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ProxTom Lymphatic Vessel Reporter Mice Reveal Prox1 Expression in the Adrenal Medulla, Megakaryocytes, and Platelets

Open AccessPublished:February 06, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.026
      Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.
      Lymphatic vessels (LVs) are important structures for antigen presentation, fluid homeostasis, and lipid metabolism and as conduits for tumor metastases. Despite their importance, research into LVs has fallen behind that of arteries and veins because these thin-walled vessels have been difficult to visualize in vivo. Only recently have specific markers for LVs become available. Ultimately, we have been frustrated by our inability to evaluate dynamic LV functions, such as lymphangiogenesis and lymph node remodeling in real time, in live mice.
      We have shown that after immunization, dramatic changes occur in LVs and high endothelial venules (HEVs) of the lymph node.
      • Liao S.
      • Ruddle N.H.
      Synchrony of high endothelial venules and lymphatic vessels revealed by immunization.
      To further understand these changes, our goal was to visualize LVs and HEVs simultaneously in a living lymph node. We decided to develop a fluorescent red lymphatic reporter mouse in C57BL/6 mice for two-photon laser scanning microscopy in vivo imaging. We chose the C57BL/6 background as particularly appropriate for cell transfer experiments requiring inbred mice.
      The goal was to cross the red LV reporter mice with Hec6stGFP mice that have green fluorescent protein (GFP) in their HEVs and have already proved suitable for in vivo imaging.
      • Bentley K.L.
      • Stranford S.
      • Liao S.
      • Mounzer R.M.
      • Ruddle F.H.
      • Ruddle N.H.
      High endothelial venule reporter mice to probe regulation of lymph node vasculature.
      The discovery of markers of lymphatic endothelium, such as Prox1 (prospero-related homeobox gene-1), LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1), and podoplanin (gp38), has accelerated lymphatic research.
      • Wigle J.T.
      • Oliver G.
      Prox1 function is required for the development of the murine lymphatic system.
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      • Ni J.
      • Wang S.X.
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      LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.
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      • Soleiman A.
      • Horvat R.
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      • Kerjaschki D.
      Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.
      • Tammela T.
      • Alitalo K.
      Lymphangiogenesis: molecular mechanisms and future promise.
      However, deletion of any of these lymphatic genes results in embryonic lethality (eg, Prox1, Sox18, and podoplanin).
      • Tammela T.
      • Alitalo K.
      Lymphangiogenesis: molecular mechanisms and future promise.
      Reporters driven by podoplanin or VEGFR3 are suboptimal because they fail to reproduce the expression pattern of the endogenous genes in LVs. The conditional expression of GFP under the control of LYVE-1 labeled a population of lymph node macrophages in addition to LVs.
      • Pham T.H.
      • Baluk P.
      • Xu Y.
      • Grigorova I.
      • Bankovich A.J.
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      • McDonald D.M.
      • Schwab S.R.
      • Cyster J.G.
      Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning.
      The expression of GFP in the macrophages of these LYVE-1–driven reporter mice makes them unsuitable for the study of lymphangiogenesis in lymph nodes.
      A transgene consisting of 4 kb of the Prox1 promoter driving GFP expresses the reporter in embryonic LVs, but it is unclear whether GFP is seen in adult LVs.
      • François M.
      • Caprini A.
      • Hosking B.
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      Sox18 induces development of the lymphatic vasculature in mice.
      This 4-kb construct has a short upstream regulatory segment that lacks two important binding sites for the transcription factor COUP-TFII. COUP-TFII is required for initiation and maintenance of Prox1 in LVs.
      • Srinivasan R.S.
      • Geng X.
      • Yang Y.
      • Wang Y.
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      • Studer M.
      • Porto M.P.
      • Lagutin O.
      • Oliver G.
      The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells.
      Recently, a different mouse line transgenic for Prox1 driving GFP was reported to have fluorescent green LVs.
      • Choi I.
      • Chung H.K.
      • Ramu S.
      • Lee H.N.
      • Kim K.E.
      • Lee S.
      • Yoo J.
      • Choi D.
      • Lee Y.S.
      • Aguilar B.
      • Hong Y.K.
      Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse.
      This mouse was originally made by the GENSAT (Gene Expression Nervous System Atlas) program, which was a large-scale effort to incorporate GFP reporters into neuronal genes. This GFP reporter mouse is on the outbred [FVB/N-Crl: CD1 (ICR)] background and is not appropriate for immunologic studies, which require a pure inbred background. We designed the “ProxTom” LV reporter mice to take advantage of the many knockout and transgenic lines, including our own Hec6stGFP mice, which are on the C57BL/6 background. To date, both of the published fluorescent lymphatic reporter mice use GFP, so we deliberately chose to make a red fluorescent LV reporter mouse that would be suitable to cross with the Hec6stGFP mouse that has green fluorescent HEVs.
      • Bentley K.L.
      • Stranford S.
      • Liao S.
      • Mounzer R.M.
      • Ruddle F.H.
      • Ruddle N.H.
      High endothelial venule reporter mice to probe regulation of lymph node vasculature.
      Furthermore, GFP has lower fluorescence intensity and a shorter half-life than does tdTomato, and it is not known whether the GENSAT mouse is suitable for in vivo imaging of LVs.
      • Choi I.
      • Chung H.K.
      • Ramu S.
      • Lee H.N.
      • Kim K.E.
      • Lee S.
      • Yoo J.
      • Choi D.
      • Lee Y.S.
      • Aguilar B.
      • Hong Y.K.
      Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse.
      To make the ProxTom LV reporter mice, we selected the gene for the homeobox-like transcription factor Prox1 to drive the red fluorescent protein (RFP) tdTomato.
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
      Prox1 is the mammalian homolog of the Drosophila homeobox gene prospero. In mammals, Prox1 is necessary for formation of the lymphatic system
      • Wigle J.T.
      • Oliver G.
      Prox1 function is required for the development of the murine lymphatic system.
      and development of the murine lens,
      • Wigle J.T.
      • Chowdhury K.
      • Gruss P.
      • Oliver G.
      Prox1 function is crucial for mouse lens-fibre elongation.
      liver,
      • Oliver G.
      • Sosa-Pineda B.
      • Wigle J.T.
      • Oliver G.
      Hepatocyte migration during liver development requires.
      and dentate gyrus.
      • Lavado A.
      • Oliver G.
