Our study demonstrates that, like the brain, hair follicles are maintained by adult stem cells that express the Hh response gene Gli1. We found two distinct domains of Gli1(+) epithelial stem cells in the quiescent telogen follicle, and demonstrated that Gli1(+) cells can regenerate the anagen follicle, self-renew for the life of the animal, and contribute to multiple lineages in the cycling follicle. In addition, we saw that Gli1-expressing mesenchymal cells maintain the DP. Finally, we showed that Gli1(+) K15(−) stem cells in the upper bulge receive Shh from sensory neurons, and are dependant on a novel perineural stem cell niche for their distinctive ability to become epidermal stem cells after wounding. Our Gli1-GIFM studies taken together with previous studies demonstrate that at least some molecularly distinct stem cell subdomains in the bulge have differing capacities to alter their stem cell lineage commitments in vivo (). These results have important implications regarding the complexity of adult epithelial stem cell populations and their roles in tissue regeneration and repair, as well as for studies of wound healing and skin disease.
Gli1-GIFM allowed us to study Hh-responding hair follicle stem cells in the context of their native environment. Two other markers that overlap with Gli1
expression in the lower telogen bulge and HG have been studied in intact skin using GIFM. The K15
-CrePR transgene was used to label a subset of bulge cells in the telogen follicle and follow their contribution to anagen regeneration, but persistence of labeling after the first hair cycle was not assessed (Morris et al., 2004
). More recently, Lgr5-GIFM identified anagen-regenerating cells capable of self-renewal for multiple hair cycles (Jaks et al., 2008
). Consistent with our results, gene expression studies on sorted Lgr5(+) skin cells indicated that at least some of these cells receive Hh signaling during telogen. Neither K15 nor Lgr5 expression, however, overlaps with Gli1 in the upper bugle, distinguishing the region as a molecularly distinct, Shh-responding subdomain in the telogen follicle. GIFM studies have also been done using Lgr6
, a marker expressed primarily in the isthmus of the adult telogen follicle, but also in the IFE (Snippert et al., 2010
). Labeled Lgr6-GIFM cells maintained the IFE, isthmus and SG with rare contributions to the cycling adult hair follicle, suggesting that Lgr6(+) stem cells largely reside outside the adult telogen bulge. However, some overlap with the upper bulge is suggested by a trend of Lgr6
expression being increased in GFP(+) cells (p=0.064) relative to the other sorted cells from Gli1eGFP/+
Observing stem cells in their native environment allows one to study both their role in homeostasis and their response to pathological states such as wounding. We found that Gli1(+) stem cells in the telogen bulge regenerate the cycling hair follicle throughout life. However, when exposed to additional signals from a fresh wound, Gli1(+) upper bulge cells will break lineage boundaries and move into the epidermis. The potency of the skin wound environment to alter stem cell behavior was recently illustrated by its ability to reprogram transplanted thymic epithelial cells into both hair follicle and epidermal stem cells (Bonfanti et al., 2010
). Intriguingly, it is a perineural microenvironment in the follicle that instills Gli1(+) upper bugle cells with the capacity to be similarly reprogrammed into epidermal stem cells. This illustrates the importance of environmental cues in both maintaining stem cell plasticity and altering stem cell behavior (Watt and Jensen, 2009
). These environmental effects should be considered when interpreting stem cell assays where cells are removed from their native environment. Moreover, understanding the environmental signals involved may help in reprogramming cells for stem cell based therapies.
Like many hair follicle stem cell markers (K15, Lgr5, and Sox9), Gli1 is expressed in the bulge and HG during the quiescent telogen phase and also in the proliferative ORS during anagen. Hh signaling has been proposed to regulate both stem cell maintenance and, at higher signaling levels, cell proliferation in many adult epithelia (Jiang and Hui, 2008
). In anagen skin, Shh is expressed at high levels in the follicle matrix and acts as a mitogen that drives anagen regeneration (Gat et al., 1998
; Oro and Higgins, 2003
). Here we found that removal of the neural source of lower-level Hh signaling in the upper telogen bugle changes the expression profile and biological potential of the stem cells. Thus, Shh appears to have multiple roles in the hair follicle, including regulation of a subpopulation of quiescent stem cells.
Shh is a critical intercellular signaling molecule during development that classically signals to adjacent cell populations after release into the extracellular space. However in the developing optic nerve, Shh protein is transported down neuronal axons where it signals to distant astrocytes (Wallace and Raff, 1999
). A recent study suggests this atypical mode of Shh signaling from neurons to distant astrocytes also occurs in the adult brain (Garcia et al., 2010
). We now report retrograde transportation of Shh down the peripheral process of adult bipolar sensory neurons, and signaling to a completely different tissue type – the follicle epithelium. Moreover, outside of the nervous system, the perineural microenvironment has not been widely considered as a stem cell niche, although proper embryonic innervation has been implicated as necessary for normal organogenesis in some epithelial structures including the skin (Knox et al., 2010
; Peters et al., 2002
). We have now demonstrated functional dependence of an adult epithelial stem cell population on a Shh-expressing perineural niche for maintaining lineage plasticity during wound healing.
Lgr5-GIFM-marked cells from the lower follicle were found in the upper bugle and isthmus after multiple hair cycles (Jaks et al., 2008
), showing that progeny from cells in the lower telogen follicle can redistribute upward after cycling through anagen and catagen. Similarly, we found that Gli1-GIFM-marked cells are preferentially retained in the isthmus and upper portions of the bulge after multiple hair cycles. This raises the possibility that the upper bulge and isthmus are important for long-term maintenance of the cycling follicle. As these regions of the follicle reside within or around the perineural niche, it is tempting to speculate that nerves play an additional role in maintaining long-lived follicle stem cells. This could explain the clinical hair loss seen in some patients with chronic peripheral neuropathies.
Comparative expression analysis of adult hair follicle stem cell markers illustrates that the telogen follicle contains multiple distinct domains, each with unique molecular signatures (Watt and Jensen, 2009
)(). Moreover, our study illustrates a difference between the lineage reprogramming potential of nerve-regulated Gli1(+) K15(−) upper bulge cells and that of the K15(+) bulge cells. It will be important to fully elucidate the complexity of the stem cell pool in the adult hair follicle, and identify any hierarchical organization among the stem cell populations, especially considering how the follicle bulge cells redistribute over the course of a hair cycle. Our molecular map of the telogen follicle will facilitate future studies to further dissect the genetic and functional anatomy of the bulge, and identify cell populations useful for regenerative medicine.