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. 2012 Apr;180(4):1715-25.
doi: 10.1016/j.ajpath.2011.12.026. Epub 2012 Feb 4.

ProxTom lymphatic vessel reporter mice reveal Prox1 expression in the adrenal medulla, megakaryocytes, and platelets

Lucy A Truman  1 , Kevin L Bentley, Elenoe C Smith, Stephanie A Massaro, David G Gonzalez, Ann M Haberman, Myriam Hill, Dennis Jones, Wang Min, Diane S Krause, Nancy H Ruddle
Affiliations

ProxTom lymphatic vessel reporter mice reveal Prox1 expression in the adrenal medulla, megakaryocytes, and platelets

Lucy A Truman et al. Am J Pathol. 2012 Apr.

Abstract

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.

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Figures

Figure 1
Figure 1
Construction 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.
Figure 2
Figure 2
LVs 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.
Figure 3
Figure 3
tdTomato 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.
Figure 4
Figure 4
Extralymphatic 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).
Figure 5
Figure 5
Prox1 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.
Figure 6
Figure 6
Prox1 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) 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.
Figure 7
Figure 7
Prox1 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.
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