      Prox1 expression patterns in the developing and adult murine brain.
      We chose Prox1 because it is a “master regulator” of LVs in ontogeny and into maturity. Prox1 is sufficient to drive veins toward a lymphatic phenotype in vitro and in vivo.
      • Kim H.
      • Nguyen V.P.
      • Petrova T.V.
      • Cruz M.
      • Alitalo K.
      • Dumont D.J.
      Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature.
      LVs develop after the embryonic blood vessels have formed by sprouting from the cardinal vein.
      • Srinivasan R.S.
      • Dillard M.E.
      • Lagutin O.V.
      • Lin F.J.
      • Tsai S.
      • Tsai M.J.
      • Samokhvalov I.M.
      • Oliver G.
      Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature.
      On day E9.5, a subset of endothelial cells lying in the wall of the cardinal vein start to express Prox1. These Prox1-expressing cells form the lymphatic sac that eventually buds off and separates from the vein to become mature LVs. This process of lymphatic differentiation is arrested in Prox1-deficient embryos, which die at day E15 with blood-filled lymphatics due to persistent connections between LVs and veins.
      • Wigle J.T.
      • Oliver G.
      Prox1 function is required for the development of the murine lymphatic system.
      Recent reports show that platelets are involved in lymphangiogenesis and are required for separation of the lymphatic sac from the cardinal vein.
      • Carramolino L.
      • Fuentes J.
      • Garcia-Andres C.
      • Azcoitia V.
      • Riethmacher D.
      • Torres M.
      Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis.
      • Bertozzi C.C.
      • Schmaier A.A.
      • Mericko P.
      • Hess P.R.
      • Zou Z.
      • Chen M.
      • Chen C.Y.
      • Xu B.
      • Lu M.M.
      • Zhou D.
      • Sebzda E.
      • Santore M.T.
      • Merianos D.J.
      • Stadtfeld M.
      • Flake A.W.
      • Graf T.
      • Skoda R.
      • Maltzman J.S.
      • Koretzky G.A.
      • Kahn M.L.
      Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.
      • Suzuki-Inoue K.
      • Inoue O.
      • Ding G.
      • Nishimura S.
      • Hokamura K.
      • Eto K.
      • Kashiwagi H.
      • Tomiyama Y.
      • Yatomi Y.
      • Umemura K.
      • Shin Y.
      • Hirashima M.
      • Ozaki Y.
      Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets.
      • Uhrin P.
      • Zaujec J.
      • Breuss J.M.
      • Olcaydu D.
      • Chrenek P.
      • Stockinger H.
      • Fuertbauer E.
      • Moser M.
      • Haiko P.
      • Fässler R.
      • Alitalo K.
      • Binder B.R.
      • Kerjaschki D.
      Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.
      • Abtahian F.
      • Guerriero A.
      • Sebzda E.
      • Lu M.M.
      • Zhou R.
      • Mocsai A.
      • Myers E.E.
      • Huang B.
      • Jackson D.G.
      • Ferrari V.A.
      • Tybulewicz V.
      • Lowell C.A.
      • Lepore J.J.
      • Koretzky G.A.
      • Kahn M.L.
      Regulation of blood and lymphatic vascular separation by signaling proteins SLP-76 and Syk.
      • Bertozzi C.C.
      • Hess P.R.
      • Kahn M.L.
      Platelets: covert regulators of lymphatic development.
      Mice that have a targeted depletion of megakaryocytes, which are the source of platelets, also fail to divide their lymphatic and venous vasculatures and have abnormal, persistent LV-venous connections that result in blood-filled lymphatics.
      • Carramolino L.
      • Fuentes J.
      • Garcia-Andres C.
      • Azcoitia V.
      • Riethmacher D.
      • Torres M.
      Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis.
      Knockout mice lacking podoplanin, C-type lectin-like receptor (CLEC)-2, or its signaling partners SYK and SLP-76 also fail to separate LVs from veins and have blood-filled lymphatics.
      • Bertozzi C.C.
      • Schmaier A.A.
      • Mericko P.
      • Hess P.R.
      • Zou Z.
      • Chen M.
      • Chen C.Y.
      • Xu B.
      • Lu M.M.
      • Zhou D.
      • Sebzda E.
      • Santore M.T.
      • Merianos D.J.
      • Stadtfeld M.
      • Flake A.W.
      • Graf T.
      • Skoda R.
      • Maltzman J.S.
      • Koretzky G.A.
      • Kahn M.L.
      Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.
      • Suzuki-Inoue K.
      • Inoue O.
      • Ding G.
      • Nishimura S.
      • Hokamura K.
      • Eto K.
      • Kashiwagi H.
      • Tomiyama Y.
      • Yatomi Y.
      • Umemura K.
      • Shin Y.
      • Hirashima M.
      • Ozaki Y.
      Essential in vivo roles of the C-type lectin receptor CLEC-2: embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets.
      This finding indicates that platelets must activate the CLEC2/SYK/SLP-76 pathway to separate the lymphatics from the cardinal vein. It is unknown how platelets contribute to this separation, but it has been suggested that platelets expressing CLEC2 can bind to podoplanin and aggregate to form a plug that physically divides the two systems.
      • Carramolino L.
      • Fuentes J.
      • Garcia-Andres C.
      • Azcoitia V.
      • Riethmacher D.
      • Torres M.
      Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis.
      • Uhrin P.
      • Zaujec J.
      • Breuss J.M.
      • Olcaydu D.
      • Chrenek P.
      • Stockinger H.
      • Fuertbauer E.
      • Moser M.
      • Haiko P.
      • Fässler R.
      • Alitalo K.
      • Binder B.R.
      • Kerjaschki D.
      Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.
      However, integrin α2−/− mice that cannot form a platelet plug have LVs that develop normally, implying that a physical platelet plug may not be critical for the separation of lymphatics from veins.
      • Bertozzi C.C.
      • Hess P.R.
      • Kahn M.L.
      Platelets: covert regulators of lymphatic development.
      So far, it has been impossible to observe these events in vivo, to visualize lymphangiogenesis in inflammation, or to visualize LVs in living mice; this drove us to develop the ProxTom mice. The high visibility of the tdTomato reporter allowed us to reliably image LVs, and we show images of HEVs and LVs captured simultaneously in vivo for the first time.
      We show that the transgene was expressed in LVs coincident with the endogenous Prox1 protein. We confirmed Prox1 expression in the adult liver, lens, and dentate gyrus as previously described.
      • Wigle J.T.
      • Chowdhury K.
      • Gruss P.
      • Oliver G.
      Prox1 function is crucial for mouse lens-fibre elongation.
      • Oliver G.
      • Sosa-Pineda B.
      • Wigle J.T.
      • Oliver G.
      Hepatocyte migration during liver development requires.
      • Lavado A.
      • Oliver G.
      Prox1 expression patterns in the developing and adult murine brain.
      We also report that Prox1 is expressed by cells in three novel places: the adrenal medulla, CD41+ megakaryocytes, and platelets. Prox1 was located in the megakaryocyte cytoplasm, from where it could be incorporated into platelets.
      Herein we describe the ProxTom transgenic mouse, with its brightly fluorescent LVs. The intense fluorescence of ProxTom LVs means that these animals have great potential for studying diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

