DEVELOPMENTAL DYNAMICS 243:957–964, 2014 DOI: 10.1002/DVDY.24129

PATTERNS & PHENOTYPES

Murine Notch1 Is Required for Lymphatic Vascular Morphogenesis During Development a

Anees Fatima,1 Austin Culver,1 Ford Culver,1 Ting Liu,1 William H. Dietz,1 Benjamin R. Thomson,1 Anna-Katerina Hadjantonakis,2 Susan E. Quaggin,1 and Tsutomu Kume1 1

Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, Illinois Developmental Biology Program, Sloan-Kettering Institute, New York, New York

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BACKGROUND: The transmembrane receptor Notch1 is a critical regulator of arterial differentiation and blood vessel sprouting. Recent evidence shows that functional blockade of Notch1 and its ligand, Dll4, leads to postnatal lymphatic defects in mice. However, the precise role of the Notch signaling pathway in lymphatic vessel development has yet to be defined. Here we show the developmental role of Notch1 in lymphatic vascular morphogenesis by analyzing lymphatic endothelial cell (LEC)-specific conditional Notch1 knockout mice crossed with an inducible Prox1CreERT2 driver. RESULTS: LEC-specific Notch1 mutant embryos exhibited enlarged lymphatic vessels. The phenotype of lymphatic overgrowth accords with increased LEC sprouting from the lymph sacs and increased filopodia formation. Furthermore, cell death was significantly reduced in Notch1-mutant LECs, whereas proliferation was increased. RNA-seq analysis revealed that expression of cytokine/chemokine signaling molecules was upregulated in Notch1-mutant LECs isolated from E15.5 dorsal skin, whereas VEGFR3, VEGFR2, VEGFC, and Gja4 (Connexin 37) were downregulated. CONCLUSIONS: The lymphatic phenotype of LECspecific conditional Notch1 mouse mutants indicates that Notch activity in LECs controls lymphatic sprouting and growth during development. These results provide evidence that similar to postnatal and pathological lymphatic vessel formation, the Notch signaling pathway plays a role in inhibiting developmental lymphangiogenesis. Developmental Dynamics C 2014 Wiley Periodicals, Inc. 243:957–964, 2014. V Key words: Notch; lymphatic vessel development; lymphangiogenesis; Prox1 Submitted 30 August 2013; First Decision 6 March 2014; Accepted 11 March 2014; Published online 22 March 2014

INTRODUCTION The formation of the blood and lymphatic vascular systems is tightly controlled by both intrinsic and extrinsic mechanisms (Tammela and Alitalo, 2010; Alitalo, 2011). While the mechanisms of blood vessel formation have been extensively studied, investigations relating to lymphatic vascular development have gained tremendous interest in recent years (Koltowska et al., 2013; Martinez-Corral and Makinen, 2013). During mouse development, the lymphatic vasculature system begins at approximately embryonic day (E) 9.75, with expression of the homeobox transcription factor Prox1 in a specific subset of the cardinal vein (Srinivasan et al., 2007; Yang et al., 2012). As the master regulator of the lymphatic vasculature, Prox1 not only regulates LEC fate specification in the veins (Wigle and Oliver, 1999; Wigle et al., 2002), but also plays an essential role in maintaining LEC identity throughout adult life (Johnson et al., 2008). Lack of Prox1 leads to loss of LEC specific markers and up-regulation of blood endothelial cell specific genes. Initial induction of Prox1 Grant sponsor: National Institutes of Health; Grant numbers: HL74121, EY019484, HL108795. Additional Supporting Information may be found in the online version of this article. *Correspondence to: Tsutomu Kume, Northwestern University School of Medicine, 303 E Chicago Ave., Chicago, IL 60611. E-mail: [email protected]

