Fig 1.
Generation of the Vegfr3-tdTomato reporter mouse.
A) Schematic representation of the modified genomic region around the initiating ATG of the mouse Flt4 gene in the BAC Vegfr3-tdTomato used for pronuclear injection. A cDNA expression cassette encoding the fluorescent protein tdTomato C-terminally fused to a CAAX-box for membrane-anchoring followed by a SV40-polyadenylation signal (pA) for positive selection was inserted into the BAC clone RP23-58E13 such that the initiating ATG of the Vegfr3 was now usurped by tdTomato. For Red/ET recombineering, 3’ and 5’ Vegfr3 homology regions derived from the genomic sequences preceding the initiating ATG and following Exon1 were added on both ends of the targeting cassette. Primers for PCR identification of recombinant clones and transgenic animals are indicated. B) Verification of the recombined BAC and successful transgenesis by PCR. The heterozygously transmitted allele is indicated by +/T. C) Differential interference contrast (DIC) and fluorescence images of primary dermal lymphatic endothelial cells (pdLECs) from Vegfr3-tdTomato transgenic mice following magnetic bead-based isolation and enrichment. Images were captured in passage 1. Magnetic beads appear a round black spheres. Scale bars = 50 μm.
Fig 2.
Characterization of the reporter expression in Vegfr3-tdTomato transgenic mice.
Tissues prepared from adult mice (A-F, I-J) and pups at postnatal day 5 (G-H, K-T) were analysed for lymphatic expression of the Vegfr3-tdTomato transgene in a fluorescence stereo dissection microscope (A-L) and an inverted fluorescence microscope (M-T). Distinct lymphatic networks were detected in various adult tissues including the diaphragm (A-C), lung (D), ear dermis (E), intestinal mucosa (F), and heart (I-J) as well as postnatal tissues including the mesentery (G-H, O-P, S-T), mesenteric lymph nodes (G, O-P), heart (K-L) and diaphragm (M-N, Q-R). White asterisk denotes the position of the LN. The white and black boxed areas are magnified in the indicated panels. Scale bars = 400 μm (M-P) and 200 μm (Q-T).
Fig 3.
Co-localization of the transgene-derived Vegfr3-tdTomato signal with bona fide lymphatic vessel markers in the dermis of the ear and diaphragm.
A-C) Maximum intensity projection (MIP) of representative confocal tile-scans from wholemount immunostained ear skin (A) and diaphragm (B-C) of adult Vegfr3-tdTomato transgenic mice. Stained antigens are indicated above each panel. The white boxed area in (A) is magnified in (B). Scale bars = 200μm (A), 100μm (A, magnification), 50μm (B,C).
Fig 4.
Lymph vessel-specific expression of the Vegfr3-tdTomato transgene in the mesentery.
A-F) Wholemount immunostained preparations of mesenteries from Vegfr3-tdTomato transgenic pups at P5. Shown are MIPs of multi-tile z-stacks stained for the antigens depicted in colour above each panel. Magnifications of the areas boxed in white dashed lines in (A) and (D) are shown in the indicated panels (B,C and E,F). RFP staining identified transgene expression in lymphatic vessels. Yellow arrow heads indicate formation of semilunar valves. Scale bars = 1000 μm (A, B), 200 μm (B-C, E-F).
Fig 5.
2-Photon laser scanning microscopy images of lymphatic vessels from transgenic Vegfr3-tdTomato reporter mice.
A) Spectral characteristics of the tdTomato fluorophore [33, 36]. 1P, single photon excitation spectrum, 2P, two photon excitation spectrum, em, emission spectrum. B) Expression of Vegfr3-tdTomato reporter construct in the superficial lymphatic vasculature of foetal skin (E14.5) imaged by 2P-LSM. The blood vasculature was contrasted with Alexa Fluor™ 647-labelled anti PECAM1 antibodies. C) Confirmation of Vegfr-3 promoter driven expression of the fluorescent reporter protein tdTomato in lymphatic vessels of the adult thoracic diaphragm. Due to its non-centrosymmetric biomolecular organization skeletal muscle of the diaphragm generates intense second harmonic generation (SHG) signals, that are visualized as green, repetitively striated signal. Shown are MIPs of 200 μm (B) and 150 μm (C) z-stacks. Scale bars = 50 μm.
Fig 6.
Light sheet microscopic visualization of the lymphatic vasculature in transgenic Vegfr3-tdTomato reporter mice after organic solvent-based tissue clearing.
A-E) Volume reconstruction of lymphatic vessels in the lung (A-C) and kidney (D+E) of Vegfr3-tdTomato reporter mice at P5. Immunostaining for RFP identified expression of the Vegfr3-tdTomato transgene. Endogenous tissue autofluorescence (AF) allowed contrasting of the overall organ volume. F+G) 3D visualization of cardiac lymphatic vessels by immunostaining for RFP indicating expression of the Vegfr3-tdTomato transgene. Blood vasculature was visualized by Alexa Fluor™ 647-labelled anti PECAM1 antibodies. The yellow boxed areas are magnified in the indicated panels.
Fig 7.
Light sheet microscopy of CUBIC-cleared tissues visualized Vegfr3-tdTomato transgene expression in lymphatic vessels of the mouse lung and heart.
A+B) 3D visualization of tdTomato positive lymphatic vessels in the lung (A) and heart (B) of Vegfr3-tdTomato reporter mice at P5. Tissue autofluorescence (AF) was used to contrast organ topography.