Supplementary Materials

Stem cell-driven lymphatic remodeling coordinates tissue regeneration

Shiri Gur-Cohen, Hanseul Yang, Sanjeethan C. Baksh, Yuxuan Miao, John Levorse, Raghu P. Kataru, Xiaolei Liu, June de la Cruz-Racelis, Babak J. Mehrara, Elaine Fuchs

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S15
  • Tables S1 to S3
  • Caption for Movies S1 to S15
  • References

Images, Video, and Other Media

Movie S1
HFSCs are associated with high diameter endothelial vessels. Whole mount imaging and 3D reconstruction of the pan endothelial marker CD31 (red) demonstrating the spatial localization and vascular diameter around the quiescent hair follicle stem cells (KRT24, green) at second telogen in cleared skin.
Movie S2
High-diameter vessels that are associated with HFSCs are not blood capillaries. Vascular architecture (CD31; pan endothelial marker, red and EMUC; blood capillaries marker, white) around the hair follicle bulge SCs (Krt42, green) at telogen. Note the high-diameter vessels in the vicinity of the bulge, which are EMUC negative.
Movie S3
Blood erythrocytes are localized exclusively in the blood vessels, but not inside the lymphatic vessels. Representative Z-stack intervals of whole mount cleared skin stain for erytrocyted blood cells (TER119, green), Lymphatic capillaries (LYVE1, white) and blood capillaries (Endomucine, red) at telogen. Note the exclusive localization of blood erythrocytes in the blood circulation.
Movie S4
Lymphatic capillaries present a novel niche for HFSCs. 3D image and surface rendering of the lymphatic capillaries (LYVE1, red; surface render, purple) around the hair follicles bulge (KRT24, green; surface render, yellow) at telogen. Note the close association between the bulge stem cells with the lymphatic capillaries base.
Movie S5
Lymphatic endothelium extensions interact with the bulge HFSCs. Representative high magnification 3D image and surface rendering of bulge SCs (SOX9, green), lymphatic capillaries (LYVE1, white) and blood capillaries (EMUC, red) at telogen. Note the endothelial protrusions, originate from the lymphatic capillary base, towards the bulge HFSCs.
Movie S6
Lymphatic capillaries asymmetrically positioned relative to the posterior arrector pili muscle. Representative Z-stack intervals for the orientation of the lymphatic capillaries (LYVE1, white) and the arrector pili (α-SMA, red) around the HFSCs (KRT24, green) at telogen.
Movie S7
Movie S8
Movie S9
Lymphatics are not tightly associated with developing embryonic HF. Representative Z-stack intervals of embryonic skin at E14.5, E16.5 and E18.5; respectively, demonstrating the lack of association between LYVE1+ (red) lymphatic capillaries with Sox9+ cells during HF embryonic development.
Movie S10
Lymphatic capillaries first emerge with the newly-formed quiescent HFSCs only after birth. Representative Z-stack intervals of the lymphatic capillaries (LYVE1, white) and VEGFR3 (lymphatic endothelium, red), demonstrating that these blind-ended vessels first emerge and oriented towards the upper outer root sheath (SOX9, green) only after birth (P1).
Movie S11
A 3D image of lymphatic endothelium around the hair follicle after birth. Dermal lymphatic vessels (LYVE1, white and VEGFR3, red) are associated with the newly-emerged HFSC only after birth. Note the localization of the LYVE1+ lymphatic capillaries in the upper dermis, far away from the dermal papilla and the growing HF.
Movie S12
Lymphatic capillaries were intimately associated with quiescent HFSCs during telogen. Representative 3D images and surface rendering demonstrating the association between the telogen KRT24+ HFSCs (green, surface rendering- purple), their progeny P-CADhigh HG (white, surface rendering- light blue) and LYVE+ lymphatic capillaries (red, surface rendering-yellow).
Movie S13
hair follicle hyperplasia under Angptl7 knock-down. 3D image and surface rendering and reconstruction of the hyperplastic hair follicles (P-CAD, green; surface rendering, white) in shAngptl7 lentiviral-transduced animals in utero (H2B-RFP, red as a marker for infection; surface reconstruction based on Z-positioning).
Movie S14
3D structure of the dilated lymphatic vessels around the hyperplastic HFSCs under Angptl7 knock-down. 3D image of whole mount cleared skin demonstrating HF hyperplasia in shAngptl7 lentiviral-transduced animals in utero (H2B-RFP as a marker for infection; surface reconstitution based on Z positioning), surrounded by massively dilated lymphatic vessel (LYVE1; surface rendering white).
Movie S15
A Z-stack interval of endothelial vessels in whole mount cleared skin under disrupted Angptl7 expression. Blood capillaries (EMUC, magenta) and lymphatic capillaries (LYVE1, green) are both perturb and closely enwrapping the hyperplastic hair follicle in shAngptl7 lentiviral-transduced animals in utero (H2B-RFP as a marker for infection).