      Materials and Methods

      Mice

      ProxTom transgenic mice were made by pronuclear microinjection of C57BL/6-fertilized ova by Animal Genomic Services, Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT. Hec6stGFP mice that express GFP in HEVs have been described previously.
      • Bentley K.L.
      • Stranford S.
      • Liao S.
      • Mounzer R.M.
      • Ruddle F.H.
      • Ruddle N.H.
      High endothelial venule reporter mice to probe regulation of lymph node vasculature.
      C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The Yale University Institutional Animal Care and Use Committee approved all animal use.

      ProxTom Transgene Construction, Genotyping, and Southern Blot Analysis

      A 143,990-bp genomic fragment containing the Prox1 gene and flanking regions was captured in pClasper by homologous recombination in yeast. The pClasper method for construction of transgenes, using a yeast-bacteria shuttle vector, has been described.
      • Bentley K.L.
      • Shashikant C.S.
      • Wang W.
      • Ruddle N.H.
      • Ruddle F.H.
      A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes.
      Genomic information necessary for designing the transgene was obtained from the National Center for Biotechnology Information Mouse Genome Resources database. The ProxTom transgene is based on a 214,895-bp BAC clone, RPCI-23-385H16 (Invitrogen, Carlsbad, CA), corresponding to nucleotides 191,782,085 to 191,996,980 of mouse chromosome 1 (National Center for Biotechnology Information Mouse Genome Build 37.1). A 143,990-bp genomic fragment, representing nucleotides 23,399 to 167,389 of RPCI23-385H16, containing the Prox1 gene and flanking regions was captured in pClasper by homologous recombination in yeast. The Prox1 gene was modified by insertion of a promoterless tdTomato RFP reporter gene (a gift from Roger Y. Tsien, University of California, San Diego, CA)
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
      • Bentley K.L.
      • Shashikant C.S.
      • Wang W.
      • Ruddle N.H.
      • Ruddle F.H.
      A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes.
      in-frame after the DNA coding for amino acid five (Asp5), which is upstream of the Prox1 nuclear localization signal (Figure 1A). Transgene-positive mice were identified by PCR of DNA from tail biopsy samples using ProxseqF and ProxseqR primers that amplify a 933-bp product. The number of copies of the transgene inserted in founder mice was analyzed by Southern blot analysis. EcoRV-digested genomic tail DNA from founder numbers 12 and 15 was probed with a 1132-bp 32P-dCTP–labeled URA3 probe that hybridized to a 5164-bp fragment in the reporter gene cassette (Figure 1, B and C). Oligonucleotides used for the construction and molecular analysis of the ProxTom transgene in transgenic mice are listed in Table 1.
      Figure thumbnail gr1
      Figure 1Construction of the ProxTom transgene, genotyping, and Southern blot analysis of ProxTom mice. A: Diagram of the ProxTom transgene. Approximately 144 kb of BAC clone RPCI23-385H16 (215 kb) containing the mouse Prox1 gene, including 80 kb of upstream flanking sequence, was captured in pClasper by homologous recombination in yeast. A promoterless tdTomato reporter gene was mated to the SV40polyA signal and the yeast gene, URA3, to select for pCLA-ProxTom yeast transformants. The reporter cassette was inserted in-frame after amino acid five (Asp5) of the Prox1 gene in pCLA-Prox1 (152,609 bp) to produce pCLA-Prox1-Tom (155,546 bp) by yeast recombination. Asp5 is upstream of the nuclear localization sequence in Prox1 and leads to default expression of tdTomato in the cell's cytoplasm. The ProxseqF and ProxseqR primers used for genotyping are shown. B: A 933-bp product was amplified in ProxTom-positive founders (4, 12, and 15) but not in negative littermates (1, 10, 23, and 27). The ProxTom transgene construct was used as a positive control (pos), and genomic DNA from C57BL/6 and no DNA were negative controls (neg). C: Southern blot analysis of EcoRV-digested genomic tail DNA from founders 12 and 15 was probed with a 1132-bp 32P-dCTP–labeled URA3 probe hybridizing to a 5164-bp fragment. Dilutions of the transgene DNA are shown to estimate the number of copies of the transgene inserted into each mouse. Founders 12 (eight to ten copies) and 15 (five to six copies) had only one integration site each.
      Table 1Oligonucleotides Used in this Study
      Oligo nameSequence and description
      Construction of ProxTom Transgene
      5PROXCNucleotides 1–50 correspond to pClasperA vector. Nucleotides 51–100 correspond to nucleotides 23,399–23,448 of RPCI23-385H16.
      5′-AGGAGTCATATTACCCTGTTATCCCTAGGCCCTCGAGGCCGGCGCGCCACCTAGATGGAGCATGTGGCGCATTCC-TGCATTGGGTATAGGGTACCCATC-3′
      5PROXCRCReverse complement of 5PROXC.
      5′-GATGGGTACCCTATACCCAATGCAGGAATGCGCCACATGCTCCATCTAGGTGGCGCGCCGGCCTCGAGGGCCTAG-GGATAACAGGGTAATATGACTCTCT-3′
      3PROXCNucleotides 1–50 correspond to nucleotides 167,340–167,389 of RPCI23-385H16. Nucleotides 51–100 correspond to pClasperA vector.
      5′-TGAACGCCTGGTTGCCAGTAGGTGGCGCTGTTGGGGAGGTTTAGGAGATGGATCCGTTTAAACGCGGCCGCTTAA-TTAATTAGGGATAACAGGGTAATTA-3′
      3PROXCRCReverse complement of 3PROXC.
      5′-TAATTACCCTGTTATCCCTAATTAATTAAGCGGCCGCGTTTAAACGGATCCATCTCCTAAACCTCCCCAACAGCG-CCACCTACTGGCAACCAGGCGTTCA-3′
      PROXTOMFNucleotides 1–50 correspond to nucleotides 80,936–80,985 of RPCI23-385H16. Nucleotides 51–75 are the TOMSmaF primer (missing CC at the 5' end).
      5′-CGAGCTTTTGAAGATGGCACAATAACTGTCCAGTGATGCCTGACCATGACGGGATGGTGAGCAAGGGCGAGGAGG-3′
      PROXRFP3Nucleotides 1–50 correspond to nucleotides 80,864–80,913 of RPCI23-385H16. Nucleotides 51–75 are the URA3R primer.
      5′-TGTCCCTACCGTCCTTTTCACTCCAATGTCAACCCTTCTCCTCTTGGTTTGCACCACAGCTTTTCAATTCAATTC-3′
      TOMSmaF5′-CCCGGGATGGTGAGCAAGGGCGAGGAGG-3′
      TOMSpeR5′-ACTAGTTTACTTGTACAGCTCGTCCATGCCG-3′
      Genotyping
      PROXSEQF5′-CCATGTTGTTGTCCTCG-3′
      PROXSEQR5′-AAATCCCAGAGCCTATGC-3′
      Generation of URA3 Probe for Southern Blot Analysis
      URA3F5′-CACACCGCATAGGGTAATAACTG-3′
      URA3R5′-ACCACAGCTTTTCAATTCAATTC-3′
      RT-PCR Primers
      PROXRTF15′-CCGCAGAAGGACTCTCTTTGTCAC-3′
      PROXRTR15′-CGTCCGAGAAGTAGGTCTTCAGC-3′
      MACT1F5′-GCTGTGCTGTCCCTGTATGCCTCT-3′
      MACT1R5′-CCTCTCAGCTGTGGTGGTGAAGC-3′

      Fluorescent Immunohistochemical Analysis

      Tissues were fixed in periodate-lysine–1% paraformaldehyde, embedded in OCT, and stored at −80°C. The 7-μm–thick frozen sections were cut and blocked with 4% serum (Sigma-Aldrich, St. Louis, MO) and 5% bovine serum albumin in PBS. Primary antibodies: rabbit anti-RFP 1:500 (Rockland Antibodies & Assays, Gilbertsville, PA), rabbit anti-Prox1 1:100 (Abcam Inc., Cambridge, MA), rabbit anti-mouse Prox1 1:100 (AngioBio, Del Mar, CA), rat anti–LYVE-1 1:250 (R&D Systems, Minneapolis, MN), fluorescein isothiocyanate rat anti-mouse CD11b 1:200 (BD Pharmingen, San Diego, CA), rabbit anti-mouse tyrosine hydroxylase 1:500 (Abcam Inc.), mouse anti-human VE-cadherin 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-mouse CD41 1:50, and rat isotype control (R&D Systems). Secondary antibodies: biotinylated goat anti-rabbit IgG 1:300 (Vector Laboratories, Burlingame, CA), streptavidin-CY3 1:500 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), streptavidin-CY2 1:500 (Jackson ImmunoResearch Laboratories, Inc.), goat anti-mouse Dylight488 (Jackson ImmunoResearch Laboratories, Inc.), donkey anti-rabbit IgG Dylight 549 1:1000 (Jackson ImmunoResearch Laboratories, Inc.), goat anti-rabbit 488 (Alexa Fluor; Molecular Probes Inc., Eugene, OR), and goat anti-rat CY2 1:100 (Jackson ImmunoResearch Laboratories, Inc.). Digital images were captured using an Axiocam camera (Carl Zeiss MicroImaging GmbH, Jena, Germany) mounted directly onto the microscope. Images were analyzed and size bars were calculated using Axiovision software (Carl Zeiss MicroImaging GmbH). Images were saved in the TIFF format. Red, green, and blue images were merged using Photoshop version 8.0 (Adobe Systems Inc., San Jose, CA).

