Title: Epidermal insulin/IGF-1 signalling control interfollicular morphogenesis and proliferative potential through Rac activation
Abstract: Article24 July 2008free access Epidermal insulin/IGF-1 signalling control interfollicular morphogenesis and proliferative potential through Rac activation Heike Stachelscheid Heike Stachelscheid Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Institute for Genetics, University of Cologne, Cologne, Germany Search for more papers by this author Hady Ibrahim Hady Ibrahim Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Linda Koch Linda Koch Institute for Genetics, University of Cologne, Cologne, Germany Search for more papers by this author Annika Schmitz Annika Schmitz Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Michael Tscharntke Michael Tscharntke Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author F Thomas Wunderlich F Thomas Wunderlich Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Jeanie Scott Jeanie Scott Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Christian Michels Christian Michels Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Claudia Wickenhauser Claudia Wickenhauser Institute of Pathology, University of Cologne, Cologne, Germany Search for more papers by this author Ingo Haase Ingo Haase Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Jens C Brüning Corresponding Author Jens C Brüning Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Carien M Niessen Corresponding Author Carien M Niessen Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Heike Stachelscheid Heike Stachelscheid Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Institute for Genetics, University of Cologne, Cologne, Germany Search for more papers by this author Hady Ibrahim Hady Ibrahim Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Linda Koch Linda Koch Institute for Genetics, University of Cologne, Cologne, Germany Search for more papers by this author Annika Schmitz Annika Schmitz Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Michael Tscharntke Michael Tscharntke Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author F Thomas Wunderlich F Thomas Wunderlich Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Jeanie Scott Jeanie Scott Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Christian Michels Christian Michels Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Claudia Wickenhauser Claudia Wickenhauser Institute of Pathology, University of Cologne, Cologne, Germany Search for more papers by this author Ingo Haase Ingo Haase Department of Dermatology, University of Cologne, Cologne, Germany Search for more papers by this author Jens C Brüning Corresponding Author Jens C Brüning Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Carien M Niessen Corresponding Author Carien M Niessen Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Department of Dermatology, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Author Information Heike Stachelscheid1,2,‡, Hady Ibrahim1,3,‡, Linda Koch2,‡, Annika Schmitz1,3, Michael Tscharntke3, F Thomas Wunderlich1, Jeanie Scott1,3, Christian Michels1,3, Claudia Wickenhauser4, Ingo Haase3, Jens C Brüning 1,2,5 and Carien M Niessen 1,3,5 1Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 2Institute for Genetics, University of Cologne, Cologne, Germany 3Department of Dermatology, University of Cologne, Cologne, Germany 4Institute of Pathology, University of Cologne, Cologne, Germany 5Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany ‡These authors contributed equally to this work *Corresponding authors: Institute for Genetics, University of Cologne, Zülpicherstrasse 47, 50674 Cologne, Germany. Tel.: +49 221 470 2467; Fax: +49 221 470 5185; E-mail: [email protected] Center for Molecular Medicine Cologne, University of Cologne, Joseph Stelzmannstrasse 9, 50931 Cologne, Germany. Tel.: +49 221 478 7738; Fax: +49 221 478 4836; E-mail: [email protected] The EMBO Journal (2008)27:2091-2101https://doi.org/10.1038/emboj.2008.141 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The lifelong self-renewal of the epidermis is driven by a progenitor cell population with high proliferative potential. To date, the upstream signals that determine this potential have remained largely elusive. Here, we find that insulin and insulin-like growth factor receptors (IR and IGF-1R) determine epidermal proliferative potential and cooperatively regulate interfollicular epidermal morphogenesis in a cell autonomous manner. Epidermal deletion of either IR or