expression in the cardinal vein is regulated by COUP-TFII and Sox18 transcription factors (Francois et al., 2008; Srinivasan et al., 2010). Following specification, LEC progenitors migrate from the cardinal vein via paracrine action of vascular endothelial growth factor (VEGF)-C expressed by the surrounding mesenchyme and then form the primitive lymph sacs (Karkkainen et al., 2004; Xu et al., 2010). LECs express VEGF receptors, VEGFR2, and VEGFR3, as well as the co-receptor Neuropilin 2 (Nrp2) (Wirzenius et al., 2007; Xu et al., 2010), and the tyrosine kinase activity of VEGFR3 upon ligand stimulation is required for lymphangiogenesis (Veikkola et al., 2001; He et al., 2005). However, it remains largely unknown whether additional transcription factors/signaling pathways control these key processes during lymphatic vessel formation. Notch signaling is an evolutionarily conserved pathway that controls many developmental events, including vascular endothelial cell fate and blood vessel sprouting. Notch receptors and ligands such as Notch1 and Dll4, respectively, are predominantly expressed in arterial endothelial cells in the developing embryo and specify arterial cell identity (Kume, 2009; Gridley, 2010). Recent studies indicate a feedback loop among Prox1, COUPTFII, and Notch to maintain the delicate balance between Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24129/abstract C 2014 Wiley Periodicals, Inc. V

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arteriovenous-lymphatic endothelial cells fates (Kang et al., 2010; Choi et al., 2012; Aranguren et al., 2013), and consistently, LEC-specific deletion of Notch1 or inhibition of Notch signaling in mice results in excessive Prox1þ LEC progenitors in the veins and enhanced LEC differentiation during vascular development (Murtomaki et al., 2013). These new findings provide evidence that Notch signaling negatively regulates lymphatic endothelial cell identity in the venous endothelial cells. Notch signaling also interacts with the VEGF pathway to regulate blood vessel sprouting by selecting tip and stalk cells (Tung et al., 2012; Blanco and Gerhardt, 2013). By analogy, Notch activity suppresses tip cell formation and sprouting in LECs in vitro (Zheng et al., 2011), and the Notch1/Dll4 pathway is important for postnatal and pathological lymphangiogenesis (Niessen et al., 2011; Zheng et al., 2011). Although Notch/Dll4 signaling has recently been shown to guide lymphatic vessel patterning along the arterial vasculature in the zebrafish embryo (Geudens et al., 2010), the role of Notch in developmental lymphangiogenesis in mammals remains to be elucidated (Simons and Eichmann, 2013). We demonstrate here that Notch1 is a key regulator of LEC sprouting and growth during lymphatic vessel morphogenesis in the developing mouse embryo. Conditional LEC-specific deletion of Notch1 in mice resulted in significant lymphatic overgrowth with dilated lymphatic vessels, and Notch1-mutant LECs exhibited increased proliferation and decreased cell death as well as enhanced sprouting. LEC-Notch1 mutants also exhibited increased filopodia formation in the lymphatic vessels. These new results significantly extend the recent observation that Notch1 activity influences LEC specification in the venous endothelium (Murtomaki et al., 2013). Furthermore, our genome-wide RNAseq analysis using Notch1-mutant LECs provides new insight into the molecular mechanisms that regulate key signaling pathways in lymphatic vessel development. Together, our new findings indicate that Notch signaling is required for proper lymphangiogenesis during embryonic development.

RESULTS AND DISCUSSION Detection of Canonical Notch Activity in Developing Lymphatic Vessels During lymphatic vascular development, formation of the primitive lymph sacs and an early lymphatic network occurs between E9.75 to E14.5. Starting from E15.5 to postnatal life, the primitive lymphatic vascular network undergoes extensive remodeling to form lymphatic capillaries and collecting lymphatic vessels (Schulte-Merker et al., 2011). Previous studies using global knockout mice for Notch1 reveal that Notch1 is required for embryonic vascular development (Huppert et al., 2000; Krebs et al., 2000; Gridley, 2010), and Murtomaki et al. have recently shown that Notch1 and its ligand (Jagged1) are expressed in the cardinal vein at the time of LEC specification (Murtomaki et al., 2013). Given that Notch activity is detected in a subset of the cardinal vein (Murtomaki et al., 2013), we sought to elucidate the localization of Notch activity during developing lymphangioigenesis using single-cell resolution Notch signaling reporter (CBF:H2B-Venus) mice (Nowotschin et al., 2013). This transgenic mouse strain allows detection of canonical (CBF1-mediated) Notch signaling activity in the embryo, and we found activation of the Notch pathway (i.e., nuclear YFP-Venus expression) in stalk cells of sprouting lymphatic vessels (Fig. 1C, D) in the dorsal

skin at E15.5 (Fig. 1A). These results suggest that canonical Notch signaling is activated in LECs during embryonic development even after LEC fate specification in the cardinal vein.

