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Lgr6 is a stem cell marker in mouse skin squamous carcinomas

The G-protein-coupled receptors Lgr4/5/6 are Wnt signalling mediators, but their functions in squamous carcinomas (SCCs) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2- and Lgr6-EGFP-CreERT2- Rosa26/Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in vivo in SCCs. We identify, by single molecule in situ hybridisation and cell sorting, rare Lgr6-positive cells in immortalised keratinocytes, and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation, with expanded lineage tracing from Lgr6+ epidermal stem cells. Surprisingly, Lgr6 germline knockout mice are predisposed to SCC development, by a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss of function mutations in WNT pathway genes RSPO1 or LGR4, who show increased susceptibility to squamous tumour development.


Introduction
The mouse skin hair follicle (HF) comprises several distinct stem cell populations [1][2][3] . Lrig1 3,4 defines a cell population in the HF junctional zone that serves to maintain the HF infundibulum [5][6][7] , while quiescent Krt15-positive stem cells in the bulge region act as a reserve stem cell pool that becomes activated in response to stress 8 . Lgr4-6 are expressed in stem cells in many tissues, including the small intestine 9 , breast 10 , ovary 11,12 and haematopoietic system 13 . In the skin, Lgr5 has been genetically linked to a network of genes that are expressed specifically in the HF 14,15 , while Lgr6 is expressed in the isthmus region, sebaceous gland and interfollicular epidermis 1617 .
The Lgr4-6 receptors bind R-spondins (Rspo1-4) to enhance Wnt/β-catenin signalling 18-21 . Wnt signalling is known to be activated in the hair germ and HF bulge during the transition from telogen to anagen phase, and is critical for the formation of new follicles [22][23][24][25] . However, several reports have also indicated a negative role for Lgr5 in Wnt signalling, including in human colorectal cancers 26,27 . Moreover, loss-of-function mutations in human RSPO1 28 and LGR4 29 increase susceptibility to skin squamous carcinoma development, and LGR6 has been implicated both as a potential tumour suppressor gene 20 , and a breast cancer germline susceptibility gene 30 . Thus, the exact roles of Lgr4-6 in Wnt signalling and tumourigenesis appear to vary depending on the specific Lgr4-6 and Rspo1-4 family members that are expressed and interact in a given cellular context 27 .
The relationships between normal tissue stem cells and "cancer stem cells" (CSCs, also known as "tumour initiating cells") are controversial and unresolved [31][32][33][34][35] . Lgr5 has been reported to be a marker of both normal stem cells and CSCs in intestinal adenoma 36 , and in gastric cancer 37 but it remains unclear whether members of this gene family are expressed, and in particular play a functional role, in CSCs in other tumour types including cutaneous SCC.
Here, we identify a specific role for Lgr6 as a cutaneous CSC marker. Expression of Lgr6, but not Lgr5, increases during tumour progression and metastases, while lineage tracing in established tumours shows that only the progeny of Lgr6-positive cells form significant epithelial colonies within SCCs. Nevertheless, germline deletion of Lgr6 predisposes mice to development of SCCs. These data underline the parallels between this mouse model and human patients with germline loss of genes within this pathway, including RSPO1 and LGR4, who also have increased propensity for SCC development.

Lgr6 is a stem cell marker in squamous carcinoma
Mouse skin SCCs can be initiated with a single dose of Dimethyl-benzanthracene (DMBA) and promoted by repeated treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) 38 . Tumours from this model have common activating mutations in Ras oncogenes, and progress through benign and malignant stages, ultimately metastasising to cause the death of the host animals 39 . While the existence of stem cells within tumours in this model has been documented 40 , the relationship between these CSCs, and markers of normal stem cells, is unknown. We first analysed published gene expression data from samples of normal skin, papillomas, primary carcinomas and matched metastatic tumours from an interspecific FVBBX backcross population 39 . While Lgr6 expression continues to rise during progression, Lgr5 expression shows a decrease, suggesting that Lgr5 may not be required for tumour maintenance (Figure 1a).
We investigated the localised expression of Lgr5 and Lgr6 in SCCs from mice carrying an EGFP reporter gene under the control of the Lgr5 or Lgr6 promoters (Lgr5-EGFP-CreERT2 and Lgr6-EGFP-CreERT2 mice 15,16 ). Staining of tumours using antibodies against GFP (Lgr6, green) or Keratin 14 (Krt14, red) (Figure 1b-e and Supp. Fig. 1) showed that Lgr6 (Fig. 1e), but not Lgr5 (Fig. 1c), is clearly expressed in distinct colonies of cells distributed through the SCCs. Patterns of co-staining for GFP and Krt14 suggest that while the vast majority of Lgr6-positive cells are in the epithelial compartment, the average expression levels of Krt14 in these cells is relatively low compared to the adjacent more differentiated cells (Supp. Fig. 1i, insert). These data are thus compatible with the hypothesis that Lgr6 may act as a stem cell marker in squamous carcinomas, whereas Lgr5 does not.
Lgr5 + cells have been reported to be capable of acting as the cells-of-origin of intestinal and gastric adenoma [41][42][43] , cutaneous BCC 44 and SCC 45 . Schepers et al. 36 exploited the Lgr5-EGFP-IRES-CreERT2 +/− mouse to activate Wnt signalling in the Lgr5-positive crypt stem cells, and showed that the resulting adenomas continued to express Lgr5. We asked whether induction of Ras mutations within Lgr5-positive stem cells in the hair follicle gives rise to SCCs that continue to express Lgr5, or if it is dispensable for tumour maintenance. We crossed Lgr5-EGFP-IRES-CreERT2 +/− or Lgr6-EGFP-IRES-CreERT2 +/− mice with Kras LSL-G12D mice carrying a floxed mutant Kras allele 46 . Activation of oncogenic Kras with topical 4-hydroxytamoxifen (4OHT) in Lgr5-positive stem cells resulted in some limited sebaceous gland hyperplasia (Supp. Fig. 2a), but when the mice were further challenged with a back wound stimulus, papillomas readily arose within 4 weeks (Supp. Fig.  2b). Similar observations were made after activation of Kras in the Lgr6-positive stem cell in the epithelial carcinoma cells (Supp. Fig. 2e). TaqMan and immunofluorescence analyses of 3 independently derived cell lines derived from these spindle carcinomas showed an absence of Lgr5 expression (Supp. Fig. 2f, g) while Lgr4 and Lgr6 expression could readily be detected (Supp. Fig. 2f), We conclude that even tumours arising from Lgr5-positive cells in the HF fail to express Lgr5 during tumour progression, and that Lgr5 is not a positive driver of SCC growth.