IGF-1R or both in mice progressively decreased epidermal thickness without affecting differentiation or apoptosis. Proliferation was temporarily reduced at E17.5 in the absence of IGF-1R but not IR. In contrast, clonogenic capacity was impaired in both IR- and IGF-1R-deficient primary keratinocytes, concomitant with an in vivo loss of keratin 15. Together with a reduction in label-retaining cells in the interfollicular epidermis, this suggests that IR/IGF-1R regulate progenitor cells. The expression of dominant active Rac rescued clonogenic potential of IR/IGF-1R-negative keratinocytes and reversed epidermal thinning in vivo. Our results identify the small GTPase Rac as a key target of epidermal IR/IGF-1R signalling crucial for proliferative potential and interfollicular morphogenesis. Introduction Continuous renewal of the interfollicular epidermis (IFE) is crucial for organisms to maintain and restore the skin barrier that protects them from external challenges and dehydration. This lifelong process of self-renewal is driven by a high proliferative capacity of progenitor cells, which under physiological conditions reside in the basal layer of the IFE (Ito et al, 2005; Levy et al, 2005; Kaur, 2006; Watt et al, 2006; Clayton et al, 2007). Hair follicle stem cells reside in the bulge of the hair follicle and have been well characterized at the molecular marker level (Morris et al, 2004; Tumbar et al, 2004). In contrast, the spatial localization and molecular identity of murine IFE progenitor cell are less clearly defined (Kaur, 2006; Jones and Simons, 2008). In human skin, interfollicular progenitor cells in the basal layer are distinguished by differential expression of markers such as the β1-integrin, the Notch ligand Delta and Desmoglein3, but none of these appear to spatially identify a progenitor cell population in the mouse IFE (Jones and Simons, 2008). Similarly, the identity of the niche of IFE progenitor cells is unknown, although it is likely that dermal fibroblasts constitute part of the niche, as has been shown for the hair follicle stem cells located in the bulge (reviewed in Jones and Wagers, 2008). In the epidermis, several downstream mediators, such as for example p63, the small GTPase Rac and c-Myc, have been implicated in the determination and/or maintenance of interfollicular epidermal progenitor cells and their proliferative potential (Arnold and Watt, 2001; Waikel et al, 2001; Koster et al, 2004; Benitah et al, 2005; Castilho et al, 2007; Senoo et al, 2007). However, the upstream extracellular signals and their receptors have remained largely elusive. In humans and mice, differential expression of α6- and β1-integrin, receptors for extracellular matrix, are hallmarks associated with differential proliferative potential of basal interfollicular cells (reviewed in Kaur, 2006). This suggests that cell–extracellular matrix adhesion may be one mechanism by which the environment can regulate progenitor cell maintenance. Another potential mechanism is through the IGF growth factors and their relative insulin. IGF-II has recently been implicated in the regulation of clonogenicity of human embryonic stem cells through the activation of the IGF-1R (Bendall et al, 2007). Overexpression of IGF-II in either mouse colon or epidermis also increased the number of proliferative units without a change in cell size, or in colon, crypt area (Ward et al, 1994; Bennett et al, 2003). In addition, both insulin and IGFs negatively regulate the Foxo family of transcription factors (Taniguchi et al, 2006), which have been shown to have a central function in the regulation of stem cells (Arden, 2007). Consistently, conventional inactivation of IGF signalling components, such as IGF-1R, IGF-1 and/or IGF-II (Liu et al, 1993), insulin substrate I (IRSI; Sadagurski et al, 2007) or both of the downstream kinases AKT1 and AKT2 (Peng et al, 2003) in mice result in a hypomorphic epidermis, although the mechanisms remain unclear. Complete inactivation of the closest relative of IGF-1R, the insulin receptor (IR), did not reveal any obvious skin phenotype in mice even though proliferation and differentiation were altered (Wertheimer et al, 2001). As IR and IGF-1R are also key regulators of growth, apoptosis, differentiation and metabolism (Pollak et al, 2004; Taniguchi et al, 2006), the observed epidermal phenotype in the IGF-1R pathway knockout mice could be caused by cell-autonomous changes in any of these processes in keratinocytes, or, more indirectly, by alterations in the dermis