Stem cell self-renewal is the process by which stem cells proliferate and generate more stem cells. This process requires control of the cell cycle and maintenance of the undifferentiated state. Embryonic stem cells are refractory to most proliferation checkpoints4
, and they typically promote self-renewal by suppressing differentiation8
. By contrast, the few established regulators of self-renewal in adult stem cells function by regulating cell-cycle progression9–12
. A prerequisite to unlocking the key to regenerative medicine is to dissect the complex mechanisms governing stem cell self-renewal in adult tissues.
With their enormous capacity for tissue regeneration, hair follicles offer an ideal system to explore these mechanisms. Hair follicle stem cells (HF-SCs) become activated early in each hair growth cycle, when a few of these cells exit their niche (called the bulge) to generate a new hair follicle. The differentiation of stem cells into lineage-restricted progeny probably requires micro-environmental stimuli that are not present in the stem cell niche, because this process happens gradually along the follicle outer root sheath13–15
. The stepwise process culminates at the base of the mature follicle, where committed transit-amplifying matrix cells differentiate into the six lineages that are involved in hair production.
Following their activation, stem cells within the bulge and its vicinity (the upper outer root sheath, which becomes the bulge in the next cycle) briefly self-renew, replenishing the expended stem cells and ensuring long-term tissue regeneration13–15
. Niche HF-SCs also proliferate following injury and repair wounds13–15
. Another feature that distinguishes HF-SCs from their committed progeny is their ability to be propagated for at least five passages in vitro
, reflecting their capacity for long-term proliferative potential6
In the current study, we surmised that there might be two sources for finding intrinsic factors responsible for maintaining ‘stemness’ inside and outside the stem cell niche: self-renewal factors that have been identified in other stem cell studies; and nuclear proteins that we found to be enriched twofold in stem cells relative to their transit-amplifying progeny (Supplementary Fig. 1a, b
). Focusing on about 400 such candidates, we devised an in vitro
RNA interference (RNAi) screen for long-term versus short-term self-renewal (). By choosing genes whose expression was enriched in stem cells relative to committed proliferative progeny, this pool of candidates should not contain housekeeping genes and general proliferation-associated genes. However, if short hairpin RNAs (shRNAs) target a gene that is essential for long-term but not short-term self-renewal, then cells expressing this gene should persist during early passages but then decrease in number or disappear with sequential passaging. Operating on this premise, we transduced purified primary HF-SCs in triplicate with a lentiviral pool encoding control (scramble) shRNAs and a pool of 2,035 candidate shRNAs (about five per gene) such that, on average, each stem cell expressed a single shRNA (Supplementary Fig. 1c
). The transduced stem cells were cultured and, at 24 h and following each passage, shRNAs were amplified from the surviving cells and subjected to high-throughput sequencing.
In vitro RNAi screen for genes involved in stem cell long-term self-renewal
Data are shown for passage 1 (P-1) and P-5 (, Supplementary Figs 2 and 3a
, and Supplementary Tables 1 and 2
). More than 96% of the initial shRNAs were detected at 24 h after transduction, and these shRNAs were used as a reference for changes in shRNA representation. Consistent with our strategic exclusion of housekeeping genes and general proliferation-associated genes, most cells that harboured shRNAs survived the first passage. By contrast, after five passages, many shRNAs were depleted or enriched, suggesting that the transduced cells had different long-term proliferative potentials. Using unsupervised hierarchical clustering, triplicates of individually transduced and passaged cells behaved strikingly similarly, suggesting that these changes reflected bona fide alterations in stem cell character.
Parallel screens with fibroblasts weeded out shRNAs corresponding to cell-survival genes such as Bcl2,
which were selected against after five passages in both HF-SCs and fibroblasts (, Supplementary Fig. 3b
and Supplementary Table 3
). Our refined short list of self-renewal candidates contained those whose cognates all showed similar trends and for which two or more shRNAs per gene displayed specific changes in P-5 stem cell cultures but not in P-1 stem cell cultures or in P-5 fibroblasts (Supplementary Fig. 2
and Supplementary Table 1
). Category I shRNAs () were maintained in P-1 stem cell cultures but were underrepresented by more than 90% at P-5, meeting the criteria for an shRNA that suppresses a long-term self-renewal gene. Representing only 3.8% of the initial pool, category I included shRNAs targeting Hmga2
, which is required for neural stem cell self-renewal10
, and Runx1
, which promotes HF-SC proliferation16
Real-time PCR (rtPCR) of transcripts targeted by six of the most effective category I shRNAs confirmed that each shRNA blocked the expression of its intended target (Supplementary Fig. 4
). Moreover, stem cells that were individually transduced with Hmga2, Runx1
shRNA were progressively selected against over time (). The transcription factor TBX1 was particularly intriguing because it has been implicated in tissue formation in other organs17,18
. We selected it as our model for in vivo
testing of the functional relevance of our RNAi screen.
