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Abnormal lymphatic vessel development in neuropilin 2 mutant mice

Li Yuan1, Delphine Moyon1, Luc Pardanaud1, Christiane Bréant1, Marika J. Karkkainen2, Kari Alitalo2 and Anne Eichmann1,*

1 INSERM U36, Collège de France, 11, place Marcelin Berthelot, 75005 Paris, France
2 Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, 00014 Helsinki, Finland



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  		Fig. 1. NRP2 expression in the vascular system of E10 mouse embryos is restricted to veins. In situ hybridization with the indicated antisense riboprobes to sections of E10 mouse embryos. (A) Nrp2 is expressed in the floor plate (asterisk), neurons of the ventral spinal cord (arrowhead), dorsal root ganglion (DRG, arrow), sympathetic ganglion (sg), somitic tissue (S) and dorsal aspect of the forelimb (Fl). (B) Overlapping, but distinct expression of Nrp1, which is expressed in neurons of the ventral spinal cord (arrowhead) and DRG (arrow). Asterisk indicates floor plate. (C) Vegfr3 expression is restricted to the vascular system. (D-F) Higher magnification of the sections in A-C. Nrp2 is expressed in the cardinal vein (cv, D), while Nrp1 shows complementary expression in the aorta (Ao, E) and surrounding mesenchymal cells. (F) Vegfr3 expression is low in EC of the aorta and cardinal vein, but high in small vessels that appear to branch from the cardinal vein (arrows in F). NT, neural tube. Scale bars: in A, 200 µm for A-C; in D, 95 µm for D-F.

 


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  		Fig. 2. Nrp2 expression becomes restricted to embryonic lymphatic vessels and, at low levels, veins. Transverse adjacent sections through the neck of an E13 embryo stained with antibodies against the indicated proteins. (A) NRP2 is expressed in the ventral part of the neural tube (NT) and sympathetic ganglia (SG), as well as in mesenchyme surrounding the developing vertebrae (V) and trachea (T). In the vascular system, arteries (A) are negative, the cardinal veins (cv) are weakly positive and jugular lymphatic vessels (asterisks) are strongly positive. (B) VEGFR3 is expressed in the jugular lymphatic vessels (asterisks). (C) PECAM staining reveals abundant blood-vascular capillaries, arteries (A) and the cardinal veins (cv). Note expression on lymphatics at this developmental stage (asterisks). (D,E) High-power magnification of the cardinal vein (cv) and the adjacent lymphatic vessel (asterisk). Note weak NRP2 expression in the cv (arrows). DRG, dorsal root ganglion; oe, oesophagus. Scale bars: in A, 175 µm for A-C; in D, 35 µm for D,E.

 


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  		Fig. 3. Nrp2 expression in adult vessels. (A-C) Transverse sections of P30 gut from a Nrp2+/– mouse stained with antibodies directed against the indicated proteins. Note co-expression of NRP2 (A) with the lymphatic markers podoplanin (B) and VEGFR3 (C) in subserosal lymphatic vessels (asterisks). Weak NRP2 expression is observed in a subserosal vein (v, arrow). NRP2 is also expressed in the smooth-muscle layer surrounding the gut (Chen et al., 1997Go), punctuate staining reflects loss of one wild-type allele. (D,E) Staining of Nrp2+/+ P0 mesentery sections with the indicated antibodies. Note co-expression of NRP2 and VEGFR3 in lymphatics (asterisks), weaker expression of NRP2 in veins (V, arrows), and absence of expression in arteries (A). (F,G) P0 skin section from a Nrp2+/– mouse double-stained with X-gal (blue staining) and VEGFR3 (F) or podoplanin (G) antibodies (brown staining). Note double-stained lymphatics (asterisks). NRP2 also labels hair-follicles (arrowheads). Scale bars: in A, 140 µm for A-C; in D, 35 µm for D,E; in F, 40 µm for F,G.

 


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  		Fig. 4. Lymphatic vessel defects in E13 Nrp2 mutant embryos. Transverse sections of E13 Nrp2+/+ (A,D,G), Nrp2+/– (B,E,H) and Nrp2–/– (C,F,I) embryos stained with antibodies recognizing the indicated proteins. Note very similar PECAM staining in the three genotypes (A-C). (D-F) VEGFR3 labels jugular lymphatic vessels (asterisks) present in all three genotypes, as well as lymphatic capillaries in the skin (arrows). Note absence of these capillaries in the Nrp2–/– embryo (F). (G-I) Higher magnification of adjacent sections corresponding to the boxed area in D-F stained with NRP2 antibodies. The jugular lymphatic vessel (asterisk) is present in all three genotypes. Skin capillaries are NRP2 positive in the Nrp2+/+ (G) and the Nrp2+/– (H) embryo. In the Nrp2–/– embryo (I), only punctuate staining is observed, reflecting the retention of mutant protein in the endoplasmic reticulum. Ao, aorta; Fl, forelimb; H, heart; NT, neural tube. Scale bars: in A, 575 µm for A-F; in G, 230 µm for G-I.