      In Vivo Two-Photon Laser Scanning Microscopy and Three-Dimensional Volume Rendering of Lymph Nodes in Living Mice

      For in vivo imaging of an exposed lymph node, progenies of a cross between Hec6stGFP and ProxTom mice (progeny of founder 12) were immunized by skin painting with 50 μL of 4% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich) in acetone on the hind leg. Seven days later, anesthesia was induced by an i.p. injection of 200 μL of 10 mg/mL ketamine and 1 mg/mL xylazine (Fort Dodge Animal Health, Fort Dodge, IA) and was maintained by an inhaled isofluorane and air mixture. The mouse was immobilized on a custom-built stage, and the right popliteal lymph node was surgically exposed as previously described.
      • Mempel T.R.
      • Scimone M.L.
      • Mora J.R.
      • von Andrian U.H.
      In vivo imaging of leukocyte trafficking in blood vessels and tissues.
      The lymph node was immersed in 0.9% saline and was covered with a glass coverslip. An Olympus BX61WI fluorescence microscope combined with an Olympus 20X, 0.95NA objective (Olympus, Melville, NY) and a two-photon microscopy system (LaVision BioTec, Bielefeld, Germany) was used for imaging of the lymph node. An autotunable titanium-sapphire multipass laser (Chameleon-XR; Coherent Inc., Santa Clara, CA) pumped by a 12-W Verdi laser source (Coherent Inc.) was used for the excitation light source. Emitted light was collected using non-descanned detectors outfitted with the following bandpass filters: 435/90 nm (for second harmonic emission of collagen fibers), 525/50 nm (GFP), and 615/100 nm (RFP). Stacks of either 96 or 150 optical sections with 1-μm z spacing were acquired with the laser set at a wavelength of 940 nm. The field of view for each x-y plane was 500 mm, acquired at a resolution of 0.976 mm per pixel. Volocity software (PerkinElmer, Waltham, MA) was used to generate volume renderings of image stacks.

      Ex Vivo Fluorescent Microscopy

      Tissues were visualized using an Axioskop fluorescent microscope (Carl Zeiss MicroImaging GmbH), and images were captured using an Axiocam camera. Axiovision software was used for analyses.

      mRNA Analysis

      Adrenal glands and livers were removed from wild-type mice, and RNA was purified by the RNeasy spin column method (Qiagen Inc., Valencia, CA). cDNA was prepared using SuperScript II enzyme (Invitrogen) and was analyzed in SYBER Green reactions (iQ supermix; Bio-Rad Laboratories, Hercules, CA) by real-time PCR (iQ5; Bio-Rad Laboratories). RT-PCR primers for Prox1 spanning exons 2 and 3 of mouse Prox1 (192 bp) and mouse β-actin (206 bp) are indicated in Table 1.

      Preparation of Bone Marrow and Blood Smears

      The mice were anesthetized, and heparinized blood was collected from the retro-orbital plexus. Smears of blood and bone marrow cells were prepared, dried, and fixed in either 4% paraformaldehyde or methanol.

      Primary Megakaryocyte Culture and Separation from Whole Bone Marrow

      Primary megakaryocytes were derived from murine bone marrow. After lysis of mature red blood cells (BD Pharm Lyse, BD Biosciences, San Jose, CA), bone marrow cells were cultured at 2 × 106 cells/mL in StemSpan serum-free expansion medium (STEMCELL Technologies Inc., Vancouver, BC, Canada), 30% serum substitute (BIT9500; STEMCELL Technologies Inc.), 50 ng/mL murine thrombopoietin (PeproTech, Rocky Hill, NJ), 2 mmol/L l-glutamine, and penicillin/streptomycin.
      • Cheng E.C.
      • Luo Q.
      • Bruscia E.M.
      • Renda M.J.
      • Troy J.A.
      • Massaro S.A.
      • Tuck D.
      • Schulz V.
      • Mane S.M.
      • Berliner N.
      • Sun Y.
      • Morris S.W.
      • Qiu C.
      • Krause D.S.
      Role for MKL1 in megakaryocytic maturation.
      To enrich for megakaryocytes, after 4 days, the cells were fractionated on a 3% discontinuous bovine serum albumin (Sigma-Aldrich) gradient. The fully mature polyploid megakaryocytes were located mainly in the pellet below the 3% fraction.
      • Drachman J.G.
      • Sabath D.F.
      • Fox N.E.
      • Kaushansky K.
      Thrombopoietin signal transduction in purified murine megakaryocytes.

      Human Erythroleukemia Cell Culture

      Human erythroleukemia (HEL) cells were purchased from American Type Culture Collection (Manassas, VA) and were cultured as described elsewhere.
      • Cheng E.C.
      • Luo Q.
      • Bruscia E.M.
      • Renda M.J.
      • Troy J.A.
      • Massaro S.A.
      • Tuck D.
      • Schulz V.
      • Mane S.M.
      • Berliner N.
      • Sun Y.
      • Morris S.W.
      • Qiu C.
      • Krause D.S.
      Role for MKL1 in megakaryocytic maturation.
      HEL cells were cultured in the presence of 1.5 × 10−8 mol/L phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 24 to 72 hours to induce differentiation. Cytoplasmic fractions were prepared from whole HEL cells by sequential homogenization in lysis buffer and were centrifuged at 1300 × g saving the supernatant. The nuclear pellet was resuspended in the same buffer, ultrasonicated to break the nuclear membranes apart, and centrifuged at 1300 × g. The nuclear fraction was in solution in the supernatant.

      Human Lymphatic Endothelial Cell Culture

      The commercially available human lymphatic endothelial cells isolated from human dermal lymphatic microvascular endothelial cells were purchased from Lonza Group Ltd. (Basel, Switzerland). Human lymphatic endothelial cells were cultured in EGM-2MV media (Lonza Group Ltd.) in cell culture dishes coated with 0.1% gelatin (Bio-Rad Laboratories) at 37°C in 5% CO2 as described previously.
      • Jones D.
      • Xu Z.
      • Zhang H.
      • He Y.
      • Kluger M.S.
      • Chen H.
      • Min W.
      Functional analyses of the nonreceptor kinase bone marrow kinase on the X chromosome in vascular endothelial growth factor-induced lymphangiogenesis.
      Confluent cells were harvested, and protein was extracted for Western blot analysis.