Lineage tracing of Lgr6-positive stem cells in squamous tumours
CSCs can give rise to progeny within tumours that continue to fuel tumour development and progression 40,49 . We carried out lineage tracing within chemically induced skin tumours from Lgr6-EGFP-CreERT2 or Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice 15,16 (hereafter Lgr6or Lgr5-reporter mice). Early stage papillomas were induced in both double transgenic lines by standard DMBA treatment followed by 8 weeks of tumour promotion using TPA (Supp. Fig. 3). At this stage, mice were treated with Tamoxifen and papillomas were harvested at different times (2 days, 3 weeks and 6 weeks) thereafter, to measure the extent of lineage tracing from either Lgr5- (Fig. 2a, c, e, g) or Lgr6-positive ( Fig. 2b, d, f, g) stem cells. Imaging of fluorescent cell populations from papillomas 2 days after Tamoxifen treatment identified rare GFP/RFP positive cells in the epithelial compartment in Lgr6-reporter mice, but not in Lgr5-reporter mice. (Fig. 2a, b).
The number of Tomato-positive traced cells increased at 3 weeks and 6 weeks, giving rise to distinct clones that showed evidence of migration from the initial Lgr6-positive cells (Fig  2d,f). Some Tomato-positive traced cells in Lgr6-reporter mice were also observed in the dermis/stromal compartment (Supp. Fig. 4b, d), but these were only a minor population, the significance of which is unknown. In contrast, papillomas from Lgr5-reporter mice displayed very few Tomato-positive traced cells, even after 6 weeks, and the majority of these were found in the dermal compartment (Fig. 2e, g and Supp. Fig. 4a, c).
We also carried out lineage tracing from Lgr6-positive cells in primary carcinomas that arose between 24-30 weeks after tumour initiation. At 2 days (Fig. 3a, with Tamoxifen showed a strong decrease in clone numbers over several weeks, but no such decrease was seen in clone number in our studies of serial biopsies of carcinomas from Lgr6-CreER/RosaRFP mice (Fig. 3). This suggests that Krt14 initially labels many transit amplifying cells with poor self-renewal capacity, whereas the Lgr6-positive population is already enriched in stem cells that are capable of long term self renewal.
The transcription factor Sox2 is expressed in papillomas and squamous carcinomas, and was reported to mark a stem cell population that is enriched in tumour-initiating cells 50 . We investigated possible overlap between Sox2-positive cells and Lgr6-positive cells in SCCs from the DMBA/TPA model by staining for Sox2 in tumours from Lgr6-EGFP-CreERT2 mice. The data in Supp. Fig 6 show that Sox2 is more widely expressed in a larger population of cells than is Lgr6, with minimal overlap between the cells expressing these two distinct markers.