Conditional Ablation of Notch1 in LECs Results in Enlarged Lymphatic Vessels To determine the specific functions of Notch1 in lymphatic vessel morphogenesis, we crossed mice with a conditional null Notch1flx mutation (Yang et al., 2004) with inducible Prox1CreERT2 mice (Srinivasan et al., 2007) to generate Notch1flx/flx;Prox1CreERT2 mice. Tamoxifen was administered to pregnant dams at E10.5, and lymphatic vessel formation in the dorsal skin was subsequently analyzed by whole mount Lyve-1 immunostaining at E15.5, a time when maximum activity of Cre-mediated recombination is detected in Prox1þ LECs (Srinivasan et al., 2007). Compared with control (Notch1flx/flx) embryos (Fig. 1E), LEC-specific Notch1 mutants (Notch1flx/flx;Prox1CreERT2) showed dilated/ enlarged lymphatic vessels (Fig. 1F). We further carried out quantitative analysis of lymphatic vessel width and branching points. Notch1-mutant lymphatic vessels at E15.5 were significantly wider but normally branched, compared to their littermate controls (Fig. 1G, H). At the same embryonic stage (E15.5), we also found that LEC-specific ablation of Notch1 increased lymphatic cell number (Lyve1þ and Prox1þ) in the dorsal skin (Fig. 1I–L). We analyzed 44 LEC-specific Notch1 mutant embryos at different embryonic stages (E12.5–E15.5). Based on our gross examination, only one mutant at E13.5 showed blood-filled dermal lymphatics, whereas no mutant embryos exhibited obvious lymphedema.

Abnormal Circumferential Growth of Lymphatic Vessels Following Conditional Notch1 Deletion Is Accompanied by Increased Proliferation and Decreased Cell Death of LECs To further determine the primary defects of the abnormally increased lymphatic growth in LEC-specific Notch1 mutants at the cellular level, we examined whether lack of Notch1 could affect LEC proliferation and survival (Fig. 2). Cell proliferation was evaluated in E12.5 control and LEC-specific Notch1 mutant embryos by BrdU staining. Deletion of Notch1 increased the number of BrdUþ cells in the lymph sacs compared to the littermate controls (Fig. 2A–C). Coupled with the increased LEC proliferation, the enlarged lymph sacs were clearly observed in E12.5 conditional Notch1 mutants (Fig. 2B), while we found there was no obvious difference in the cell death rate between control and Notch1 mutant LECs at E12.5 by performing terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining (data not shown). Consistent with the enhanced proliferation in the lymph sacs at E12.5, loss of Notch1 also increased LEC proliferation in the dorsal skin at E15.5 (Fig. 2D–F). To determine whether impaired Notch activity has effects on lymphatic cell survival during lymphangiogenesis, we carried out TUNEL staining on E15.5 dorsal skins. We observed reduced cell death in Lyve1þ LECs of Notch1 mutants (Fig. 2G–I). Moreover, Lyve-1þ/CD31þ LECs isolated from E15.5 control and LECspecific Notch1 mutant embryos were stained with Annexin V to assess cell death (Fig. 2J–L). Notch1-deficient LECs showed the reduced rate of cell death compared to the controls (Fig. 2L). Thus, the dilated lymphatic vessel phenotype appeared to, at least