that perturb dermal–epidermal communication. Here, we show that cell-autonomous insulin and IGF-1 receptor signalling cooperatively regulate epidermal morphogenesis. Surprisingly, epidermal-specific loss of IR and/or IGF-1R did not affect epidermal differentiation or survival in vivo, whereas proliferation was only temporarily affected in epidermal development but only upon deletion of IGF-1R not IR. The data identify insulin and IGF-1 receptors as key upstream activators of the small GTPase Rac in the epidermis through which they regulate proliferative potential of keratinocytes in vitro and interfollicular morphogenesis in vivo. Results Cell-autonomous IR/IGF-1R signalling regulate epidermal morphogenesis To examine the cell-autonomous role of insulin and IGF signalling in the epidermal compartment of skin, we specifically inactivated their receptors, either the IR (IRepi−/−), the IGF-1 receptor (IGF-1Repi−/−) or both receptors (double knockout or dkoepi), in the epidermis. This was achieved by crossing mice homozygous for the respective floxed alleles (Brüning et al, 1998) for these receptors with mice expressing the Cre recombinase under the K14 promotor (Hafner et al, 2004) and carrying one floxed allele for either the IR and/or IGF-1R (Figure 1A). This results in the deletion of the floxed region in IR or IGF-1R or both at the genomic level (Figure 1B) and the absence of protein expression (Figure 1C) in the epidermal compartment. Mice with epidermal loss of IR were viable and exhibited no macroscopically detectable defects either in the epidermis (Figure 1D) or in hair follicles, as was expected based on the total IR knockout mice (Wertheimer et al, 2001). In contrast, inactivation of either the IGF-1R or the combination of IGF-1R and IR (dkoepi) in the epidermis resulted in a fragile, translucent skin (Figure 1D), with a more severe appearance in the dkoepi. All dkoepi died perinatally, whereas around 55% of the IGF-1Repi−/− mice survived, but showed occasional hair loss. Figure 1.Epidermal inactivation of insulin receptor, IGF-1 receptor affects epidermal morphogenesis. (A) PCR analysis on genomic DNA isolated from tail biopsies showing the different genotypes of the mice: 1, K14-Cre; 2, IRfl/+;IGF-1Rfl/+; 3, IRfl/fl;IGF-1Rfl/fl; 4, K14-Cre; IRfl/fl;IGF-1R+/+; 5, K14-Cre; IR+/+;IGF-1Rfl/fl; 6,K14-Cre; IRfl/fl;IGF-1Rfl/fl. (B) PCR analysis on genomic DNA from split epidermis showing the efficiency of deletion of the floxed region in either the IR or the IGF-1R locus in the presence of keratin 14-driven Cre. (C) Western blot analysis on epidermal lysates using antibodies against either the IR receptor or the IGF-1R receptor. (D) Macroscopic appearance of control mice and mice with an epidermal deletion of IR, IGF-1R or both (dko). Download figure Download PowerPoint Histochemical analysis revealed a striking hypoplastic epidermis in the IGF-1Repi−/− mice (Figure 2A), showing that the previously observed hypomorphic epidermis of the total IGF-1R knockout mice (Liu et al, 1993) results from a direct signalling defect in the epidermis itself. Surprisingly, even though the IRepi−/− mice displayed no obvious macroscopic phenotype (Figure 1D), the IFE was significantly thinner than that of control mice (Figure 2B). Deletion of both IR and IGF-1R resulted in a further decrease (Figure 2), revealing that cell-autonomous insulin and IGF-1 signalling cooperatively regulate epidermal thickness. This hypomorphic phenotype was also observed in other stratifying epithelia that express K14, such as palate or tooth anlagen (Supplementary Figure 1A) and did not deteriorate further in surviving IGF-1Repi−/− mice (Supplementary Figure 1A) or IRepi−/− mice (not shown). Thus, insulin and IGF-1 receptor signalling cooperatively regulate the number of suprabasal cell layers and thereby interfollicular epidermal morphogenesis, with a more significant contribution of IGF-1R compared to IR signalling. As the IRepi−/− mice showed no obvious hair follicle defects and the dkoepi mice died within the first 2 days, we focused our study on the role of IR/IGF-1R in regulating interfollicular morphogenesis. Figure 2.Cooperative and cell-autonomous regulation of epidermal thickness by epidermal insulin and IGF-1 receptor signalling. (A) H&E staining of paraffin sections from newborn back skin. Scale bar is 50 μm. (B) Quantification of the thickness of the epidermis (without the stratum corneum) using H&E stained sections of back skin. N=7 for each genotype. Download figure Download PowerPoint