Single cell analysis of Lgr5 and Lgr6 co-expression in normal keratinocytes and tumour cell lines
Analysis of gene expression at the single cell level is important in the context of CSCs, only a small and variable number of which may be present in solid tumours or tumour cell lines. We carried out gene expression microarray analysis of Lgr4, 5 and 6 levels in mouse cell lines representing various stages of chemically induced tumours 51,52 . Lgr5 was not expressed above background levels in any of these cell lines, while Lgr4 and Lgr6 were expressed at varying levels (Supp. Fig. 7). Taqman analysis (Fig. 4a), confirmed that Lgr5 was not expressed above the limits of detection except in positive control back skin samples.
To address the possibility that Lgr5 may be expressed in rare single cells in keratinocyte cultures, we performed Lgr4-6 multiplex in situ hybridisation 53 . We also analysed a BCC cell line (Asz001) 54 , since Lgr5 has been reported to be expressed in BCC cells. Expression of Lgr5 was detected in the BCC cells by Taqman and qPCR analyses, albeit at very low levels ( Fig. 4b arrow). In situ hybridisation analysis revealed that while Lgr4 was broadly expressed in Asz001 cells, Lgr5 and Lgr6 were more restricted in expression. Interestingly, all three family members were co-expressed in rare single cells ( Fig. 4c and insets) at a frequency of about 0.75% (4 in 533 cells). This observation raises the intriguing possibility that these rare Lgr5, 6 and 4 triple-positive cells are CSCs in the BCC cell line, although further work would be needed to test this hypothesis.
In contrast to the BCC cell line, none of the SCC and keratinocyte cell lines showed significant Lgr5 expression (Fig. 4d, e, and Supp.  Fig. 7). These data therefore demonstrate that, at the single cell level, there appears to be a hierarchy in expression patterns of the Lgr gene family. BCCs have distinct Lgr5-positive cells, some of which are triple-positive cells that express all three family members. Immortalised keratinocytes lack Lgr5, but have rare double-positive Lgr6 and Lgr4 cells, and these appear to increase in number in more advanced tumours.

Multiplex analysis of Lgr family gene expression using PCR-activated cell sorting (PACS)
To determine the levels of co-expression of Lgr4-6 at a more quantitative level, we used a novel PCR-activated Cell Sorting (PACS) technology [55][56][57] , (see Materials and Methods). With this method, we were able to identify and sort single cells by performing single-cell TaqMan RT-PCR reactions in >100,000 cells targeting Lgr4, 5 and 6 mRNA sequences. We validated this approach using a subset of the cell lines that were analysed for Lgr gene expression by in situ hybridisation and microarrays. Figure 4f shows that almost 100% of the single NK (immortalised keratinocytes), E4 (metastatic squamous carcinoma cells), and ASZ001 (BCC) cells expressed Lgr4, in agreement with the results of in situ hybridisation. In contrast, about 1% of the BCC cells expressed Lgr5, however, there was no significant expression in NK cells (Fig. 4f). Interestingly, this extremely sensitive approach also identified a small population of E4 cells (around 0.4%) that expressed some Lgr5 (Fig. 4f) despite the fact that expression analysis by TaqMan (Fig. 4a), microarrays (Supp. Fig. 7), or in situ hybridisation (Fig. 4e) of the whole cell population did not detect any significant Lgr5 transcripts. Lgr6 was expressed in over 90% of the E4 cells, and in about 10% of BCC cells, but only in about 1-2% of the NK cells, again in line with the in situ data (Supp. Fig. 8). We also analysed expression of the same genes in keratinocytes from normal mouse skin. Lgr4 was quite widely expressed (20-30% of keratinocytes), in agreement with previous data on expression in mouse and human skin 16,58 , while Lgr6, known to be expressed in the upper isthmus as well as the interfollicular epidermis 16 , was found in around 18% and Lgr5 in around 2% of single keratinocytes (Fig. 4f).
Further analysis of these data ( Fig. 4g) confirmed the presence of Lgr4, 5 and 6 triple positive cells in vivo in normal keratinocytes. Around 1.5-2% of normal dorsal telogen skin keratinocytes expressed Lgr5 (Fig. 4f), and of these about 20% (0.3% of the total population) were triple positive (Fig. 4g). Comparable results were seen for the BCC cell line, where of the 1% of the total cell population that expressed Lgr5 (Fig. 4f) about 18% (0.18% of the total) were triple positive for all three genes (Fig. 4g). The concordance between in situ hybridisation and PACS sorting data for the cell lines indicates that in vivo there are specific subpopulations of Lgr5-expressing cells that also co-express the other family members. Whether these co-expression patterns are involved in cell fate decisions in normal skin homeostasis in vivo remains to be addressed. Interestingly, PACS analysis of primary squamous carcinoma also revealed the presence of a small population of triple positive cells (Supp. Fig. 8d, e). These cells however do not appear to be clonogenic in tumours, as the lineage tracing studies using the Lgr5-EGFP-CreERT2 allele failed to identify a significant population of traced epithelial tumour cells.

Lgr6 is enriched in keratinocyte cells with stem cell properties
To test the possibility that Lgr6-positive cells represent a stem cell subpopulation, we utilised the Aldefluor assay [59][60][61][62] to isolate Aldefluor + and Aldefluor − subpopulations from C5N and D3 cultures. Lgr6 expression was elevated in the Aldefluor + fraction compared to the Aldefluor − fraction (Fig. 5a, b), and Aldefluor + cells sorted from C5N cultures were capable of forming more and larger colonies in vitro than Aldefluor − cells (Fig. 5c). We conclude that Lgr6, and not Lgr5, acts as a marker of a stem-like cell population in squamous epidermal cells.