      Western Blot Analysis

      Cells were lysed in radioimmunoprecipitation assay buffer (Bio-Rad Laboratories) containing protease inhibitors (Roche Applied Sciences, Indianapolis, IN). Protein was boiled, resolved in 4% to 20% polyacrylamide gels (Mini-Protean TGX; Bio-Rad Laboratories), and transferred to nitrocellulose membranes (Bio-Rad Laboratories). Membranes were then blocked with either 5% milk or bovine serum albumin in PBS containing 0.05% Tween 20. Primary antibodies: rabbit anti-mouse–Prox1 1:1000 (Chemicon, Temecula), rabbit anti-human–Prox1 1:1000 (Abcam Inc.), mouse anti-mouse–actin 1:10,000 (Sigma-Aldrich), and rabbit anti-human–glyceraldehyde-3-phosphate dehydrogenase 1:10,000 (Sigma-Aldrich). Secondary horseradish peroxidase–conjugated antibodies: goat anti-rabbit Ig 1:5000 and goat anti-mouse Ig 1:5000 (Santa Cruz Biotechnology). Bands were detected using an enhanced chemiluminescence detection system (GE Healthcare Bio-Sciences Corp, Piscataway, NJ).

      Results

      Transgenic Mice Express the RFP tdTomato Under the Control of the Prox1 Gene

      ProxTom transgenic mice were made on the C57BL/6 background using the pClasper technique.
      • Bentley K.L.
      • Shashikant C.S.
      • Wang W.
      • Ruddle N.H.
      • Ruddle F.H.
      A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes.
      A large genomic DNA fragment was isolated that contained the entire mouse Prox1 gene and its known regulatory elements (Figure 1A). Three mice from 27 progenies of microinjected embryos were positive for the transgene by PCR (Figure 1B). Southern blot analysis confirmed that founder mice 12 and 15 contained approximately ten or six copies of the transgene, respectively (Figure 1C). Founder 12, with the highest copy number, had brighter fluorescence than did founder 15. Both mice were healthy, germline transmission was achieved in C57BL/6 mice with normal mendelian transmission, and lines 12 and 15 were established from founders 12 and 15, respectively. Mice from both lines were used in this study.

      Prox1-Driven tdTomato Is Expressed in LVs Coincident with Endogenous Prox1 and LYVE-1 in Lymph Nodes

      Lymphatic endothelial cells from the lymph nodes of transgenic mice had a robust red signal in their cytoplasm that was not seen in littermate controls (Figure 2). The tdTomato signal was apparent by direct fluorescence in progenies of founder 12. Line 12 was brighter than line 15, most likely because it carries a higher number of copies of the transgene (Figure 1C). Although the signal was less bright in line 15, it could be intensified using an anti-RFP antibody that binds tdTomato. We co-stained LVs with an anti-Prox1 antibody to check the fidelity of transgene expression. In both lines, we observed tdTomato in the same cells as endogenous Prox1. tdTomato was detected mainly in the cytoplasm, whereas endogenous Prox1 was predominantly found in the nuclei of LVs (Figure 2A).
      Figure thumbnail gr2
      Figure 2LVs can be visualized in ProxTom lymph nodes. A: tdTomato and Prox1 were co-expressed in lymphatic endothelial cells. tdTomato (red) is in the cytoplasm, and endogenous Prox1 (green) is in the nuclei of lymphatic endothelial cells. Nuclei were counterstained with DAPI (blue). B: tdTomato (red) is expressed in the cytoplasm of lymphatic endothelial cells co-incident with LYVE-1 (green). Antibodies to LYVE-1 also bind to macrophages in the lymph node (green; arrowhead). Nuclei were counterstained with DAPI (blue). C: CD11b-positive macrophages (green) do not express the tdTomato transgene (red) in the ProxTom lymph node. D: An in vivo three-dimensional image of a popliteal lymph node acquired by two-photon laser scanning microscopy. Progeny from a cross between a ProxTom mouse and a Hec6stGFP mouse with green fluorescent HEVs was imaged, showing tdTomato in LVs (red), HEVs (green), and a blue capsule. All the data are from ProxTom line 12.
      LYVE-1 is a marker of LVs that is also expressed by macrophages.
      • Pham T.H.
      • Baluk P.
      • Xu Y.
      • Grigorova I.
      • Bankovich A.J.
      • Pappu R.
      • Coughlin S.R.
      • McDonald D.M.
      • Schwab S.R.
      • Cyster J.G.
      Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning.
      We saw that tdTomato co-localized with LYVE-1 in LVs (Figure 2B) but not in macrophages. Lymph node macrophages, defined by staining with antibody to CD11b, also expressed LYVE-1 but not tdTomato (Figure 2B). These data demonstrate that Prox-1–driven tdTomato is a more specific and useful marker for lymph node LV imaging than is LYVE-1 because CD11b-positive macrophages from transgene-positive mice do not express tdTomato (Figure 2C).

      LVs Can be Visualized in Living ProxTom Mice

      Our goal was to develop a mouse whose LVs could be visualized in a living animal to conduct immunologic studies. We wanted to observe the dynamic interaction of LV with HEV lymph node in real time because of the close interactions between these vessels that we have observed after immunization.
      • Liao S.
      • Ruddle N.H.
      Synchrony of high endothelial venules and lymphatic vessels revealed by immunization.
      We crossed a red fluorescent ProxTom mouse (from line 12) with an Hec6stGFP mouse that has green fluorescent HEVs, and their progenies were immunized with 4% oxazolone. Seven days later, the popliteal lymph node of a ProxTom × Hec6stGFP mouse was surgically exposed and examined in vivo using a two-photon laser scanning microscope. The LVs were fluorescent red and were seen clearly, and the HEVs were green (Figure 2D). The blue color on the edge of the lymph node results from “second harmonic” emission generated by photons interacting with collagen in the capsule. We imaged tissue immediately beneath the capsule to a depth of 96 μm and obtained stacks of 1-μm-thick optical sections (see Supplemental Video S1 at http://ajp.ampathol.org). We also imaged the medulla of the lymph node to a depth of 150 μm (see Supplemental Video S2 at http://ajp.ampathol.org). The distribution of LVs relative to HEVs in the subcapsular tissues was revealed by a three-dimensional reconstruction of these image stacks (Figure 2D). These data confirm the suitability of ProxTom mice for in vivo two-photon laser scanning microscopy.

      Prox1-Driven tdTomato Is Expressed in LVs in a Variety of Tissues and Is Highly Expressed in Lymphatic Valves

      tdTomato fluorescence was seen in LVs in many organs of mice aged 10 to 16 weeks (Figure 3), and fluorescence continued to be observed in tissues from older mice (>1 year old). We saw tdTomato in LVs in whole mounts of the following tissues: ear skin, the lacteals and collecting vessels of the ileum, esophagus, mesentery, diaphragm, tongue, mucosal and serosal surfaces of a Peyer's patch, and bladder (Figure 3). The strongest tdTomato signal was seen in the valve leaflets in the large collecting vessels found in the mesentery and draining the esophagus (Figure 3, C and D). There was relatively lower expression of tdTomato in the segments between the valves. This finding confirmed previous observations that Prox1 expression is concentrated in the valve leaflets of collecting LVs.
      • Norrmén C.
      • Ivanov K.I.
      • Cheng J.
      • Zangger N.
      • Delorenzi M.
      • Jaquet M.
      • Miura N.
      • Puolakkainen P.
      • Horsley V.
      • Hu J.
      • Augustin H.G.
      • Ylä-Herttuala S.
      • Alitalo K.
      • Petrova T.V.
      FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1.
      The higher expression of tdTomato in the valves compared with that of the intervalve segments suggests that the transgene is regulated in the same way as the endogenous gene.
      Figure thumbnail gr3
      Figure 3tdTomato is expressed in LVs and valves. Whole mounts of ex vivo tissues from ProxTom mice aged 10 to 16 weeks showing tdTomato (red) expression in LVs in different tissues. A: Ear skin. B: Central lacteals and submucosal lymphatics of the ileum. C: An LV encircling the distal esophagus showing the valve leaflets (arrowhead). D: Mesentery of the small intestine with valves (arrowhead). E: The lymphatics follow the course of the muscle fibers in the diaphragm. F: The tongue. G and H: The mucosal (G) and serosal (H) surfaces of a Peyer's patch. I: Urinary bladder. All the data are from ProxTom line 12.