 


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  		Fig. 5. Lymphatic vessel defects in the skin of developing Nrp2 mutant mice. (A,B) Whole-mount X-gal staining of E15 dorsal skin. Note the absence of X-gal-positive vessels in the Nrp2–/– (B). (C-H) Transverse sections through the dorsal skin of E17 embryos stained with anti-VEGFR3 (C,E,G) and PECAM (D,F,H). Note the absence of lymphatic vessels in the Nrp2–/– dermis (C, bottom) compared with Nrp2+/+ and Nrp2+/– dermis (C, top; G). PECAM staining shows no difference between the three genotypes (D,H). (E,F) Comparison of VEGFR3 and PECAM staining of a Nrp2–/– dermis at higher magnification. Note weaker PECAM staining of VEGFR3-positive vessels (asterisks) as compared with arteries (A) and veins (V). Note also that the lymphatic vessels that form are located in a slightly deeper position at the border of the dermis and the connective tissue (compare E with C, G). (I,J) The comparison of VEGFR3 and {alpha}-actin staining shows that lymphatic vessels (asterisks) are not surrounded by smooth-muscle cells, while arteries and veins are (A, V, arrows). Scale bars: in A, 230 µm for A,B; in C, 180 µm for C,D,G,H; in E, 70 µm for E,F; in I, 20 µm for I,J.

 


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  		Fig. 6. BrdU incorporation in lymphatic vessels is decreased in mutant skins. (A,B) Bright-field images showing BrdU-positive (arrows) and -negative (asterisks) nuclei in lymphatic EC from sectioned E17 embryos. (C,D) Lymphatic EC were identified by double-staining with VEGFR3 antibody. Note the decreased number of BrdU-positive nuclei in the Nrp2–/– (arrows in B,D) Scale bar: 30 µm.

 


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  		Fig. 7. Lymphatic vessel defects in the heart and diaphragm of Nrp2 mutants. (A,B) Whole-mount X-gal staining of P0 hearts showing the reduction of capillaries in the Nrp2–/– heart (B). (C) Double-staining of the X-gal-positive vessels present at the epicardial surface of Nrp2+/– embryos (arrowhead) with anti-VEGFR3 (brown staining). (D,E) Transverse sections of E15 hearts stained with anti-VEGF-C antibodies. Note staining of the superficial epicardium in both Nrp2+/– and Nrp2–/– (D,E, arrows). (F,G) Whole-mount X-gal staining of P0 diaphragms (thoracic view). Note the reduction of peripheral X-gal-positive lymphatic capillaries (arrowheads) in the Nrp2–/– diaphragm. Brackets outline the central portion of the diaphragm, which is unaffected. Weaker NRP2 expression in veins is also indicated (arrows). H. Double-staining of the X-gal-positive vessels present in the diaphragm of Nrp2+/– embryos with anti-VEGFR3 (brown staining) confirms that they are lymphatics. (I) Higher magnification of the boxed region in H. Scale bars: in A, 580 µm for A,B; in C, 50 µm; in D, 360 µm for D,E; in F, 1400 µm for F,G; in H, 270 µm; in I, 40 µm.

 


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  		Fig. 8. Lymphatic vessel defects in the lung and the gut of Nrp2 mutants. (A-G) Transverse sections of the lung of E17 (A-D) and P0 (E-G) mice stained with antibodies directed against the indicated proteins. (A,B) Note presence of lymphatics of the thoracic duct in both Nrp2+/+ and Nrp2–/– embryos (asterisks), while VEGFR3-positive capillaries in the Nrp2–/– lung are almost completely absent (arrowheads, A,B). (C,D) PECAM staining of the alveolar blood-vascular capillaries is uniform in both genotypes, arteries (A) and veins (V) are also present in both genotypes. (E,F) Co-expression of VEGFR3 and NRP2 in larger lymphatics (asterisks), as well as in numerous capillaries (E,F, arrowheads). (G) Lymphatic capillaries are absent in Nrp2–/– lung, while lymphatics surrounding the bronchi are present (asterisks). (H,I) Transverse sections through the gut of E17 embryos. Note numerous subserosal VEGFR3-positive vessels in the Nrp2+/+ gut, which are reduced in the Nrp2–/– gut (H, I, arrowheads). Scale bars: in A, 275 µm for A-D,H,I; in E, 325 µm for E-G. Ao, aorta.

 


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  		Fig. 9. Lymphatic vessels of adult Nrp2 mutant mice. (A,B) FITC-dextran injection into ears of P30 mice. (C,D) Whole-mount X-gal staining of P30 hearts. Note the abnormal lymphatic vessels in the Nrp2–/– heart. (E,F) Milk-uptake after feeding in the mesenteries of Nrp2+/+ and Nrp2–/– mice. (G,H) Whole-mount VEGFR3 immunostaining of P30 guts. Note enlarged lymphatic vessels in the Nrp2–/– gut. Scale bars: in A, 430 µm for A,B; in C, 1010 µm for C,D; in E, 390 µm for E,F; in G, 575 µm for G,H.

 

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© The Company of Biologists Ltd 2002