Lgr6 suppresses epidermal cell growth in vitro and in vivo
We next overexpressed Lgr6 in immortalised keratinocytes (NK cells), which have low endogenous levels of Lgr6 mRNA (Supp. Figs. 7, 8). Transfection of NK cells with an inducible Lgr6 expression construct (Fig. 5d) led to growth retardation after Doxycycline treatment (Fig. 5e). To validate this growth suppressive effect of Lgr6 in epidermal cells, we knocked down Lgr6 in an SCC cell line (E4), which expresses relatively high endogenous levels of Lgr6 ( Fig. 4e and Supp. Fig. 7). We transduced E4 cells with an inducible Lgr6 shRNA construct, which, upon doxycycline administration, elicits efficient knockdown of Lgr6 without altering the levels of either Lgr4 or Lgr5 (Fig. 5f). Consistent with a growth suppressive role for Lgr6 in epidermal cells, doxycycline treatment of transduced E4 cells led to increased growth but had no effect on unmodified E4 cells (Fig. 5g).

Lgr6 knockdown in vivo expands lineage tracing in the skin
Lgr6 is a marker of infundibulum stem cells, but a possible functional role in control of homeostasis in vivo has not been investigated. Although the Lgr6−/− mouse is viable 63 , developmental adaptation to loss of expression could lead to compensatory mechanisms for controlling skin homeostasis in the adult mouse. We therefore generated Lgr6 KD mice, which possess an Lgr6 shRNA construct knocked into the Rosa26 locus that can be induced by doxycycline treatment (Fig. 6a). Adult Lgr6 KD mice were treated with doxycycline water for 10 days, leading to an approximately 60% reduction in Lgr6 expression levels in the back skin, while Lgr4 and Lgr5 levels were not significantly affected (Fig. 6b).
Immunofluorescence analysis showed that doxycycline administration for this period resulted in an increase in proliferation in the epidermis of Lgr6 KD mice, as shown by BrdU labelling (Fig. 6c). These results therefore agree with the in vitro data shown in Fig. 5g.
We also tested the effect of Lgr6 downregulation in vivo on patterns of lineage tracing from Lgr6 + stem cells. We generated Lgr6 KD +/− /Lgr6-EGFP-IRES-CreERT2 +/ /R26RLacZ +/− triple transgenic mice, and administered doxycycline to 8-week-old triple transgenic animals and controls for 10 days, followed by activation of Cre recombinase by topical 4OHT treatment. Skin samples were collected 7 days thereafter, and X-Gal analysis revealed a striking increase in the number of LacZ-positive cells in the epidermis (Fig. 6d,e,f) in doxycycline-treated mice. This expanded epidermal lineage tracing was accompanied by an increase in the clone size (Fig. 6g), suggesting that Lgr6 downregulation led to increased proliferation within the Lgr6 progeny compartment in the epidermis, in line with our observations above (Fig. 6c). It is not possible at this stage to determine whether the increase in traced cells in the epidermis is due to migration from the Lgr6-positive cells in the isthmus region, or to independent proliferation of Lgr6-positive cells that have been identified within the epidermis 17 . Interestingly, we also saw evidence for Lgr6 lineage tracing within the dermis, which was particularly elevated in the doxycycline-treated animals (Fig. 6d, lower panel) and has not been previously reported. Control skin from non-4OHT treated adult Lgr6-EGFP-IRES-CreERT2 +/− /R26RLacZ +/− mice showed no staining in either the HFs or the IFE, indicating the non-leakiness of the Lgr6-EGFP-IRES-CreERT2 +/− promoter construct (Supp. Fig. 9).