Normal differentiation in the absence of epidermal IR/IGF-1R The appearance of a hypo- or hypermorphic epidermis is often associated with impaired differentiation. This was indeed reported to be abnormal in three-dimensional skin co-culture systems with keratinocytes and fibroblasts both deficient in IGF-1R (Sadagurski et al, 2006). Using different IFE markers, we thus examined whether epidermal loss of IR or IGF-1R impaired differentiation in vivo. In the IRepi−/−, IGF-1Repi−/− or dkoepi mice, appropriate keratin 10 and loricrin expression was still observed, with keratin 10 marking the suprabasal layers and loricrin marking granular layer. However, due to the reduction in spinous and granular layers in knockout mice, the domain was increasingly smaller (Supplementary Figure 2). The basal layer marker keratin 14 was also still confined to the basal layer in all mice examined (Supplementary Figure 2). Moreover, no obvious difference in integrin α6-staining, marking the epidermal basement membrane zone, was observed, suggesting normal polarization of basal keratinocytes and contact to the basement membrane (Supplementary Figure 2). These results show that the intrinsic differentiation program is not directly affected by the loss of epidermal insulin and/or IGF-1 signalling. IR/IGF-1R signalling do not affect apoptosis in newborn epidermis Both insulin and IGF-1 can promote cell survival by the activation of AKT (Pollak et al, 2004; Taniguchi et al, 2006). In vitro studies using keratinocytes negative for either IR or IGF-1R revealed changes in AKT signalling and increased apoptosis (Wertheimer et al, 2001; Sadagurski et al, 2006). However, no change in apoptotic activity was seen in the epidermis upon inactivation of either IR or IGF-1R alone or both as assessed by either TUNEL assays (Figure 3A) or cleaved caspase 3 protein levels (not shown). Although IGF-1 signalling can stimulate AKT in keratinocytes (Haase et al, 2003; Sadagurski et al, 2006), relatively little activated AKT was detected under steady-state conditions in isolated control newborn epidermis (Figure 3F) and this was similar or even slightly increased in the absence of IR, IGF-1R or both. Strikingly, an increase in the total levels of AKT was seen upon deletion of IR, IGF-1R or both, suggesting that epidermal keratinocytes attempt to compensate for the loss of IR and/or IGF-1R. Deletion of IR, IGF-1R or both also did not obviously alter phosphorylation of a downstream target of AKT, GSK3β (Figure 3F). Figure 3.Apoptosis and proliferation in the epidermis in the absence of IR/IGF-1R. (A) TUNEL staining (green) on sections isolated from back skin of newborn mice. Nuclei were counterstained using DAPI (blue). Scale bar is 100 μm. (B) Ki67 staining on sections of back skin isolated from newborn mice. Scale bar is 100 μm. (C) Quantification of Ki67 staining in the basal cells of IFE of control and dko mice in back skin, palate or tongue epithelium. N=5 and P>0.05 for each genotype. (D) Quantification of Ki67 staining of the epidermis in embryos. N=4 mice/group, P>0.05 for E15.5 and 16.5, P<0.01 for E17.5 (E) H&E staining on paraffin sections from E15.5, E16.5 and E17.5 embryos. Scale bar is 50 μm. (F) Western blot analysis for the indicated proteins on epidermal lysates of newborn mice. Download figure Download PowerPoint IGF-1R but not IR regulates epidermal proliferation in vivo and in vitro In the epidermis, overexpression of IGF-1 or IGF-II results in hyperproliferation (Bol et al, 1997; Bennett et al, 2003), whereas MAPK activation is altered in keratinocytes deficient for IGF-1R (Sadagurski et al, 2006), thus providing a potential explanation for the hypomorphic epidermis. Surprisingly, no obvious change could be detected in staining for the proliferation marker Ki67 between control, IRepi−/−, IGF-1Repi−/− and dkoepi newborn epidermis (Figure 3B and C). As an even stronger decrease in layers was seen in the stratifying epithelia of palate and tongue, which may thus more obviously reveal alterations in proliferation, we also examined Ki67 staining in these tissues. Again, no significant difference in Ki67-positive cells was observed in control versus dkoepi palate and tongue epithelium (Figure 3C). We next wanted to determine at which developmental stage loss of IR and IGF-1R affects interfollicular epidermal morphogenesis and whether this relates to temporary changes in proliferation. As the phenotype is most obvious in the dkoepi epidermis, we focused on these embryos. E15.5 dkoepi mice showed