      Prox1-Driven tdTomato Is Expressed in Some Extralymphatic Tissues

      Prox1 has been described in the lens,
      • Wigle J.T.
      • Chowdhury K.
      • Gruss P.
      • Oliver G.
      Prox1 function is crucial for mouse lens-fibre elongation.
      in hepatocytes
      • Oliver G.
      • Sosa-Pineda B.
      • Wigle J.T.
      • Oliver G.
      Hepatocyte migration during liver development requires.
      and in the dentate gyrus of the brain,
      • Lavado A.
      • Oliver G.
      Prox1 expression patterns in the developing and adult murine brain.
      and we confirmed reliable expression of the transgene in these tissues. The control liver had some background autofluorescence (Figure 4A). However, ProxTom hepatocytes from line 12 fluoresced more brightly than did those of littermate controls (Figure 4B). To verify that the red fluorescence seen in ProxTom liver was not simply autofluorescence, we used an anti-RFP antibody on transgenic line 15 liver. The anti-RFP antibody did not bind to control liver (Figure 4C), but it did detect tdTomato in hepatocytes from line 15 ProxTom mice (Figure 4D). These results confirmed transgene expression in the liver.
      Figure thumbnail gr4
      Figure 4Extralymphatic expression of tdTomato and Prox1. A: Background red autofluorescence in the liver of a transgene-negative littermate. B: Bright tdTomato signal (red) is above background in ProxTom (line 12) hepatocytes. C: Anti-RFP antibody (red) does not bind to transgene-negative littermate control liver. D: Anti-RFP antibody (red) labels tdTomato in ProxTom liver (line 15). E: Dentate gyrus of the brain. Anti-Prox1 antibody (red) labels endogenous Prox1 in the nuclei of the neurons of transgene-negative littermates. F: Anti-RFP antibody (red) labels tdTomato in the cytoplasm of neurons in ProxTom dentate gyrus (line 15). In C to F, nuclei were counterstained with DAPI (blue). G: ProxTom in the lens (line 12). H: ProxTom (arrowhead) in the adrenal gland medulla (line 12). I: Endogenous Prox1 (green) is co-expressed with tdTomato (red) in cells of the adrenal medulla ProxTom (line 12). J: Neuroendocrine cells of the adrenal medulla co-expressed tyrosine hydroxylase (green) and tdTomato (red) in ProxTom (line 12).
      Endogenous Prox1 was observed in the nuclei of cells of the dentate gyrus of wild-type mice (Figure 4E), and we confirmed cytoplasmic expression of tdTomato in ProxTom neurons using an anti-RFP antibody (Figure 4F) but not in those of littermate controls. The ProxTom lens was brightly fluorescent (Figure 4G) compared with that of a littermate control. The bright red fluorescence of the lens makes it easy to screen transgene-positive mice under UV light.
      Herein, we report for the first time the presence of Prox1-driven tdTomato in the adrenal medulla of adult mice. We looked at whole mounts of the adrenal gland and saw tdTomato in the medulla but not in the cortex (Figure 4H). Endogenous Prox1 was expressed together with tdTomato in cells of the adrenal medulla (Figure 4I). Tyrosine hydroxylase is an enzyme important for the synthesis of adrenaline and noradrenaline in the neuroendocrine (chromaffin) cells of the adrenal medulla. tdTomato was co-expressed in cells labeled by an antibody against tyrosine hydroxylase, confirming Prox1 expression in the neuroendocrine cells of the adrenal medulla (Figure 4J). To confirm the expression of endogenous Prox1 mRNA in whole adrenal gland and liver, we isolated RNA from wild-type C57BL/6 mice and analyzed it by quantitative RT-PCR. We show that Prox1 mRNA is expressed in adrenal glands and liver (Figure 5).
      Figure thumbnail gr5
      Figure 5Prox1 mRNA expression in liver and adrenal glands. A: Expression levels of endogenous Prox1 mRNA in mouse adrenal gland and liver were quantified by quantitative RT-PCR relative to mouse β-actin and are expressed as the fold difference in threshold cycle values (2−ΔCT) between Prox1 and β-actin. B: Amplification products from the quantitative RT-PCR analysis were separated on a 2% agarose gel. The composite figure shows the 192-bp Prox1 and 206-bp β-actin products from adrenal gland (Adr) and liver (Liv). M, size marker.

      Prox1 Is Expressed in Megakaryocytes

      We saw that ProxTom bone marrow contained tdTomato-positive cells, including large multinucleate cells with the appearance of megakaryocytes (Figure 6A). Having shown tdTomato expression in megakaryocytes using the transgenic reporter, we looked for Prox1 in wild-type megakaryocytes by immunohistochemical analysis. We double stained bone marrow with anti-Prox1 and anti-CD41 antibodies because CD41 is a marker of megakaryocytes and also of platelets. The results showed that endogenous Prox1 was detected in the cytoplasm of megakaryocytes of C57BL/6 mice (Figure 6, B–D). We confirmed Prox1 expression in megakaryocytes by Western blot analysis (Figure 6, E and F). The predicted molecular weight of Prox1 is 83 kDa, and, as expected, we saw a band of this size in lymph node stromal cells. After culturing C57BL/6 bone marrow to stimulate megakaryocyte maturation, we used a bovine serum albumin gradient to enrich for large, polyploid megakaryocytes. We saw a band at approximately 83 kDa in the fraction that was enriched for megakaryocytes and a much less intense band in the megakaryocyte-depleted bone marrow (Figure 6E). Thus, by means of three separate methods—transgene reporter fluorescence, endogenous gene immunofluorescence, and Western blot analysis—we demonstrated unequivocally that megakaryocytes express Prox1 protein.
      Figure thumbnail gr6
      Figure 6Prox1 in megakaryocytes. A: Bone marrow smear showing ProxTom (line 12) megakaryocyte expression of tdTomato (red) in the cytoplasm; nuclei were counterstained with DAPI (blue). B: Anti-Prox1 antibody (red) labels endogenous Prox1 in the cytoplasm of megakaryocytes from a transgene-negative C57BL/6. C: The same megakaryocyte stained with anti-CD41 antibody (green). D: Merge showing Prox1 and CD41 (yellow) in the cytoplasm; nuclei were counterstained with DAPI (blue). E: Western blot of Prox1 expression in megakaryocyte (MK)-depleted and MK-enriched bone marrow (BM). Anti-Prox1 antibody identifies a band at 83 kDa in lymph node stromal cells. Prox1 is seen in the MK-enriched fraction and a lesser amount in the MK-depleted fraction. F: Human lymphatic endothelial cells (HLECs)
      • Mounzer R.H.
      • Svendsen O.S.
      • Baluk P.
      • Bergman C.M.
      • Padera T.P.
      • Wiig H.
      • Jain R.K.
      • McDonald D.M.
      • Ruddle N.H.
      Lymphotoxin-α contributes to lymphangiogenesis.
      and HEL cells express Prox1. Prox1 is found predominantly in the cytoplasm of HEL cells. HEL cells that have differentiated into megakaryocytes after culture with phorbol 12-myristate 13-acetate (TPA) for 0 to 72 hours express Prox1 in their cytoplasm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      Prox1 Is Detected in Megakaryocyte Cytoplasm