Lgr6 activates Wnt signalling but is a germline tumour suppressor gene
Lgr5 has been reported to be overexpressed in hepatocellular 64 , colon and ovarian cancers 65 , and has also been demonstrated to promote the tumourigenicity of BCC cells 66 . Paradoxically, however, Lgr5 has also been shown to reduce the growth of colorectal cancer cells 26,27 , and tumour suppressive roles have been suggested for Lgr4 29 and Lgr6 20 .
Germline loss of RSPO1, the upstream ligand for the Lgr family of receptors, or of LGR4, also causes predisposition to squamous carcinoma development in human patients 28,29 . We therefore investigated the potential role of Lgr6 as an inducer of Wnt signalling in squamous cells, and as a germline tumour suppressor gene.
We assessed the effects of Lgr6 deregulation on Wnt signalling in keratinocyte cell lines using the well characterised TOPFlash assay 67 . Overexpression of Lgr6 in multiple epidermal cell lines consistently led to activation of this pathway (Fig. 6h). Finally, analysis of human TCGA gene expression data on head and neck SCCs (Supplementary Tables 1 and  2) showed a strong connection to the WNT pathway only for LGR6, and not to any significant extent with LGR5 or LGR4. This supports the notion that LGR6 is linked to Wnt signalling and may play a unique role in tumours of squamous origin.
To assess the effect of germline inactivation of Lgr6 on tumour susceptibility, we bred Lgr6−/− mice through multiple generations on to a pure FVB/N genetic background, and exposed them to the DMBA/TPA chemical carcinogenesis protocol. The knockout mice showed a highly significant increase in susceptibility to papilloma development (Fig. 6i), as well as a shortened carcinoma-free survival (Fig. 6j). We conclude that Lgr6, in spite of its activity as a positive stimulator of Wnt signalling, acts as a germline suppressor of SCC development, thus mimicking the functions of human WNT activators RSPO1 and LGR4 as SCC suppressor genes. One possible explanation for these apparently contradictory results is that germline loss of Lgr6 could cause a developmental upregulation of other pathway components that may compensate for loss of Lgr6 activity. Stem cells in normal skin or intestine display considerable plasticity, such that ablation of Lgr5 positive cells can stimulate their regeneration from alternative stem cell populations 68 . To investigate possible mechanisms of compensation, we derived a series of cell lines from Lgr6−/− skin SCCs, and analysed expression levels of the other Lgr family members. The wild type mouse carcinomas showed variable activation of the Wnt pathway, as demonstrated by the differences in levels of Phospho-β-catenin 69 . Of the three carcinomas tested, highest Wnt activity is seen in the cell line (168 cells) that also has the highest level of Lgr6 expression (left panel, Fig. 6k). Variable levels of Wnt activity are also seen in the carcinomas from Lgr6 knockout mice (right panel, Fig. 6k, Supp. Fig. 11), but in 3 of 4 cases with high activity, the level of Lgr5 expression is strongly upregulated (arrows). This is particularly significant as all of our expression data show that in SCCs from wild type mice, Lgr5 is switched off even at the single cell level and plays no significant role as an epithelial cancer stem cell marker. These data therefore identify a mechanism by which cancer stem cells in Lgr6−/− mice can be regenerated through a feedback mechanism resulting in upregulation of Lgr5.

Discussion
Using a combination of gene expression analysis, lineage tracing, single molecule in situ hybridisation, and PACS analysis of single tumour cells we have shown that Lgr6 satisfies many of the criteria of an SCC stem cell marker. Lineage tracing identified a subpopulation of Lgr6+ tumour cells that give rise to progressively growing epithelial clones. Lgr6 was enriched in aldefluor-positive cells with stem cell properties, and its inhibition resulted in increased proliferation and lineage tracing in the epidermis. Although Lgr5 was not significantly associated with squamous tumour development or progression, it was expressed in rare single cells in a BCC cell line 66 , some of which were triple positive for all 3 Lgr family members. The detection of these triple positive cells in vivo in basal keratinocyte cells, as well as in primary carcinomas, suggests that they may have a distinct role in stem cell homeostasis, but this question will require further investigation. We believe the model that best explains our results is shown in Supp. Fig. 10. In this model, an early stem cell population may express Lgr5 and Lgr6 as well as Lgr4, with the initial cell fate choice being determined by competitive interactions of these GPCRs with their Rspondin ligands and other potential effector molecules such as Rnf43 70 and Znrf3 71 . Lgr5 expression is maintained in some cells that can give rise to BCCs, but lost in cells that commit to the epidermal cell fate. In this stem cell compartment, Lgr6 functions as a brake towards further epidermal commitment and differentiation, such that loss of Lgr6 leads to increased epidermal lineage differentiation and proliferation. It is possible that loss of Lgr6 expression (but continued expression of Lgr4) may be linked to commitment to a more differentiated keratinocyte state. Indeed, the highest levels of Lgr4 expression are seen in cell lines with the most "epithelial" characteristics such as the immortalised keratinocytes C5N and NK, or the differentiated papilloma cell lines MscP1 or Mscp5 (Supp. Fig. 7).
Germline deletion of Lgr6 in the mouse caused predisposition to SCC development, as also seen in human patients with germline loss of LGR4 29 or RSPO1 28 . Tumours that arose in Lgr6−/− mice in fact showed upregulation of Lgr5, particularly in those tumours with the highest levels of Wnt signalling. These data highlight the plasticity and complex control of Wnt/B-catenin signalling in the skin, where activation of the pathway can maintain hair follicle [72][73][74] and epidermal 75 stem cell growth, but impairment in hair follicle stem cells stimulates commitment to the epidermal lineage 73,[76][77][78] . Elevated expression of β-catenin is negatively associated with normal epidermal proliferation, but its localisation in the nucleus is positively associated with growth of Hras-transformed cells 79 . Successful targeting of the Wnt pathway for SCC therapy will require resolution of the complex mechanisms that distinguish signalling in these different biological contexts.