rtPCR and epigenetic chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analyses19
of purified hair follicle populations revealed that Tbx1
was transcribed at higher levels in stem cells than in any of their progeny ( and Supplementary Fig. 5a
). In vivo,
the developmental expression of TBX1 protein most closely resembled that of two essential HF-SC transcription factors, SOX9 and LHX2 (). The adult pattern of expression resembled that of CD34, which is a cell surface marker of HF-SCs (). Nuclear TBX1 was not detected in self-renewing transit-amplifying cells or in terminally differentiating cells (). Notably, in contrast to some other HF-SC transcription factors, TBX1 was also maintained in stem cells in long-term cultures.
The transcription factor TBX1 is highly enriched in stem cells in vivo and in vitro
To evaluate TBX1 function in vivo
, we conditionally targeted Tbx1
cKO) in the skin epithelium of embryonic day 15.5 mice20,21
cKO mice were viable, and hair follicle morphogenesis appeared to be normal (Supplementary Fig. 5b, c
). We tested for possible defects in stemness by analysing tissue regeneration during the normal hair cycle (Supplementary Fig. 6
). For this purpose, same-sex littermates were shaved at the normal onset of the first hair cycle (postnatal day 21, P21) and the second hair cycle (P60). In both cases, Tbx1-
cKO hair follicles remained quiescent longer than normal, but they eventually cycled. Maturation/differentiation was unaffected, as shown by the development of normal hair types and lengths.
Self-renewal occurs briefly after anagen onset, replenishing the stem cells that are used during initiation15
. To challenge stem cells to sustain long-term tissue regenerative potential, we repeatedly depilated the hair coat, a process that removes the old hair along with tightly adhering niche signalling cells that maintain stem cell quiescence14,15
(). After depilation, more than 80% of the stem cells remained viable in their niche, where they became activated to enter a new hair cycle (Supplementary Fig. 7
Tbx1-null stem cells fail in an in vivo assay for stem cell self-renewal and long-term tissue regeneration
Wild-type (WT) HF-SCs survived each round of depilation-induced hair regeneration, indicating the robust ability to sustain self-renewal and long-term tissue regeneration. By contrast, after five rounds, the Tbx1-
cKO stem cell numbers had declined by more than 70% (P
<0.001) (). Their steady depletion was accompanied by a thinning of the hair coat and a reduction in hair follicle density ( and Supplementary Fig. 8
). Histological analysis revealed that many Tbx1
-cKO hair follicles were dormant and had lost their stem cell niche, retaining only sebaceous glands and dermal papillae. However, the few hair follicles that cycled appeared morphologically normal, reflecting the presence of active stem cell niches ().
Similar results were obtained when hair cycles were monitored during natural ageing. Although the intervals between the hair cycles were longer than those in the depilation procedure, the Tbx1-cKO stem cell niche residents had declined by about 30% after 1 year (). Thus, Tbx1-null stem cells seem to be specifically impaired in their long-term ability to replenish their niche during normal and depilation-induced tissue regeneration.
We used 5-bromodeoxyuridine (BrdU) incorporation to define the brief window of bulge stem cell proliferation that occurs following depilation. WT stem cell proliferation peaked at day 3 after depilation, and the cells returned to quiescence by day 7. Tbx1-cKO stem cells also proliferated but to a lesser extent during this time period (). Within 2 to 3 days of depilation, only about 25% of Tbx1-null stem cells were BrdU-positive, whereas about 70% of WT stem cells were BrdU-positive (). This proliferative decrease was verified by DNA-content-based cell-cycle analysis of purified stem cells, which showed that the decrease was accompanied by fewer stem cells being present in the niche (). As discussed earlier, most hair follicles eventually produced hairs of WT length, reflecting an otherwise normal lineage program.
