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First published online 4 December 2008
doi: 10.1242/dev.025353

Development 136, 191-195 (2009)
Published by The Company of Biologists 2009
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Phospholipase C{gamma}2 is necessary for separation of blood and lymphatic vasculature in mice

Hirotake Ichise1,*,{dagger}, Taeko Ichise1,*, Osamu Ohtani2 and Nobuaki Yoshida1

1 Laboratory of Gene Expression and Regulation, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
2 Department of Anatomy, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Toyama 930-0194, Japan.


Figure 1
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  		Fig. 1. A spontaneous mutant mouse strain, abnormal lymphatics (al), and positional candidate cloning. Chylous ascites (A) and blood-filled vessels (B) in mutant newborns. Blood-filled vessels were Lyve1+ lymphatic vessels (C, arrows). Arrowheads indiacte blood vessels. Scale bar: 100 µm. (D) Genotyping of the 139 affected mice (n=2,4,5,128) using SSLP markers mapped the al locus to a B6-derived region between D8Ims10 and D8Ims18. (E) Sequencing of PCR-amplified genomic DNA showed that a G-to-A transitional mutation (an asterisk in D) in the Plcg2 gene occurred in the affected mice. (F) Western blot analysis of newborn heart tissue lysates showed that PLC{gamma}2 was not expressed in al/al mice. PLC{gamma}1, loading control.

 

Figure 2
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  		Fig. 2. Aberrant lymph sacs in PLC{gamma}2-null embryos. (A-F) Transverse sections of E13.5 embryos immunostained for Lyve1 (green) and CD31 (red). D-F are higher magnifications of A-C, respectively. (C,F) Aberrant separation of a lymph sac from a cardinal vein. Blue, DAPI-stained nuclei; A, carotid artery; V, cardinal vein; L, jugular lymph sac. Arrow indicates a site at which ECs of the two vessels are in contact. Arrowheads indicate connections between BECs and LECs. Scale bars: 200 µm in A-C; 50 µm in D-F.

 

Figure 3
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  		Fig. 3. Double immunostaining for endogenous Plcg2 gene-driven EGFP and cell markers. EGFP (green), cell markers (red) and DAPI (blue) staining are shown. (A) Transverse sections of Plcg2EGFP/EGFP E14.5 embryos. Arrows indicate EGFP+ F4/80+ monocytes. (B) Transverse sections of Plcg2EGFP/EGFP E13.5 embryos. V, cardinal vein; L, lymph sac. Arrows indicate EGFP+ platelets lack nuclei. Arrowhead indicate venous EGFP+ ECs. (C) Transverse sections of Plcg2EGFP/EGFP E14.5 embryos. Arrows indicate a subpopulation of EGFP+ ECs. (D) Transverse sections of thoracic walls of E15.5 embryos. Arrows indicate lymphatic vessels; arrowhead indicates Lyve-1+ EGFP+ monocytes. EGFP+ ECs were not LECs. Scale bars: 50 µm in A-D. (E) Transverse sections of E13.5 embryos. A, carotid artery; V, cardinal vein; L, jugular lymph sac. Panels c and d are higher magnification images for a and b, respectively. Arrows indicate EGFP+ non-ECs. Arrowhead indicates a venous EC. Scale bars: 200 µm in a,b; 50 µm in c,d.

 

Figure 4
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  		Fig. 4. A BM reconstitution study using PLC{gamma}2-null mice. (A) Appearance of small intestines (a,b,d,e,g,i) and Lyve1+ intestinal lymphatic vessels (brown; c,f,h,j). a-c, PLC{gamma}2-null BM-reconstituted wild-type mice; d-f, wild-type BM-reconstituted PLC{gamma}2-null mice. b is a higher magnification of a. (d,e) Two representative segments from one mouse. Plcg2+/EGFP (g,h) and Plcg2EGFP/EGFP (i,j) mice not subjected to BM transplantation are shown for comparison. Scale bars: 1 mm. (B) Sections of small intestines of BM-reconstituted mice immunostained for Lyve1 (green) and CD31 (red). The type of transplantation is indicated at the top of each panel. Merged images are shown. Arrows indicate connections between CD31+, Lyve1- blood vessels (red) and CD31- or low, Lyve1+ lymphatic vessels (green/yellow). Scale bar: 100 µm.

 

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