Mouse treatments, chemical carcinogenesis and lineage tracing in skin
Lgr6 KD mice were generated in a similar fashion to previously reported doxycyclineinducible knockdown mice 80 , using the Lgr6 shRNA sequence GCATGACACTGGAAGACAACT. Recombinant ES cells were injected into blastocysts and chimeric animals were obtained at the UCSF Cancer Center Transgenic and Targeted Mutagenesis Core Facility. Doxycycline water was prepared by dissolving 2 g of doxycycyline (RPI) in 1 l of water containing 50 g of sucrose (Affymetrix). For BrdU studies, mice were injected intraperitoneally with BrdU labelling reagent (Life Technologies) at a concentration of 10 µL/g of body weight and sacrificed 3 hours postinjection. For chemical carcinogenesis experiments, 8-to 12-week-old male and female mice were randomly assigned to control or treatment groups. Initiation was carried out using a single dose of DMBA (25 ug per mouse in acetone) applied to back skin. One week later, the mice were administered biweekly topical treatments of TPA (200 uL of a 10-4 M solution in acetone) for 20 weeks. The animals were then monitored for papilloma number and progression to carcinoma. Mice were sacrificed when their first carcinoma reached 1-1.5 cm in diameter. For fluorescent Lgr5/6GFP-Tomato lineage tracing experiments, papillomas were established following the chemical carcinogenesis protocol. After 8 weeks of TPA treatment, one topical dose of Tamoxifen (200 ul of 25 mg/ml in 100% ethanol) was administered to shaved back skin and papillomas were then surgically removed 2 days, 3 weeks and 6 weeks after the initial Tamoxifen treatment for analysis. Similar lineage tracing was carried out with mice bearing carcinomas that arose after 24-30 weeks post initiation with DMBA. To preserve Lgr5/6GFP & Tomato reporter expression, prior to embedding, excised tumours were briefly fixed in 4% paraformaldehyde for 2 hours and then placed in 30% sucrose overnight at 4 °C before freezing in OCT for subsequent cryosectioning. Center. Age-matched animals of the required genotypes were randomly assigned to treatment groups (for instance, wounded or unwounded, Supp. Fig. 2; vehicle-or doxtreated, Fig. 6c-g). When assessing experimental outcomes in animal samples (for instance, BrdU staining in Fig. 6c; X-Gal staining in Fig. 6d-g), investigator was blinded through the use of unique animal IDs for the samples that do not indicate treatment conditions.

X-Gal staining
Back skin samples were surgically removed from 4OHT-or vehicle-treated mice and cut into small pieces. These were added to 4% formaldehyde diluted in PBS, and placed on a shaker at room temperature for 10 min. After 3 washes with PBS, the skin samples were incubated in X-Gal staining solution (1 mg/ml X-Gal, 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , and 2 mM MgCl 2 in PBS) at 37 °C in the dark. The next day, the samples were washed with PBS, transferred to 70% ethanol and made into paraffin blocks. 6 µm sections were cut on a microtome and finally counterstained with nuclear fast red (Vector Laboratories).

Multiplex in situ hybridisation
ViewRNA ISH Cell Assay probes for murine Lgr4-6 were obtained from Panomics and in situ hybridisation was performed, according to the manufacturer's protocol, on cells grown in poly-lysine coated, glass-bottom 96-well plates (In Vitro Scientific). Images were acquired on the 6D high throughput microscope in the UCSF Nikon Imaging Center and analysed using the NIS Elements and ImageJ software packages.

Single-cell RT-PCR analysis
Single cell analysis of Lgr4, Lgr5 and Lgr6 expression was performed using the Mission Bio, Inc. PACS platform [55][56][57] . Briefly, single keratinocyte suspensions of disaggregated back skin, NK cells, E4 SCC cells, BCC cells or primary squamous carcinoma cells were stained with a calcein violet viability dye and encapsulated into microfluidic droplets. Droplets containing individually lysed cells were further processed with the PACS microfluidic workflow to enable droplet-based TaqMan RT-PCR detection of target transcripts. The multiplex RT-PCR reactions consisted of FAM labeled Lgr5, HEX labeled Lgr6 and Cy5 labelled Lgr4 TaqMan probes. Thermocycling conditions were as follows: 15 min 50 °C, 93 °C for 2 min, 35-40 cycles of 92 °C for 15 s and 60 °C for 1 min. Following thermocycling, the droplets were reinjected into a microfluidic device for fluorescence analysis using four coincident lasers (405 nm, 473 nm, 532 nm, and 640 nm, CNI lasers) to excite the cell viability stain and TaqMan probes. The resulting fluorescence channels (centered at 440 nm, 510 nm, 572 nm, and 680 nm) were separated from the lasers and each other with dichroic filters (Semrock) before being detected by four PMTs (Thorlabs). The detection was processed in real time using LabVIEW and the droplet scatter plots were generated using MATLAB.
Single-cells were analysed with the multiplexed TaqMan assay (7,334 back skin cells, 14,454 NK cells, 21,596 E4 SCC cells, 16,914 BCC cells and 2,617 primary SCC cells). Three independent replicates were performed for each cell/sample type.

Immunofluorescence
First, deparaffinisation and antigen retrieval were performed on 6 µm back skin sections using the Trilogy pretreatment solution (Cell Marque). Then, the sections were incubated in 10% donkey serum (Abcam) diluted in PBS containing 0.3% Triton X-100, at room temperature for 1 hour. Subsequently, the sections were incubated with primary antibody against BrdU (rat, Abcam) or GFP (rabbit, Santa Cruz) at 4 °C overnight. After washing 3 times with PBS containing 0.1% Tween-20, the sections were incubated with the appropriate Alexa 488/555 conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. After washing 3 times with PBS containing 0.1% Tween-20, the samples were finally mounted in Vectorshield hardset mounting medium with DAPI (Vector Laboratories). Lgr5/6GFP and Krt14 expression in primary SCC cryosections was visualised by incubation with anti-GFP (chicken, Abcam) and anti-Krt14 (rabbit, Covance) antibodies, respectively.