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Fig. 1. LEC-specific Notch1 deletion leads to enlarged lymphatic vessels. A: E15.5 wild-type (WT) embryo. Black dotted line represents the area of the dorsal skin isolated for Lyve-1 immunostaining. B–D: Whole mount Lyve-1 immunostaining in the dorsal skin of E15.5 wild-type (WT) and CBF: H2B-Venus transgenic embryos showing canonical (CBF1-mediated) Notch signaling activity in LECs. Note that Notch activity was detected in stalk cells but not in a tip cell (yellow arrowhead) in sprouting lymphatic vessels. Scale bar ¼ 50 mm (B, C); 20 mm (D). E, F: Analysis of subcutaneous lymphatic vessels by whole mount Lyve-1 immunostaining of the dorsal skins in control and Notch1 mutant embryos at E15.5. Note dilated/enlarged Lyve1þ lymphatic vessels in Notch mutant embryo (F). Arrows indicate Lyve-1þ macrophages. Scale bar ¼ 50 mm. G, H: Quantitative analysis of lymphatic vessel width (G) and branching points (H). n¼5. ns, non-significant. I, J: Histological analysis of Lyve-1þ/DAPIþ LECs at E15.5. Scale bar ¼ 50 mm. K, L: Quantitative analysis of Lyve-1þ/DAPIþ LECs (K) and Prox1þ LECs (L) at E15.5. n¼3.

in part, result from a significant increase in LEC proliferation and survival. These findings provide further support for the recent data showing that LEC-specific conditional Notch1 mutants exhibit lymphatic overgrowth (Murtomaki et al., 2013). While Murtomaki et al. have recently shown that Notch1 deficiency leads to excessive differentiation of Prox1þ lymphatic progenitor cells arising from the cardinal vein (Murtomaki et al., 2013), our new findings further demonstrate that Notch signaling also regulates LEC proliferation and survival during developmental lymphangiogenesis.

Loss of Notch1 in LECs Results in Increased Lymphatic Vessel Sprouting Notch signaling plays an essential role in blood vessel sprouting by controlling tip and stalk cell fates (Tung et al., 2012; Blanco and Gerhardt, 2013). To further elucidate the nature of Notch1 function in lymphatic vessel development, we investigated LEC sprouting from the jugular lymph sacs. Significantly, the formation of filopodia in the lymph sacs at E12.5 was increased in LEC-specific Notch1 mutants (Fig. 3B, C), and loss of Notch activity at E13.5 enhanced LEC sprouting (Fig. 3E, F) compared to the

littermate controls (Fig. 3D, F). At E14.5, we also observed increased filopodia formation and lymphatic branching in Notch1-mutant LECs in the dorsal skin (Fig. 3G–L). Consistently, Notch1 deficiency continued to augment filopodia formation in LECs at E15.5 (Fig. 3M–O), whereas Notch-dependent lymphatic branching in the dorsal skin appears to have reached a stable level as seen in the control littermates (Fig. 1H). These results suggest that analogous to blood vessel sprouting, Notch signaling is critical for controlling excessive LEC sprouting during normal lymphatic vessel development. Moreover, this observation accords with recent evidence that the Notch pathway is essential for postnatal lymphangiogenesis by regulating LEC sprouting (Niessen et al., 2011; Zheng et al., 2011). It should be noted that conditional activation of Notch signaling in Prox1þ cells shows no obvious effects on LEC migration from the cardinal vein at E10.5 (Murtomaki et al., 2013). Although the reason for the discrepancy in Notch function for LEC migration/sprouting is currently unclear, it may be due to the phenotypic differences in lymphatic vessel development between gain-of-function and loss-of-function mouse models for Notch1, as described recently (Murtomaki et al., 2013).

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Fig. 2. Conditional Notch1 deletion in LECs results in increased proliferation and survival of LECs. A–C: BrdU staining in Lyve-1þ LECs in E12.5 control and Notch1 mutant embryos showing an increase in the number of Lyve-1þ/BrdUþ LECs in Notch1 mutants. Note the enlarged lymph sac (LS) in Notch1 mutant embryo (B). Scale bar ¼ 100 mm. C: Quantitative analysis of proliferating LECs at E12.5 showing a statistically significant difference between control and Notch1 mutants. D–F: BrdU staining in Lyve-1þ LECs in dorsal skin (DS) of E15.5 control and Notch1 mutant embryos showing an increase in the number of Lyve-1þ/BrdUþ LECs (yellow arrowheads) in Notch1 mutants. Ep, epthelium. Scale bar ¼ 50 mm. F: Quantitative analysis of proliferating LECs at E15.5 showing a statistically significant difference between control and Notch1 mutants. G–I: TUNEL staining in Lyve-1þ LECs in the dorsal skin of E15.5 control and Notch1 mutant embryos showing a decrease in the number of Lyve-1þ/ TUNELþ LECs (yellow arrowheads) in Notch1 mutants. Scale bar ¼ 100 mm. I: Quantitative analysis of apoptotic LECs at E15.5 showing a statistically significant difference between control and Notch1 mutants. Ten-consecutive, 10-mm sections were used to count Lyve-1þ/BrdUþ or Lyve1þ/TUNELþ LECs that were normalized against the total number of LECs counted (C, F, I). n¼3. J, K: Representative dot-plots from E15.5 control (J) and Notch1 mutant (K) embryos. Q3 represents a Lyve-1þ/CD31þ LEC population, whereas Q4 represents Annexin-Vþ early apoptotic LECs. L: Quantitative analysis of cell death in Lyve-1þ/CD31þ LECs at E15.5 showing a statistically significant decrease in Annexin-Vþ LECs of Notch1 -mutants compared to the littermate controls. n¼3.