the expected 2–3 epidermal layers that are indistinguishable from control mice (Figure 3E), with no change in proliferation (Figure 3D). The first signs of a hypomorphic epidermis in the dkoepi mice became apparent at E16.5. Whereas control mice have formed a 4–6 layer epidermis at this stage, dkoepi epidermis remained a 3–4 layered epidermis more resembling the E15.5 mice (Figure 3E). Surprisingly, no changes in Ki67 (Figure 3D) or TUNEL staining (not shown) were found at E16.5, indicating that at this stage the inability to increase the number of suprabasal layers is not due to changes in proliferation or apoptosis. At E17.5, both the control and dkoepi mice showed epidermal stratification, even though the dkoepi epidermis remained hypomorphic. This coincided with impaired proliferation (Figure 3D) but not apoptosis (not shown). In contrast, E17.5 IRepi−/− embryos, which exhibit the mildest phenotype, showed no change in proliferation even though the epidermis was hypomorphic (Supplementary Figure 3A and B). Defects in proliferation in vivo may be partially masked by mitogenic signals coming from the dermis. To directly examine the consequences of IR or IGF-1R signalling for proliferation, growth assays were performed with primary keratinocytes. Whereas IGF-1R keratinocytes did not grow in the absence of fibroblast feeders, no growth impairment was observed for the IR−/− keratinocytes in comparison to control keratinocytes isolated from littermates (Supplementary Figure 3C and D). Thus, cell-autonomous IGF-1R signalling regulates proliferation in keratinocytes but in vivo signals from the dermis most likely compensate at most time points. Recently, it was demonstrated that inactivation of Mek1/2, upstream kinases of MAPK, in the epidermis almost completely abrogates MAPK activation in newborn mice. This, as observed in dkoepi mice, is associated with a hypomorphic epidermis and changes in proliferation only during embryogenesis (Scholl et al, 2007). However, we could not detect any differences in either total or active MAP kinase levels in the epidermis of control, IR-, IGF-1R- or IR/IGF-1R-negative epidermis (Figure 3F). This demonstrates that IR and IGF-1R are not the crucial activators of the MAPK pathway in murine epidermis, consistent with results in human keratinocytes (Haase et al, 2003). Taken together, the results show that IGF-1R but not IR regulates proliferation of keratinocytes, both in vivo and in vitro, and this most likely contributes to the hypoplastic epidermis in the IGF-1Repi−/− and dkoepi mice. However, more importantly, proliferative defects cannot solely be responsible as IR epidermis is hypomorphic without proliferative changes and the dkoepi hypomorphic epidermis is already obvious at E16.5 independent of proliferation. IR and IGF-1R signalling regulate proliferative potential of primary keratinocytes Insulin and IGF may directly regulate the proliferative potential of epidermal progenitor cells, similar to that observed for IGF-II using human embryonic stem cells (Bendall et al, 2007). Therefore, we assessed the colony-forming capacity of primary keratinocytes isolated from the control, IRepi−/− or IGF-1Repi−/− mice. A strong reduction in the size of the colonies, indicative of the number of cell divisions, was observed in IGF-1R−/− keratinocytes compared to control, with an almost complete loss of large-sized colonies (Figure 4A and B). A reduction in colony size was also observed in IR−/− keratinocytes compared to control (Figure 4A and B), although not as dramatic as the IGF-1R−/−. This is in line with the in vivo results showing that loss of the epidermal IR affects the thickness of the epidermis less severely than the loss of IGF-1R (Figure 2). When increasing colony size is plotted as a continuum against accumulative percentage of colonies, one could identify two different curves in control keratinocytes, one with a relative flat slope that represents 90% of all colonies and another where the steepness of the slope dramatically increases over the last 5–10% (Figure 4C). This indicates the presence of two different cell populations, one that represent over 90% of the colonies that have a similar, relatively low proliferative potential, and a second one, representing around 5–10% of the colonies with a much higher proliferative potential. This population likely represents epidermal progenitor cells. When comparing the curve for IGF-1R−/− cells the overall angle of the initial slope is less than in control, indicating that proliferative