Western blot and RT-qPCR analysis
Lgr6 WT and Lgr6 KO cell lines were derived from SCC generated by DMBA/TPA treatment of WT FVB mice or FVB Lgr6 −/− mice 67 . SCC were harvested and digested in 4mg ml-1 Collagenase A (Sigma Aldrich) at 37°C for 60 min with agitation, followed by 30 min incubation in 0.25% trypsin-EDTA solution. Dissociated cells were then passed through a 40 µm filter (Greiner Bio-One) and seeded into a 10 cm dish with regular DMEM. For western blot analysis, cells were prepared with lysis buffer (CST) supplemented with protease inhibitors, 100 µM NAF and 100 µM Na 3 VO 4 . Protein lysates were run by SDS-PAGE and transferred onto PVDF membranes (Biorad). After blocking, membranes were incubated overnight at 4°C with anti phospho β-catenin (#8814; CST) or anti β-actin (#4967; CST) antibodies. Anti Rabbit HRP-linked was used as secondary antibody (#7074; CST). Enhanced Chemoluminescence was used for detection. Phospho β-catenin membrane was stripped for 30 min at room temperature in Restore Plus Stripping buffer (Thermo Fisher), re-blocked and incubated with anti β-catenin (#1247-1; Epitomics) antibody. For Lgr gene expression analysis, RNA was extracted from Lgr6 WT and Lgr6 KO cells with TRIzol reagent (Thermo Fisher) and reverse-transcribed using Quantitect RT kit (Qiagen). qPCR expression analyses were performed with the indicated primer sets (Table S3.) by using Power-Up SYBR green mastermix (Thermo Scientific, #A25742) and run on an AB7900HT instrument (Applied Biosystems). Relative gene expression was calculated by using L19 as housekeeping genes to generate ΔCt. ΔCt values from 1425-L1 Lgr6 WT cells were used as reference to generate ΔΔCt, and relative gene expression was calculated as 2 (−ΔΔCt) , or presented as copy number/1,000 copies of L19 to calculate gene expression. All reactions were run in duplicate. For primer sequences, see table S3.

Cell culture treatments, MTT and TOPFlash assays
Doxycycline was administered, where appropriate, at a concentration of 1 µg/mL of culture medium. The shRNA sequence used to target Lgr6 in E4 cells is identical to the one used to generate the Lgr6 KD mice (see above) and this was cloned into the doxycycline-inducible F1HtUTG lentiviral vector 81 . For MTT assays, 2000 (Lgr6 ox and null NK) or 4000 (Lgr6 shRNA and null E4) cells were seeded in each well of a 96-well plate and the culture medium was replaced every third day. The Vybrant MTT Cell Proliferation Assay (Life Technologies) was performed according to the manufacturer's protocol at the indicated time points after initial seeding. For TOPFlash assays, cells grown in 6-well plates were first transfected with 2 µg of the M50 Super 8x TOPFlash plasmid and 0.1 µg of the pRL-TK renilla luciferase plasmid per well, using Lipofectamine 2000 (Invitrogen). After 6 hours, the medium was replaced and doxycycline was added, where appropriate. 2 days later, luciferase activity was determined for each well using the Dual-Luciferase Reporter Assay System (Promega) with the GloMax Luminometer, according to the manufacturer's instructions.

Cell Lines and Microarray analysis
The cell lines shown in Supplementary Figure 7 were described previously 55,51,82,83 (and references therein). NK cells and C5 cells are immortalised keratinocytes derived from wild type (C5) or Trp53 null mice (NK). Hras null cells were derived from a carcinoma induced by DMBA/TPA in Hras knockout mice 57 . All of the other lines carry initiating mutations in the Hras gene. Cells were grown in serum supplemented media into the log phase, then TRIzol RNA extraction was then performed, followed by purification by Qiagen kit. Gene expression analysis of RNA samples from these lines was carried out using Affymetrix M430 2.0 arrays. Microarray CEL files were background corrected and normalised with RMA using oligo in R and then log2 transformed prior to analysis. Cell lines were routinely tested for mycoplasma contamination.