TBX1 controls stem cell proliferation in part by fine-tuning the response to BMP signalling
We observed a similar trend when we monitored the normal hair cycle. In this case, fewer stem cells were expended than during depilation; thus, the demand for self-renewal was lower, as reflected by the natural bulge niche having a lower proliferative activity than the depilation-induced WT bulge niche. Consistent with a role for TBX1 in HF-SC self-renewal, the overall proliferative activity within the Tbx1-
null niche was less than in the WT niche, and that in the natural niche was less than in the depilation-induced niche (Supplementary Fig. 9
To understand how these differences arise, we transcriptionally profiled messenger RNAs that were isolated from purified HF-SCs 2 days after depilation (, Supplementary Fig. 10
and Supplementary Table 4
). Bioinformatic analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional gene annotation tool uncovered mostly cell-cycle regulators in the 123 genes that were downregulated by 1.8 fold or more in Tbx1-
null stem cells compared with WT stem cells. The 188 genes that were upregulated by 1.8 fold or more were enriched for genes implicated in bone morpho-genetic protein (BMP) signalling (, red), including Bmp2
, which encodes a secreted ligand for BMP receptors, and Id
genes, which are major targets of the transcriptional effectors of BMPs, namely SMAD4 in complex with phosphorylated SMAD1 (pSMAD1), pSMAD5 or pSMAD8 (denoted pSMAD1/5/8). Using rtPCR, Tbx1-
cKO HF-SCs upregulated Id1 and Id2
(refs 22, 23
In cardiomyocytes, TBX1 seems to suppress BMP signalling by competitively interfering with SMAD4 for pSMAD1/5/8 binding24
. Consistent with this idea, overexpression of a TBX1–green fluorescent protein (GFP) fusion protein in WT keratinocytes significantly suppressed BMP-induced Id1
transcription in vitro
(). Similar effects, albeit slower and weaker, were observed for Id2.
Transgenic overactivation of the BMP circuitry results in hair coat thinning with age25
. If TBX1-deficient HF-SCs have a heightened sensitivity to BMP signalling, then BMP inhibitors might ameliorate the proliferative defect. We tested this hypothesis by plucking hair follicles from mice and then injecting them intradermally with beads soaked in the BMP antagonist noggin26,27
. Within 3 days, Tbx1
-cKO HF-SC proliferation had been restored to near WT levels (, P <0.001, and Supplementary Fig. 11
). As reflected by the bulge size, the expanded HF-SC pool was sustained throughout the hair cycle. However, additional treatments with noggin were necessary to maintain the stem cell pool through multiple rounds of depilation-induced hair regeneration. Interestingly, the self-renewal of TBX1-deficient stem cells was also elevated in vitro when BMP signalling was impaired by ablation of the BMP receptor BMPR1A (). Together, these findings are consistent with a BMP-induced proliferative defect in Tbx1-null stem cells.
Given these inverse links between TBX1 and BMP signalling, it seemed paradoxical that Smad1
shRNAs surfaced in our self-renewal screen (Supplementary Table 1
). Further analyses revealed that even though these shRNAs depleted Smad1
transcripts and SMAD1 protein, the SMAD1/5/8 target genes Id1
were still expressed. Moreover, the transduced cells still responded to BMP signalling, as judged by reporter assays (Supplementary Fig. 12
HF-SCs reside in a WNT-restricted, BMP-high micro-environment28,29
in which they must self-renew to replenish the stem cell pool. Therefore, HF-SCs must have an intrinsic mechanism to lower the BMP signalling threshold, and this mechanism fails to occur in the absence of TBX1. Entering the hair cycle also necessitates decreased BMP signalling; however, in this case, the proliferation is fuelled by early progeny (hair germ) with naturally low TBX1 levels that are present at the bulge base6,30
. Because, paradoxically, hair cycle initiation was delayed in Tbx1
-null mice, we surmise that the hair germ may be negatively influenced by BMP2 or other local effectors that are secreted by Tbx1-
In summary, we have discovered that TBX1 functions in the replenishment of HF-SCs during tissue regeneration. Our RNAi screens excluded roles for TBX1 in cell cycling, housekeeping and survival. The finding that once initiated, Tbx1-null hair cycles have a normal progression also ruled out roles for TBX1 in cell-fate determination or lineage progression. Instead, the TBX1 defect seems to be rooted in diminished stem cell self-renewal, coupled with enhanced sensitization to intrinsic BMP signalling. Together, these result in progressive HF-SC depletion and thinning of the hair coat. Although the effects of TBX1 are likely to be more complex than we have shown, its ability to intrinsically control these features poises it in the middle of a balancing act with the micro-environment to control stem cell behaviour in tissue homeostasis.