Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development
Fu-Jung Lin, Xinpu Chen, Jun Qin, Young-Kwon Hong, Ming-Jer Tsai, Sophia Y. Tsai
Fu-Jung Lin, Xinpu Chen, Jun Qin, Young-Kwon Hong, Ming-Jer Tsai, Sophia Y. Tsai
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Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development

Abstract

The lymphatic system plays a key role in tissue fluid homeostasis. Lymphatic dysfunction contributes to the pathogenesis of many human diseases, including lymphedema and tumor metastasis. However, the mechanisms regulating lymphangiogenesis remain largely unknown. Here, we show that COUP-TFII (also known as Nr2f2), an orphan member of the nuclear receptor superfamily, mediates both developmental and pathological lymphangiogenesis in mice. Conditional ablation of COUP-TFII at an early embryonic stage resulted in failed formation of pre-lymphatic ECs (pre-LECs) and lymphatic vessels. COUP-TFII deficiency at a late developmental stage resulted in loss of LEC identity, gain of blood EC fate, and impaired lymphatic vessel sprouting. siRNA-mediated downregulation of COUP-TFII in cultured primary human LECs demonstrated that the maintenance of lymphatic identity and VEGF-C–induced lymphangiogenic activity, including cell proliferation and migration, are COUP-TFII–dependent and cell-autonomous processes. COUP-TFII enhanced the pro-lymphangiogenic actions of VEGF-C, at least in part by directly stimulating expression of neuropilin-2, a coreceptor for VEGF-C. In addition, COUP-TFII inactivation in a mammary gland mouse tumor model resulted in inhibition of tumor lymphangiogenesis, suggesting that COUP-TFII also regulates neo-lymphangiogenesis in the adult. Thus, COUP-TFII is a critical factor that controls lymphangiogenesis in embryonic development and tumorigenesis in adults.

Authors

Fu-Jung Lin, Xinpu Chen, Jun Qin, Young-Kwon Hong, Ming-Jer Tsai, Sophia Y. Tsai

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Figure 5

Inducible inactivation of COUP-TFII in adult mice, with Tam treatment at 2 months, does not affect normal function of lymphatic vessels but suppresses lymphangiogenesis in an animal model of cancer.

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Inducible inactivation of COUP-TFII in adult mice, with Tam treatment at...
(A) LYVE1 staining (green) of whole-mount ear preparations of 3-month-old controls and mutants. (B) COUP-TFII (green) and LYVE1 (red) double immunofluorescence was performed on ear sections of 4.5-month-old controls and mutants. (C) Fluorescent dextran lymphangiography of the tail. Levels of FITC-dextran uptake are similar in tail lymphatic vessels of controls and mutants. (D–G) Lymphangiography performed by injection of Evans blue dye into the hind limb foot pad of 4.5-month-old controls and mutants. Dye perfused large lymphatic vessels of the legs (F and G) and draining iliac (arrows) and renal (arrowheads) lymph nodes (D and E) of the abdomen to a similar extent in controls mutants. Insets in F and G are higher-magnification images of the boxed regions. K, kidney. (H and I) Immunofluorescence microscopy of mammary gland tumor sections from PyMT/+;F/F control or PyMT/+;CRE-ERT2/+;F/F mutant littermates at 4.5 months, using an antibody against LYVE1 (green). Insets in H and I show LYVE1 staining (green) in tissue sections from a lymph node adjacent to the tumor. Inactivation of COUP-TFII results in the reduced tumor lymphangiogenesis but does not affect the preexisting lymphatic vessels in lymph nodes. n = 3 for each group. (J) Statistical summary of LYVE1-positive areas in mammary gland tumor sections. Error bars indicate SD. ***P < 0.01. (K and L) Immunofluorescence microscopy reveals that COUP-TFII proteins (red, K) are detected in LYVE1- (green, K), VEGFR3- (green, L), and Prox1-expressing (red, L) tumor-associated lymphatics of control mice. Scale bars: 200 μm (A, H, and I); 20 μm (B, K, and L); 500 μm (C); 2 mm (D–G).