Data Availability
The cell line gene expression microarray data have been deposited in GEO at the time of submission with accession number GSE88742

Aldefluor, colony formation assays and qPCR
Cells cultured in vitro were trypsinised and stained with Aldefluor (and DEAB) (Stem Cell Technologies), according to the manufacturer's instructions. The cells were then sorted into Aldefluor + and Aldefluor − fractions on the BD FACSAria III. For expression analyses, RNA was extracted from the sorted cells using TRIzol (Invitrogen), and reverse transcription was performed using the High Capacity Reverse Transcription Kit (Life Technologies). qPCR reactions were subsequently run on the AB7900HT, using Taqman gene expression assays for Lgr4 (ID Mm00554385_m1), Lgr5 (ID Mm00438890_m1 and Mm01251804_m1) and Lgr6 (ID Mm01291336_m1) (Life Technologies). Within each sample, the Ct value for betaactin was used as a reference to calculate delta Ct values for genes of interest. For each qPCR experiment, all samples were run in technical triplicates and the mean delta Ct value amongst the technical triplicates was determined for genes of interest; all samples were further run in biological triplicates and the data presented show the mean and s.e. amongst these biological triplicates.
For colony formation assays, 2000 cells were seeded in each well of a 6-well plate and grown in a 37 °C cell culture incubator for 19 days. The cells were then fixed in ice-cold methanol for 10 min and finally stained with crystal violet for 10 min.

Statistical analysis
Statistical analyses were carried out using the Prism software package (Graphpad). Significance of difference between two groups was determined using the unpaired, twotailed Student's t test and labelled as follows: * for p < 0.05; ** for p < 0.01; and *** for p < 0.001. F-tests indicate no evidence of a statistically significant difference in variances of all groups being compared, except in Fig. 5d, 5g (null samples), 6c, 6e and 6h (H11 samples), where Welch's correction is applied. Significance analysis for multiple groups was analysed by ANOVA.
As we could not anticipate an effect size prior to performing our initial experiments, rather than performing a power calculation we defined cohort size by estimating from prior work. We evaluated our prior experimental evidence from unrelated experiments to define a size that allowed us to detect a both statistically as well as practically meaningful difference between the genotypes. In our experiments, using the two-stage carcinogenesis protocol, this is generally between 10 and 20 mice, assuming a less than 5% dropout rate during carcinogenesis.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  Lgr5-and Lgr6-derived lineage tracing was performed within chemically induced skin tumours from Lgr6-EGFP-CreERT2 or Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 8 weeks of tumour promotion using TPA. Mice were then treated with Tamoxifen (TAM) and papillomas were harvested at 2 days, 3 weeks and 6 weeks thereafter, to measure the extent of lineage tracing from either Lgr5GFPor Lgr6GFP-positive stem cells. (a) At 2 days post-TAM, no Lgr5derived Tomato+ cells (red) were readily observed in papilloma epithelium, however, in Lgr6-reporter mice, rare Lgr6GFP+Tomato+ cells were localised in the epithelial compartment (b). (c, e, g) Over time, rare Lgr5-derived, Tomato+ cells (arrow) were observed in papilloma epithelium, in contrast, Lgr6-derived, Tomato+ traced cell numbers increased at 3 weeks (g) and 6 weeks, giving rise to distinct epithelial clones (d,f). Yellow dotted box demarcates the magnified regions in the figure insets. White dotted line indicates epithelial-dermal border. DAPI staining (blue) localises cell nuclei. Scale bar = 50µm.

Figure 3. Lgr6-derived clonal expansion in primary SCCs
Lgr6-derived lineage tracing was performed within chemically induced primary (1°) SCCs from Lgr6-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 20 weeks of tumour promotion using TPA. Once SCCs were readily visible, mice were treated with Tamoxifen (TAM) and a biopsy was taken observed within the epithelial tumour tissue. Yellow dotted box demarcates the magnified regions in the figure insets. White line indicates epithelial-dermal border. DAPI (blue) staining was used to visualise cell nuclei. (a) H&E sections of images depicted in (b) to provide tumour histology. Scale bar = 50µm.  expression level was determined in the sorted Aldefluor + and Aldefluor − populations using Taqman analysis. (c) Normal epidermal cells (C5N) were sorted into Aldefluor − and Aldefluor + subpopulations and these were separately seeded at low density (2000 cells per well) in 6-well plates. Colonies were allowed to grow for 19 days before being visualised by staining with crystal violet. (c) Representative wells showing colony formation by Aldefluor − cells (left) and Aldefluor + cells (right). For each well, the colonies were manually counted and the cell area was calculated in ImageJ to determine the colony formation efficiency and average colony area (c). Data are presented as mean ± s.e. *p < 0.05, **p < 0.01 and ***p < 0.001. n = 5 for (a) and (b), and n = 3 for (c). (d,e) Immortalised keratinocytes (NK) were transduced with an inducible Lgr6 expression construct (Lgr6 ox cells) or left unmodified (null cells). The cells were then grown either in the presence of absence of doxycycline, and growth was monitored at various timepoints using the MTT assay.  Adult Lgr6 KD +/− mice were administered doxycycline water or vehicle for 10 days. The animals were then intraperitoneally injected with BrdU and skin samples were harvested 3 h later. (a) Cartoon depicting mechanism of Lgr6 knockdown induced by doxycycline treatment of Lgr6 KD +/− mice. In the absence of doxycycline, the tet repressor sits on the H1-tetO promoter 80 and prevents the transcription of the Lgr6 shRNA. In the presence of doxycycline, the tet repressor is bound by the drug and prevented from associating with the H1-tetO promoter; this in turn allows for the transcription of the Lgr6 shRNA. (b) Lgr4-6