Extended Data Fig. 11: Validation of scRNA-seq data with RNA in-situ hybridization and immunohistochemical staining of LEC clusters.
From: Increased CSF drainage by non-invasive manipulation of cervical lymphatics
a, Uniform manifold approximation and projection (UMAP) plot showing expression of Xist, a female-specific non-coding RNA, in LEC clusters from 3 male and 3 female Prox1-GFP mice shown in Extended Data Fig. 10a. Equal distributions of Xist positive and Xist negative cells are consistent with the sample being representative. b,c, Representative RNA in-situ hybridization images and signal counts comparing Nos3 mRNA expression in superficial cervical lymphatics scLV-1 (white dashed lines) in whole mounts from adult (8 weeks) and aged (96 weeks) Prox1-GFP mice. Scale bars, 20 μm. In c, each dot is the value for one side of scLV-1 from one mouse. n = 6 mice (adult) and n = 5 mice (aged) from three independent experiments. Error bars indicate mean ± s.e.m. P values calculated by two-tailed Welch’s t-test. d,e, Confocal microscopic images and measurements of immunofluorescence staining for eNOS and phosphorylated-eNOS (phos-eNOS) in superficial cervical lymphatics scLV-1 in tissue whole mounts from adult (8 weeks) and aged (96 weeks) Prox1-GFP mice. Scale bars, 100 µm. Each dot is the value for one side of scLV-1 from one mouse. n = 7 (adult, eNOS), n = 6 (adult, Phos-eNOS) mice and n = 5 (aged, eNOS), n = 6 (aged, Phos-eNOS) mice from three independent experiments. Error bars indicate mean ± s.e.m. P values calculated by two-tailed Welch’s t-test. f,g, Confocal microscopic images of immunofluorescence staining (f) and uniform manifold approximation and projection (UMAP) plot (g) of FOXP2 expression in superficial cervical lymphatics scLV-1 (white dashed lines) in tissue whole mounts from adult (8 weeks) Prox1-GFP mice. Green dashed line boxes in f mark regions enlarged in the right three panels. Diverse features of FOXP2 expression in LEC are marked by colored arrowheads: red (Foxp2high LEC), pink (Foxp2low LEC), and yellow (Foxp2high LEC in valve). Scale bars, 50 μm. Representative of n = 3 mice from three independent experiments. FOXP2 immunofluorescence was variably strong in luminal LEC but consistently strong in valve LEC (f), which fits with the UMAP plot that shows two separate Foxp2high LEC clusters (g). h-k, Immunofluorescence images of whole mounts showing staining for Prox1-GFP and LYVE1 in initial lymphatics (with blunt ends, red arrowheads) and pre-collecting lymphatics (with valves, yellow arrowheads) of regional cervical lymphatics (h). White dashed line box marks the region enlarged in the left lower corner. Scale bar, 500 μm. Representative of n = 3 mice from three independent experiments. Immunofluorescence images of whole mounts showing Prox1-GFP in all lymphatics but not in a facial vein; αSMA staining of smooth muscle in a collecting lymphatic and facial vein but not in a pre-collecting lymphatic; and LYVE1 staining in a pre-collecting lymphatic (white arrowheads and dashed line outline) but not in the other vessels (i). Scale bar, 200 μm. Representative of n = 3 mice from three independent experiments. Dot plot graph showing superficial cervical collecting lymphatics that are larger in diameter than pre-collecting lymphatics in adult Prox1-GFP mice (j). Each dot is the value for one lymphangion from one side of cervical lymphatics. n = 6 mice from three independent experiments. Error bars indicate mean ± s.e.m. a.u., arbitrary unit. P values calculated by two-tailed Welch’s t-test. The immunofluorescence staining (h,i) fits with the strong Lyve1 mRNA expression restricted to the regional cervical (initial and pre-collecting) LEC cluster in the uniform manifold approximation and projection (UMAP) plot (k).
