Netrin-4 overexpression in mouse skin results in an increased lymphatic density. (A) Schematic of the Netrin-4 overexpression system and genotyping of the various mice. Netrin-4 cDNA was inserted into the constitutively active Rosa26 locus downstream of a LoxP-flanked transcriptional stop signal. In presence of a cre recombinase driven by the keratin14 promoter (indicated as Prom.), the stop signal is excised allowing expression of Netrin-4. (B) Expression of the LacZ gene reporter (Rosa26 ^LacZ/+) in mouse skin keratinocytes in presence of the Keratin14 cre recombinase (K14 cre/+). Scale bars: 200 and 50 μm. (C) Quantification of Netrin-4 mRNA by qRT-PCR in the skin of Netrin-4–overexpressing mice versus littermate controls. (D) Three-week-old skin-overexpressing Netrin-4 mice are viable but hairless, smaller, and redder than controls (scale bar: 1 cm). Pictures were taken on an Olympus IX71 microscope, at 100× and 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software. (E) Skin sections of 3-week-old Netrin-4–overexpressing and littermates control mice costained with an anti–LYVE-1 and an antipodoplanin (indicated as podo.) antibody (dotted box represents the enlarged area shown; scale bars: 200 and 20 μm). (F) Quantification of the LYVE1⁺/podoplanin⁺ staining density using ImageJ and normalized to the control group. Pictures in panel E are representative of the quantification reported in panel F.