      We used the HEL cell line to examine the subcellular location of Prox1. HEL cells can be differentiated into megakaryocytes by the addition of phorbol 12-myristate 13-acetate.
      • Cheng E.C.
      • Luo Q.
      • Bruscia E.M.
      • Renda M.J.
      • Troy J.A.
      • Massaro S.A.
      • Tuck D.
      • Schulz V.
      • Mane S.M.
      • Berliner N.
      • Sun Y.
      • Morris S.W.
      • Qiu C.
      • Krause D.S.
      Role for MKL1 in megakaryocytic maturation.
      We differentiated HEL cells into megakaryocytes for 0 to 72 hours and then separated the cytoplasmic and nuclear fractions. We found that Prox1 protein was predominantly located in the cytoplasmic fraction of HEL cells and that a smaller amount was present in the nuclear fraction (Figure 6F). We also examined HEL cells by immunohistochemical analysis and, again, showed that Prox1 was predominantly located in the cytoplasm of HEL cells with a ploidy of 2N to 8N.

      Prox1 Is Located in Platelets

      Having shown that primary megakaryocytes and HEL cells express Prox1 in their cytoplasm and knowing that platelets form by budding off from megakaryocyte cytoplasm, we next looked for Prox1 in platelets. We double stained platelets in whole blood (Figure 7A) and in platelet-rich plasma (Figure 7, B–D) with antibodies to Prox1 and CD41 and discovered that platelets carry Prox1, suggesting that megakaryocytes incorporate Prox1 into platelets during platelet formation.
      Figure thumbnail gr7
      Figure 7Prox1 in platelets. A: Whole blood from C57BL/6 mice showing anti-Prox1 antibody labeling of platelets (red), erythrocytes (green), and nuclei counterstained with DAPI (blue). B: Platelet-rich plasma from C57BL/6 mice showing that anti-Prox1 antibody (red) labels endogenous Prox1 in platelets. C: The same platelets stained with anti-CD41 antibody. D: Merged image of B and C.