Normal Recruitment of Smooth Muscle Cells (SMCs) in LEC-Specific Notch1 Mutants Active lymph transport is controlled by SMC contractions in collecting lymphatic vessels, whereas lymphatic capillaries are not covered by vascular SMCs. Notably, aberrant recruitment of SMCs to lymphatic capillaries is observed in mice lacking the genes required for lymphatic vessel remodeling and maturation such as Foxc2 (Petrova et al., 2004), angiopoietin 2 (Ang2) (Dellinger et al., 2008), ephrinB2 (Makinen et al., 2005), and reelin (Lutter et al., 2012). However, the precise mechanisms underlying abnormal distribution of SMCs in the lymphatic system remain unknown. A recent study shows that Notch signaling is critical for retinal blood vessel remodeling and maturation and that lack of Notch activity in endothelial cells results in impaired recruitment of mural cells to retinal blood vessels (Ehling et al., 2013). We therefore

investigated whether SMCs are abnormally allocated to lymphatic capillaries in LEC-specific Notch1 mutant embryos (Fig. 3P, Q). We conducted whole mount co-immunostaining of a-smooth muscle actin (SMA) and Lyve-1 to examine localization of SMAþ blood vessels and Lyve1þ lymphatic capillaries in the dorsal skin. We found that ablation of Notch1 in LECs did not lead to abnormal SMC accumulation to lymphatic capillaries at E15.5 (Fig. 3Q) and E17.5 (data not shown). These results suggest that lymphatic Notch1 activity is dispensable for controlling SMC recruitment to lymphatic capillaries.

Genome-Wide Gene Expression Profiling (RNA-seq) of Notch1-Mutant LECs To comprehensively characterize genes differentially regulated in Notch1-mutant LECs, we performed RNA-seq analysis using RNA

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Fig. 3. LEC-specific Notch1 deletion results in increased lymphatic sprouting and tip-cell numbers during early lymphatic development. A–C: Lyve1/Prox1/DAPI-immunostained LECs bearing filopodia in the lymph sacs at E12.5. Note that filopodia formation (arrowheads) was enhanced in Notch1 mutant embryo (B) compared to the control (A). Scale Bar ¼ 20 mm. C: Quantitative analysis of filopodia formation in E12.5 lymphatic vessels (n¼3). D–F: Lyve-1 immunostaining of control and Notch1 mutant embryos at E13.5. Note increased lymphatic sprouting (arrowheads) from the jugular lymph sacs in Notch1 mutant (E) compared to the control (D). LS, lymph sacs; S, skin. Scale bar ¼ 100 mm. F: Quantitative analysis of lymphatic vessel sprouts emerging from the jugular lymph sacs at E13.5 showing a statistically significant difference between control and Notch1 mutant embryos. The number of lymphatic vessel sprouting was counted from 15 consecutive transverse sections through the neck. G–L: Lyve-1 stained lymphatic vessels in the dorsal skin at E14.5. H, J: High-magnification images of the boxed areas shown in G, I, respectively. Note that the number of filopodia (arrows) was increased in Notch1 mutant embryo (J) compared to the control (H). Scale bar ¼ 100 mm (G, I); 50 mm (H, J). K: Quantitative analysis of filopodia formation in E14.5 lymphatic vessels (n¼3). L: Quantitative analysis of the number of lymphatic vessel branches in E14.5 dorsal skins (n¼3). M–O: Lyve-1 stained lymphatic vessels in the dorsal skin at E15.5 embryos. Note that the number of filopodia (arrows) was increased in Notch1 mutant embryo (N) compared to the control (M). Scale bar ¼ 100 mm (M, N). O: Quantitative analysis of filopodia formation in E15.5 lymphatic vessels (n¼3). P, Q: Co-immunostaining of Lyve-1 and a-SMA in the dorsal skins at E15.5 showing normal distribution of a-SMAþ SMCs to blood vessels in both Notch1 mutant and control embryos. Note that no SMC accumulation was observed in Lyve-1þ lymphatic capillaries (dotted lines) of LECspecific Notch1 mutant embryo (Q) as in those of the control (P). LV, lymphatic vessel; BV, blood vessel. Scale bar ¼ 50 mm