potential is reduced in all cells. In fact, for these cells the steepness of the curve remained unchanged, indicating that the population with high proliferative potential is almost completely absent in these cells (Figure 4C). Figure 4.Insulin/IGF-1R signalling affects the in vitro proliferative potential of keratinocytes. (A) Colony-forming assay using primary keratinocytes isolated from control, IRepi−/− or IGF-1Repi−/− mice. (B) Quantification of the colony-forming assays shown in (A). (C) Colonies of the control versus IGF-1R−/− keratinocytes plotted as increasing colony size against accumulating percentage of colonies. Download figure Download PowerPoint Regulation of progenitor markers by IR and IGF-1R To examine whether alterations in proliferative potential were affecting epidermal progenitor cells, we used the progenitor cell marker keratin 15 (K15). Both western blot analysis (Figure 5A) and real-time PCR analysis (Figure 5B) showed a dramatic reduction in the overall K15 protein and mRNA levels in dkoepi epidermis compared with control. Indeed, other epidermal stem/progenitor cell markers, such as Igfbp-5 (Blanpain et al, 2004) were reduced in dko epidermis compared with control (Figure 5B), providing further evidence that insulin and IGF-1 receptor signalling regulate an epidermal progenitor cell compartment. Figure 5.Expression of epidermal progenitor cell markers. (A) Western blot analysis for keratin 15 on lysates isolated from epidermis of newborn mice. Same amount of lysates was run on a separate gel and probed for actin to control for loading. (B) Real-time PCR analysis of epidermis for the indicated markers. N=5 for both control and dko. (C) Keratin 15 (green) staining on back skin of newborn mice. Nuclei were counterstained using propidium iodide (red). Scale bar is 100 μm. Inset shows high magnification of basal cell layer. (D) Quantification of K15-positive basal IFE cells and HF in a 500 μM area of control, IRepi−/−, IGF-1Repi−/− and dko epidermis. N=4 independent mice per genotype with 5–6 sections per mouse. (E) FACS analysis of CD34 expression on ctr, IGF-1R−/− and dko primary keratinocytes. The average expression of four independent experiments is shown. See Supplementary Figure 4A for a representative profile. Download figure Download PowerPoint A recent study suggested that the colonies formed in the clonogenic assay are derived from the HF stem cells (Langton et al, 2008). Although they used number, and not size, of colonies as a read-out, this suggests that IR/IGF-1R may exert their effect on proliferative potential by affecting HF stem cells. Unlike IFE progenitor cells, HF stem cells are well characterized at the molecular level (Morris et al, 2004; Tumbar et al, 2004). Indeed, K15 is commonly used as a marker for hair follicle stem cells, although in newborn mice K15 is also expressed in basal interfollicular keratinocytes (Liu et al, 2003). We therefore assessed the epidermal compartment in which the loss of K15 occurred. A strong decrease in K15 staining was observed in IR-negative IFE compared to hair follicles (Figure 5C and D). In the absence of IGF-1R or in the dkoepi mice, K15 was strongly reduced in both compartments (Figure 5C). However, in these two mutants the decrease in K15 was also more pronounced in the IFE compared to hair follicles (Figure 5D). This suggested that IR mainly affects the interfollicular compartment, whereas IGF-1R signalling affects both progenitor cell compartments. Nevertheless, several other HF-specific stem cell markers such as CD34, Tcf4 and CdKn1b (Blanpain et al, 2004; Morris et al, 2004), were not changed in dkoepi compared to control, implying that the number of HF stem cells remained similar (Figure 5B). In addition, using FACS analysis, similar levels of expression were found for CD34 on control, IGF-1R or dko primary keratinocytes, suggesting that also in vitro no obvious loss of HF stem cells occurs (Figure 5E). Reduction in IFE label-retaining cells in IGF-1Repi−/− mice Progenitor cells are characterized by their ability to retain 5-bromo-2′-deoxyuridine (BrdU) for prolonged times after injection, both in the IFE and hair follicles. As dko mice die within the first 2 days, we assessed this property in IGF-1Repi−/− and control mice by injecting them for three consecutive days with BrdU and examining BrdU-positive cells after a chase of 15, 40 and 70 days. Compared to control a 80% reduction of BrdU-positive basal cells were found in the IFE of IGF-1Repi−/− mice after 15 days, whe