). Reprod Biol Endocrinol 11:43. Kezele PR, Nilsson EE, Skinner MK. 2002. Insulin but not insulin-like growth factor-1 promotes the primordial to primary follicle transition. Mol Cell Endocrinol 192:37–43. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M. 2001. Tie2-Cre transgenic mice: A new model for endothelial celllineage analysis in vivo. Dev Biol 230:230–242. Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, Bohlen P, Kerbel RS. 2000. Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105:R15–R24. Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark KL, Gridley T. 2004. Haploinsufficient lethality and formation of arteriovenous malformations in Notch pathway mutants. Genes Dev 18:2469–2473. Krebs LT, Starling C, Chervonsky AV, Gridley T. 2010. Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants. Genesis 48:146–150. Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, Smith GH, Stark KL, Gridley T. 2000. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev 14:1343–1352. Liu J, Lobe CG. 2007. Cre-conditional expression of constitutively active Notch1 in transgenic mice. Genesis 45:259–265. Matos MH, Lima-Verde IB, Bruno JB, Lopes CA, Martins FS, Santos KD, Rocha RM, Silva JR, Bao SN, Figueiredo JR. 2007. Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro. Reprod Fertil Dev 19:677–684. Nilsson E, Parrott JA, Skinner MK. 2001. Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol 175:123–130.
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Plendl J. 2000. Angiogenesis and vascular regression in the ovary. Anat Histol Embryol 29:257–266. Risau W. 1997. Mechanisms of angiogenesis. Nature 386:671–674. Sarao R, Dumont DJ. 1998. Conditional transgene expression in endothelial cells. Transgenic Res 7: 421–427. Shawber CJ, Das I, Francisco E, Kitajewski J. 2003. Notch signaling in primary endothelial cells. Ann NY Acad Sci 995:162–170. Shawber CJ, Kitajewski J. 2004. Notch function in the vasculature: Insights from zebrafish, mouse and man. Bioessays 26:225–234. Stouffer RL, Martinez-Chequer JC, Molskness TA, Xu F, Hazzard TM. 2001. Regulation and action of angiogenic factors in the primate ovary. Arch Med Res 32: 567–575. Tamanini C, De Ambrogi M. 2004. Angiogenesis in developing follicle and corpus luteum. Reprod Domest Anim 39:206–216. Uyttendaele H, Ho J, Rossant J, Kitajewski J. 2001. Vascular patterning defects associated with expression
of activated Notch4 in embryonic endothelium. Proc Natl Acad Sci USA 98:5643–5648. Venkatesh DA, Park KS, Harrington A, Miceli-Libby L, Yoon JK, Liaw L. 2008. Cardiovascular and hematopoietic defects associated with Notch1 activation in embryonic Tie2-expressing populations. Circ Res 103:423–431. Viloria-Petit A, Miquerol L, Yu JL, Gertsenstein M, Sheehan C, May L, Henkin J, Lobe C, Nagy A, Kerbel RS, Rak J. 2003. Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors. EMBO J 22:4091–4102. Vorontchikhina MA, Zimmermann RC, Shawber CJ, Tang H, Kitajewski J. 2005. Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expr Patterns 5:701–709. Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, Gridley T. 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 8:723–730.