extracted from sorted Lyve-1þ/CD31þ LECs isolated from E15.5 dorsal skins of Notch1 mutants and littermate controls (Fig. 4 and see Supplementary Tables S1–3, which are available online). Remarkably, our pathway analysis revealed that in addition to the genes related to angiogenesis, expression of the chemokine and cytokine signaling molecules was significantly upregulated in Notch1-mutant LECs (Fig. 4D). This observation indicates a possible role for Notch1 in inflammatory responses of LECs through regulation of inflammatory mediators. Although there was no obvious difference in the number of Lyve-1þ macrophages located in E15.5 dorsal skins between control and Notch1 mutant embryos (55.25 6 10.08 versus 43.75 6 9.2, P ¼ 0.4335), our new finding is likely to explore a new area of research. We also found that VEGF receptors (VEGFR2 and VEGFR3) as well as VEGFC were downregulated (Fig. 4C). This

may be due to the developmental stage (E15.5) and location (dorsal skin) to isolate LECs as lymphatic sprouting appears to have reached a plateau at this stage. However, we found that loss of Notch1 reduced the expression of the gap junction molecule Gja4 (Connexin 37) (Fig. 4C). Given recent evidence that Connexin 37 is regulated by Prox1 and Foxc2 transcription factors and is required for lymphatic valve formation (Sabine et al., 2012; Sabine and Petrova, 2014), it is possible that Notch signaling plays a role in this process. Notably, Notch1 deficiency increased the expression of Tgfbr1 (Alk5), and TGFb signaling is critical for lymphangiogenic sprouting (James et al., 2013). Together, further studies need to be performed to elucidate the molecular mechanisms underlying Notch-dependent lymphatic vascular morphogenesis, including cross-talk with other signaling pathways.

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Fig. 4. Schematic generated by PANTHER, a gene ontology-based pathway database, showing pathway analysis of differentially expressed genes in Lyve1þ/CD31þ LECs isolated from Notch1 mutant embryos at E15.5 compared to littermate controls. A, B: Dot plots showing the gating used for sorting Lyve-1þ/CD31þ LEC, where Gate1 and Gate 2 indicate a total live LEC population and a Lyve1þ/CD31þ LEC population acquired following gating of individual CD31þ and Lyve-1þ populations, respectively. C: Bar graph showing RPKM (reads per kilobase of coding sequence per million mapped) values from RNA-seq of Notch1 mutant and control LECs. Levels of VEGFR3, VEGFR2, VEGFC, and Gja4 were significantly downregulated in Notch1 mutant LECs. D: Panther pathway analysis of upregulated genes in Notch1-mutant LECs. Note upregulation of chemokine and cytokine signaling pathway genes that may influence the lymphatic phenotype of Notch1 mutant embryos. E: Panther pathway analysis of downregulated genes in Notch1-mutant LECs.

