Phospholipase C2 is necessary for separation of blood and lymphatic vasculature in mice

DEV025353 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Schematic representation of the mapping strategy for positional candidate cloning of a mutated gene at the al locus.

• Supplemental Figure S2 -

  Fig. S2. Generation and phenotypic characterization of Plcg2/EGFP knock-in mice. (A) Schematic representation of the EGFP knock-in in exon 2 of the Plcg2 gene. (B) Western blot analysis of PLCγ2 expression in Plcg2/EGFP knock-in mice. Newborn heart tissue lysates were examined. PLCγ1 expression is shown as a loading control. (C) Immunostaining for EGFP and PLCγ2. EGFP is expressed in the spleen, instead of endogenous PLCγ2. Scale bar: 200μm. (D) Plcg2^EGFP/EGFP embryos exhibit blood-filled lymphatic vessels (lower panels). Plcg2^+/EGFP embryos are shown for comparison (upper panels). (E) Plcg2^al/al embryos exhibit blood-filled lymphatic vessels (arrows), as shown by Lyve1 immunostaining (red). Scale bar: 100 μm.

• Supplemental Figure S3 -

  Fig. S3. PLCγ2 in ECs in vitro. (A) PLCγ2 was expressed in human ECs (HUVECs and HdMLECs) and mouse ECs (conditionally-immortalized mesenteric BECs and LECs). (B) VEGFA induced VEGFR2 and PLCγ1 phosphorylation, but not PLCγ2 phosphorylation, in HUVECs. Phosphorylation of PLCγ1, but not of PLCγ2 or VEGFR2, was induced by VEGFC. (C) PLCγ2 was phosphorylated in PDGF-BB-treated primary mouse embryonic fibroblasts (MEFs), but not in human BECs or LECs treated with VEGFA, VEGFC or PDGF-BB. (D) Plcg2 gene-driven GFP expression was induced in a subpopulation of Plcg2^+/GFP LECs in vitro.

• Supplemental Figure S4 -

  Fig. S4. BM-derived cells expressing Plcg2/EGFP in the intestine of BM-reconstituted mice. (A) BM-derived EGFP-expressing cells accumulated in the mucosa and submucosa of the intestine, in which F4/80⁺ and/or CD11b⁺ monocytes/macrophages were abundant. Scale bar: 25 μm. (B) Double immunostaining for GFP and F4/80 showed that the most of GFP-expressing cells were F4/80⁺ monocytes/macrophages. Scale bar: 25 μm. Intestinal sections of Plcg2^EGFP/EGFP BM-reconstituted wild-type mice were shown in A and B, and those of Plcg2^+/EGFP BM-reconstituted wild-type mice also showed similar results (data not shown). (C) Plcg2^EGFP/EGFP BM-derived ECs contributed to blood and lymphatic vessels at a low frequency. Double immunostaining was performed on intestinal sections of Plcg2^EGFP/EGFP BM-reconstituted wild-type mice. Plcg2^EGFP/EGFP BM-derived cells contributed to ECs of blood vessels and fused blood-lymphatic vessels (arrows) in the intestinal submucosa. However, the frequency of BM-derived ECs expressing GFP was very low (two or three GFP-expressing ECs were found in ∼100 submucosal vessels in these mice (n=2). Scale bar: 50 μm.