Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells

Abstract

Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a follicle-linking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFP-expressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening.

Fig. 1.

View from the dermis side of the dorsal skin in ND-GFP transgenic mice. (a) Phase-contrast microscopy. Sebaceous glands (blue arrows) are located around the hair shaft (yellow arrows). (b) Phase-contrast microscopy plus GFP fluorescence. ND-GFP cells are visualized in the follicular bulge area (white arrows) and blood vessels (red arrows). The follicular bulge area is located beneath the sebaceous gland. (c) GFP fluorescence. The ND-GFP blood vessels (red arrows) are connected to ND-GFP hair follicles (white arrows). (d) Schematic of telogen hair follicle showing position of ND-GFP hair-follicle bulge areas (black arrows) and blood vessel network (red arrows). (e) GFP fluorescence. The ND-GFP blood vessels (red arrows) are associated with ND-GFP hair-follicle bulge areas (white arrows). (Scale bars, 100 μm.)

Fig. 2.

Transplantation of an ND-GFP vibrissa follicle into the subcutis of a nude mouse. (a) Phase-contrast micrograph of follicle 28 days after transplantation. (b) Phase-contrast micrograph plus GFP fluorescence. (c) GFP fluorescence. In b and c, ND-GFP blood vessels (arrows) are seen growing from the transplanted ND-GFP hair follicle and associating with preexisting blood vessels in the nude-mouse skin. (d and e) Higher magnification of the ND-GFP vessels of b and c, respectively. (f and g) Colocalization of GFP and the endothelial cell marker CD31 (arrows). f is a fluorescent image, and g shows the same field air-dried and immunohistochemically stained with CD31. (Scale bars, 100 μm.)

Fig. 3.

Transplantation of ND-GFP vibrissa follicles under the kidney capsule of a nude mouse. The ND-GFP vessels are visualized to form a network at day 14 after transplantation. (a) Phase-contrast micrograph. (b) Phase-contrast micrograph plus GFP fluorescence. (c) GFP fluorescence. (Scale bars, 100 μm.)

Fig. 4.

Transplantation of an isolated ND-GFP vibrissa follicle into wounded nude-mouse skin. (a) The isolated ND-GFP vibrissa follicle before transplantation. (b) Day 10 after transplantation of the ND-GFP vibrissa follicle into wounded nude-mouse skin. The ND-GFP vessels (arrows) are growing from the ND-GFP vibrissa follicle toward the healing wound. (c and d) Higher magnification of the area in b indicated by the white dashed box. (e) Schematic of transplantation of the ND-GFP vibrissa follicle into wounded nude-mouse skin. (Scale bars, 100 μm.)