Summary We demonstrate here that Notch1 regulates early development of the lymphatic vasculature in mice. Conditional LEC-specific deletion of Notch1 results in significant lymphatic overgrowth, and, most importantly, we report the novel finding that lack of Notch1 results in enhanced lymphatic sprouting along with increased LEC proliferation/survival. Taken together, these results suggest that the Notch signaling pathway is required for developmental lymphangiogenesis. The results obtained from our RNA-seq analysis indicate that multiple signaling pathways control the complex process of lymphatic growth and maturation, and we expect these data will be useful to define the precise roles of Notch signaling in lymphatic vessel formation during embryonic development as well as various pathological conditions in adults.

EXPERIMENTAL PROCEDURES Mice Notch1flx/flx (Yang et al., 2004) (The Jackson Laboratory, Bar Harbor, ME) and Prox1CreERT2 (Srinivasan et al., 2007)

(a generous gift from G. Oliver, St. Jude Children’s Research Hospital, Memphis) were used. Conditional Notch1 mutant (Notch1flx/ flx ;Prox1CreERT2) mice were generated for analysis by crossing Notch1flx/flx females with Notch1flx/flx;Prox1CreERT2 males. Embryonic age was determined by defining noon on the day of vaginal plug as embryonic day 0.5 (E0.5). Tamoxifen (5 mg/40 g body weight) dissolved in corn oil was injected intraperitoneally into pregnant dams at E10.5. Genotyping of conditional Notch1 mutants has been described previously (Srinivasan et al., 2007; Tammela and Alitalo, 2010). CBF:H2B-Venus reporter mice (Nowotschin et al., 2013) were used to analyze canonical Notch activity in developing lymphatic vessels. All procedures were approved by Northwestern University’s Institutional Animal Care and Use Committee (IACUC).

Immunohistochemical Analyses The lymphatic vasculature was analyzed by whole mount Lyve-1 immunostaining of the dorsal embryonic skin (shown in Fig. 1A) at E14.5 and E15.5. Embryos were harvested in cold PBS and  fixed overnight in 4% paraformaldehyde (PFA) at 4 C. Following

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fixation, the embryos were stored overnight in 100% methanol at  20 C. The skins were then obtained under a dissection microscope, rehydrated, and blocked in 10% donkey serum before staining with either anti-Lyve-1 antibody (Abcam, Cambridge, MA) or anti-Prox1 (R&D Systems, Minneapolis, MN). The dorsal skin samples were counterstained with DAPI (Sigma Aldrich, St. Louis, MO) to count the total number of LECs in Notch1 mutants and littermate controls. For immunohistochemistry using paraffin sections, embryos at E13.5 were fixed in 4% PFA and embedded in paraffin blocks, and 8-mm sections were made on a microtome. The transverse sections through the neck were de-paraffinized and stained with anti-Lyve-1 antibody (Abcam) to analyze the jugular lymph sacs as described (Xu et al., 2010). Fifteen consecutive sections were examined to quantify lymphatic vessel sprouting from the jugular lymph sacs. For analysis of LEC proliferation, pregnant dams received 150 mg/kg BrdU dissolved in DMSO 2 hr prior to dissection of embryos. The embryos were then fixed in 4% PFA, and ten serial cryosections (10 mm thick) of the lymph sacs were stained with anti-BrdU (BD Pharmingen, San Jose, CA) and anti-Lyve-1 (Abcam) antibodies to detect proliferating LECs. Images of the entire lymph sacs were captured at 20 magnification. Lyve-1þ/ BrdUþ/DAPIþ LECs were manually counted throughout the lymph sacs. The total number of BrdUþ LECs was then normalized against the total number of LEC counted. Additionally, LEC cell death was analyzed in E15.5 dorsal skins using the TUNEL assay kit (Roche, Indianapolis, IN). Briefly, 10 serial (10 mm thick) dorsal skin sections collected at the level of heart were stained with antiLyve-1 antibody and TUNEL. The sections were finally counterstained with DAPI. Images of LV were captured at 20 and 40 magnifications. Lyve-1þ/TUNELþ/DAPIþ LEC were manually counted and normalized to the total number of LECs counted. Whole mount immunostaining was documented by Zsectioning using a Zeiss (Thornwood, NY) UV-LSM-510 confocal microscope; immunostaining on tissue sections was examined and recorded using a Zeiss axiovision fluorescent microscope.