      Discussion

      Our goal was to make a transgenic mouse with red fluorescent LVs suitable for in vivo imaging by two-photon laser scanning microscopy. We showed that ProxTom mice have brightly fluorescent LV that can be imaged directly, without the need for external manipulation or the injection of fluorescent tracer dyes. Our eventual goal is to visualize the dynamic changes in the vasculature of the lymph node after immunization in real time. ProxTom mice were successfully crossed with Hec6stGFP mice that have green fluorescent HEVs, which allowed us to image red LVs and green HEVs simultaneously in a living lymph node for the first time. The ready visibility of cells expressing the tdTomato reporter also allowed us to detect Prox1 expression in novel places, leading to the discovery of Prox1 in the neuroendocrine cells of the adrenal gland and in megakaryocytes and platelets.
      We used the pClasper technique to construct ProxTom mice because it permits the creation and modification of transgenes over a wide range of sizes, from a few kilobase pairs to entire BAC inserts, accommodating most genes. pClasper is a shuttle vector capable of propagation in yeast and bacteria. Constructs are fashioned precisely by homologous recombination in yeast, whereas transgene expansion, purification, and storage are performed in bacteria.
      • Bentley K.L.
      • Shashikant C.S.
      • Wang W.
      • Ruddle N.H.
      • Ruddle F.H.
      A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes.
      We used pClasper to capture 155 kb of the Prox1 gene, including 80 kb of upstream regulatory sequences, which is a much larger segment than previous LV reporters.
      • François M.
      • Caprini A.
      • Hosking B.
      • Orsenigo F.
      • Wilhelm D.
      • Browne C.
      • Paavonen K.
      • Karnezis T.
      • Shayan R.
      • Downes M.
      • Davidson T.
      • Tutt D.
      • Cheah K.S.
      • Stacker S.A.
      • Muscat G.E.
      • Achen M.G.
      • Dejana E.
      • Koopman P.
      Sox18 induces development of the lymphatic vasculature in mice.
      The 155-kb ProxTom construct contains the coding regions and control elements necessary for the recapitulation of normal patterns of Prox1 expression (Figure 1A). Integration site position effects are ameliorated by the fact that the entire gene, surrounded by a large amount of genomic DNA, likely harboring insulators, is included in the construct. In our experience, normal expression is seen in more than 90% of founders using the pClasper technique.
      • Bentley K.L.
      • Shashikant C.S.
      • Wang W.
      • Ruddle N.H.
      • Ruddle F.H.
      A yeast-based recombinogenic targeting toolset for transgenic analysis of human disease genes.
      The reliability of pClasper meant that we were confident that the transgene would faithfully reproduce endogenous gene expression. In fact, we could confirm previous reports of Prox1 expression in adult liver, dentate gyrus, and lens, and we also discovered Prox1 in megakaryocytes, platelets, and the adrenal medulla.
      • Wigle J.T.
      • Chowdhury K.
      • Gruss P.
      • Oliver G.
      Prox1 function is crucial for mouse lens-fibre elongation.
      • Oliver G.
      • Sosa-Pineda B.
      • Wigle J.T.
      • Oliver G.
      Hepatocyte migration during liver development requires.
      • Lavado A.
      • Oliver G.
      Prox1 expression patterns in the developing and adult murine brain.
      Prox1 has been described in the sympathetic ganglia of murine embryos and also in neuroblastomas, which are tumors of the adrenal medulla.
      • François M.
      • Caprini A.
      • Hosking B.
      • Orsenigo F.
      • Wilhelm D.
      • Browne C.
      • Paavonen K.
      • Karnezis T.
      • Shayan R.
      • Downes M.
      • Davidson T.
      • Tutt D.
      • Cheah K.S.
      • Stacker S.A.
      • Muscat G.E.
      • Achen M.G.
      • Dejana E.
      • Koopman P.
      Sox18 induces development of the lymphatic vasculature in mice.
      • Becker J.
      • Wang B.
      • Pavlakovic H.
      • Buttler K.
      • Wilting J.
      Homeobox transcription factor Prox1 in sympathetic ganglia of vertebrate embryos: correlation with human stage 4s neuroblastoma.
      The sympathetic ganglion and the adrenal medulla are derived from the neural crest. Neuronal-derived peptides and transmitters are important for lymphatic development.
      • Xu Y.
      • Yuan L.
      • Mak J.
      • Pardanaud L.
      • Caunt M.
      • Kasman I.
      • Larrivée B.
      • Suchting S.
      • del Toro R.
      • Medvinsky A.
      • Yang J.
      • Kolodkin A.
      • Thomas J.L.
      • Koch A.
      • Alitalo K.
      • Eichmann A.
      • Bagri A.
      Neuropilin-2 mediates VEGF-C induced lymphatic sprouting together with VEGFR3.
      It will be interesting to see whether the neuroendocrine (chromaffin) cells of the adrenal medulla release Prox1 after stimulation of the sympathetic nervous system.
      One advantage of the transgenic approach was that we could verify tdTomato expression by comparing it with the normal distribution of endogenous Prox1. It was especially important to check the fidelity of tdTomato fluorescence when we saw it in novel places such as in the bone marrow and the adrenal medulla. When we constructed ProxTom, we inserted the reporter gene in-frame after the DNA coding for amino acid five (Asp5) in the Prox1 gene (Figure 1A). This meant that the tdTomato stop codon was upstream of the Prox1 nuclear localization signal and directed tdTomato into the cytoplasm.
      The observation that Prox1 is located in megakaryocytes and platelets is interesting given the reported role of platelets in lymphangiogenesis in the embryo. The finding that megakaryocytes express Prox1 is consistent with an earlier report that Prox1 mRNA was observed in some human leukemias and in the megakaryocyte lines HEL, CMK86, and MEG01.
      • Nagai H.
      • Li Y.
      • Hatano S.
      • Toshihito O.
      • Yuge M.
      • Ito E.
      • Utsumi M.
      • Saito H.
      • Kinoshita T.
      Mutations and aberrant DNA methylation of the PROX1 gene in hematologic malignancies.
      At first, we were puzzled to find that Prox1 was located predominantly in the megakaryocyte's cytoplasm because Prox1 is a transcription factor that is usually found in the nucleus. However, similar to Drosophila prospero, Prox1 can also be in the cytoplasm under certain circumstances.
      • Sousa-Nunes R.
      • Somers W.G.
      Phosphorylation and dephosphorylation events allow for rapid segregation of fate determinants during Drosophila neuroblast asymmetric divisions.
      • Demidenko Z.
      • Badenhorst P.
      • Jones T.
      • Bi X.
      • Mortin M.A.
      Regulated nuclear export of the homeodomain transcription factor Prospero.
      • Duncan M.K.
      • Cui W.
      • Oh D.J.
      • Tomarev S.I.
      Prox1 is differentially localized during lens development.
      • Laerm A.
      • Helmbold P.
      • Goldberg M.
      • Dammann R.
      • Holzhausen H.J.
      • Ballhausen W.G.
      Prospero-related homeobox 1 (PROX1) is frequently inactivated by genomic deletions and epigenetic silencing in carcinomas of the bilary system.
      • Bosco A.
      • Cusato K.
      • Nicchia G.P.
      • Frigeri A.
      • Spray D.C.
      A developmental switch in the expression of aquaporin-4 and Kir4.1 from horizontal to Muller cells in mouse retina.
      • Baxter S.A.
      • Cheung D.Y.
      • Bocangel P.
      • Kim H.K.
      • Herbert K.
      • Douville J.M.
      • Jangamreddy J.R.
      • Zhang S.
      • Eisenstat D.D.
      • Wigle J.T.
      Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein.
      We speculated that Prox1 might be in the cytoplasm of megakaryocytes because these cells undergo endomitosis, which is an unusual mode of DNA replication without cell division. The amino acid sequence of Prox1, 623WFSNFR628, includes four residues that are conserved in the consensus penetratin sequence that is also shared by other homeobox proteins, eg, Pax6 and Engrailed.
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      Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein.
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      A short region of its homeodomain is necessary for engrailed nuclear export and secretion.
      The penetratins are hydrophilic “cell-penetrating” peptides that can cross lipid membranes and are necessary for nuclear export and secretion, suggesting that Prox1 may move more freely between the nucleus and cytoplasm than was initially appreciated. A previous publication has shown that single point mutations of this 623WFSNFR628 region can alter the intracellular distribution of Prox1, indicating its importance in this function.
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      Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein.
      Platelets are required for lymphangiogenesis in embryonic development.
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      It is not known how they contribute to the separation of the lymphatic sac from the cardinal vein, but it has been suggested that platelets aggregate to form a plug that physically divides the blood and the LVs. Platelets' adherence to endothelial cells expressing podoplanin, by the platelet receptor CLEC2, activates the SYK/SLP76 pathway in vitro. When bound to endothelial cells, CLEC2-activated platelets may signal to the endothelium either through direct cell-to-cell contact or indirectly via released microvesicles.
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      • Hayek F.
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      • Ratajczak M.Z.
      Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.
      Platelets can release granules and microvesicles containing transcription factors, microRNAs, and growth factors that can influence endothelial cells.
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      • Wilber A.
      • Hall K.L.
      • Iwata C.
      • Miyazono K.
      • Nisato R.E.
      • Pepper M.S.
      • Zawieja D.C.
      • Ran S.
      Inflammation induces lymphangiogenesis through up-regulation of VEGFR-3 mediated by NF-κB and Prox1.
      • Boilard E.
      • Nigrovic P.A.
      • Larabee K.
      • Watts G.F.
      • Coblyn J.S.
      • Weinblatt M.E.
      • Massarotti E.M.
      • Remold-O'Donnell E.
      • Farndale R.W.
      • Ware J.
      • Lee D.M.
      Platelets amplify inflammation in arthritis via collagen-dependent microparticle production.
      The observation that Prox1 is in platelets suggests the possibility that these cells may contribute to lymphangiogenesis in part through delivery of their contents to target endothelial cells in lymphangiogenesis in ontogeny and in inflammation.
      • Mounzer R.H.
      • Svendsen O.S.
      • Baluk P.
      • Bergman C.M.
      • Padera T.P.
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      Lymphotoxin-α contributes to lymphangiogenesis.
      In summary, we developed the ProxTom mouse, whose LVs express red fluorescent tdTomato. The high visibility of the reporter allows for reliable in vivo imaging. The extent and content of DNA surrounding the gene has allowed fidelity of its expression in multiple tissues and has revealed that the adrenal medulla, megakaryocytes, and platelets contain Prox1. Additional insights into the immune response and regulation of LV function in health and disease will be derived from the analysis of ProxTom mice.

      Note Added in Proof

      After acceptance of this manuscript, a paper was published that used Prox1 to drive morange2 with expression in lymphatic vessels.
      • Hägerling R.
      • Pollmann C.
      • Kremer L.
      • Andresen V.
      • Kiefer F.
      Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse.

      Supplementary Data

      • Supplemental Video S1

        A three-dimensional volume rendering of LVs in the capsule of the lymph node. A stack of 1-μm-thick optical sections representing 96 μm of tissue immediately beneath the capsule was acquired in a surgically exposed lymph node of a live ProxTom × Hec6stGFP mouse 7 days after immunization with oxazolone. Prox1-expressing cells (red) line the subcapsular sinus and extend down through the lymph node cortex as fine vessels. HEVs (green) can be seen beneath the capsule.

      • Supplemental Video S2

        A three-dimensional volume rendering of LVs in the medullary cord of the lymph node. A stack of 1-μm-thick optical sections representing 150 μm of tissue positioned 100 μm beneath the capsule was acquired in a surgically exposed popliteal lymph node of a live ProxTom × Hec6stGFP mouse 7 days after immunization with oxazolone. Prox1-expressing cells (red) can be seen to line a medullary sinus adjacent to HEVs (green).

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