Fluorescent Activated Cell Sorting (FACS) Cells isolated from E15.5 control (Notch1flx/flx) and conditional Notch1 mutant (Notch1flx/flx;Prox1CreERT2) embryos were stained for Lyve-1 and CD31 and subjected to FACS. Briefly, E15.5 embryos were harvested in Hank’s balanced salt solution (HBSS, Sigma-Aldrich) and then chopped for an overnight digestion with collagenease I/II. The colleagenase-treated cell suspension was incubated with RBC (Red blood cell) lysis buffer (StemCell Technologies, Vancouver, Canada). Following centrifugation, cell pellets were incubated with anti-Lyve-1 antibody (Abcam) for 20 min at  4 C. After washing with PBS, the cells were then stained with PEconjugated anti-CD31 antibody (BD Pharmingen) and Alexa 488conjugated donkey anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA). After gauze filtration with a cell strainer (40 mm BD Biosciences) to obtain a single cell suspension, the cells were subjected to staining with Annexin V-APC (BD Pharmingen) to assess cell death in the sorted LECs using Flow Fortessa.

RNA-seq (RNA Sequencing) The RNA-seq was performed on RNA extracted from sorted Lyve1þ/CD31þ LECs isolated from E15.5 dorsal skins of Notch1

mutants and littermate controls at the University of Chicago Genomics Core. Two separate pools of RNA samples were created: one pool of LECs isolated from two Notch1flx/flxand a second pool isolated from three Notch1flx/flx;Prox1CreERT2 embryos. A twolane 50-bp single end RNA-seq was performed according to a protocol on an Illumina HiSeq2500 instrument. Briefly, RNA quality was checked by determining the RNA Integrity Number (RIN) on a bioanalyzer. All the RNA samples for the sequencing experiments had RIN>9. Libraries from the above RNA samples were created using RiboZero depletion Kit. Each sample (biological replicates) was then given a unique ID “Barcode” during this process. Lastly, the pooled samples (biological replicates) were sequenced and stored in a FASTQ format. The data were then analyzed at the Bioinformatic Core at the Northwestern University Comprehensive Transplant Center using cufflinks software to test the differential regulation of genes between Notch1 mutants and control embryos.

Measurement of Lymphatic Vessel Width (LVW) To measure LVW, we created new software based on the same fundamental algorithm for distance determination as ImageJ using MATLAB; however, we built in batch image processing, which allows the user to queue a number of images for analysis by designating a directory in which they are located. It is a more streamlined method of LVW measurement to cut down on the time required for analysis. When utilizing the MATLAB software, we found no difference between previously and newly recorded LVWs, and users noted significantly easier use and decreased time expenditure. The details of the use of the software will be published elsewhere (Fatima et al., unpublished data).

Statistical Analysis Statistical analysis was performed using GraphPad Prism, and statistical significance was determined by Student’s t-test. Data are presented as mean 6 SEM.

ACKNOWLEDGMENTS We thank Dr. Olga Volpert for a critical reading for the manuscript. We also thank other members of the Kume lab and the Feinberg Cardiovascular Research Institute for technical help and thoughtful discussions. We thank Dr. G. Oliver for providing us with the Prox1CreERT2 mouse line. This work was supported by the National Institutes of Health (HL74121, EY019484, and HL108795 to T.K.).

REFERENCES Alitalo K. 2011. The lymphatic vasculature in disease. Nat Med 17: 1371–1380. Aranguren XL, Beerens M, Coppiello G, Wiese C, Vandersmissen I, Lo Nigro A, Verfaillie CM, Gessler M, Luttun A. 2013. COUP-TFII orchestrates venous and lymphatic endothelial identity by homoor hetero-dimerisation with PROX1. J Cell Sci 126:1164–1175. Blanco R, Gerhardt H. 2013. VEGF and Notch in tip and stalk cell selection. Cold Spring Harb Perspect Med 3:a006569. Choi I, Lee S, Hong YK. 2012. The new era of the lymphatic system: no longer secondary to the blood vascular system. Cold Spring Harb Perspect Med 2:a006445. Dellinger M, Hunter R, Bernas M, Gale N, Yancopoulos G, Erickson R, Witte M. 2008. Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice. Dev Biol 319:309–320.

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