Epidermal Deletion of Rac1 Causes Stem Cell Depletion, Irrespective of whether Deletion Occurs during Embryogenesis or Adulthood

SA Benitah and FM Watt Epidermal Deletion of Rac1

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Epidermal Deletion of Rac1 Causes Stem Cell Depletion, Irrespective of whether Deletion Occurs during Embryogenesis or Adulthood Journal of Investigative Dermatology (2007) 127, 1555–1557. doi:10.1038/sj.jid.5700738; published online 15 February 2007

TO THE EDITOR We recently reported that the Rho GTPase Rac1 is essential for maintenance of the adult epidermal stem cell compartment (Benitah et al., 2005). We topically applied 4-hydroxy-tamoxifen (4OHT) to 8-week-old mice to induce deletion of Rac1 in epidermal cells expressing Cre recombinase fused to a mutant estrogen receptor under the control of the keratin 14 promoter (K14CreER; Lo´pez-Rovira et al., 2005). As illustrated in Figure 1a–d, the interfollicular epidermis underwent a welldefined series of changes, with an initial increase in proliferation (early phenotype; Figure 1a, b), followed by premature terminal differentiation (middle phenotype; Figure 1c), and then local loss of all viable cell layers (late phenotype; Figure 1d; Benitah et al., 2005). In addition to the progressive and irreversible changes in the interfollicular epidermis, the sebaceous glands and hair follicles degenerated. We showed that the changes were a consequence of stem cell depletion, as judged by loss of cells with several stem cell characteristics (clonogenicity, DNA label retention, expression of CD34 and keratin 15). Treatment of Rac1flox/flox or K14CreER/Rac1wt/wt control mice with 4OHT or acetone (vehicle) did not produce any changes in epidermal histology (Figure 1a and data not shown; Benitah et al., 2005). In contrast to our results, it has recently been reported that deletion of

Rac1 via Cre recombinase expressed under the control of the keratin 5 promoter (K5Cre) has no effect on the interfollicular epidermis, even though hair follicle integrity is compromised (Chrostek et al., 2006). We found this result surprising because the K5 and K14 promoters have very similar strengths and patterns of expression (Byrne et al., 1994; Ramı´rez et al. 2004; Lo´pez-Rovira et al., 2005). Both promoters are strongly upregulated by E14.5 of embryonic development and we would have predicted that K5Cre deletion of Rac1 in the embryo would have a more severe effect than deletion in adulthood (Lo´pez-Rovira et al., 2005). There are certainly precedents for gene deletion resulting in different phenotypes in different laboratories, and in some cases this can be accounted for by differences in the genetic background of the knock out mice (Sibilia et al., 1998). A further issue is the potential for artefacts arising from the use of 4OHT-dependent Cre. Chrostek et al. (2006) reported that 4OHT treatment caused changes in the histological appearance of wildtype mice. Although this has never been our experience (Figure 1a; Frye et al., 2003; Benitah et al., 2005; Silva-Vargas et al., 2005), topical 4OHT can cause changes in gene expression in wild-type mice (Frye et al., 2003; Watt, unpublished observations). K14CreER is leaky in older mice (Vasioukhin et al., 1999;

Abbreviations: 4OHT, 4-hydroxy-tamoxifen; Tam, tamoxifen

Bleul et al., 2006) and, conversely, 4OHT treatment is not always 100% effective at deleting the floxed gene via CreER (Lo´pez-Rovira et al., 2005; SilvaVargas et al., 2005). As the phenotype of the K14CreER/ Rac1flox/flox mice has led us to propose that Rac1 and Myc represent a global stem cell regulatory axis (Benitah et al., 2005), we felt it was important to establish whether this was true irrespective of the method used to express Cre in the basal layer of the epidermis. To determine whether the epidermal Rac1 deletion phenotype was affected by the chemical composition or delivery route of the Cre inducing agent, we compared mice that had received topical applications of 4OHT (Figure 1a–d) with mice that received intraperitoneal injections of 5 mg of tamoxifen (Tam) dissolved in peanut oil. Tam was administered on days 0 and 7. Four mice were killed at day 7, and four at day 9. Mice could not be examined at later time points because their health deteriorated, as a consequence of cumulative effects of deleting Rac1 in the epidermis and internal epithelia (data not shown). Intraperitoneal injection of Tam caused very similar changes to those induced by topical application of 4OHT (Figure 1e–h). The only difference was that the phenotype appeared more rapidly, and by 7 days 6/8 mice had developed the middle and late phenotypes. One mouse exhibited complete loss of all epidermal structures, including the cornified layers, in large areas of the skin (Figure 1h). Intraperitoneal administration of Tam www.jidonline.org 1555

SA Benitah and FM Watt Epidermal Deletion of Rac1

to control Rac1flox/flox mice did not affect the histological appearance of the epidermis (Figure 1e). Experiments on mice were subject to ethical approval by the Cancer Research UK London Research Institute and performed under the terms of a UK Government Home Office license. As K14CreER is leaky in older mice (Vasioukhin et al. 1999; Bleul et al., 2006), we examined whether any mice aged 6–8 months had evidence of the

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mice had regions in which the interfollicular epidermis had been lost, with attendant inflammation, similar to the late phenotype induced by topical application of 4OHT (Figure 1k). We next investigated the effects of Rac1 deletion during epidermal embryonic development. We crossed Rac1flox/flox mice with the same K5Cre transgenic mouse line (Ramı´rez et al., 2004) used by Chrostek et al. (2006). We intercrossed K5Crehomo/Rac1flox/wt mice to obtain K5Crehomo/Rac1flox/flox animals. Only 21 out of 224 mice born from the crosses were K5Cre/Rac1flox/flox, corresponding to 9% of live births, rather than the expected Mendelian ratio of 25%. This suggests that the genotype resulted in some embryonic lethality. Eight out of 21 mice (38%) died before reaching 6 weeks of age, and a further 11 had to be killed between 6 and 12 weeks after birth because of the severity of their phenotype. A high percentage of K5Cre/ Rac1flox/flox mice (90%) were runted and never developed hair, and 71% developed skin inflammation and spontaneous wounds. Lesions were most frequent around the neck and eyes, ears, and upper abdomen. In all K5Cre/Rac1flox/flox mice, there was marked thickening of the interfollicular epidermis with hyperkeratosis and increased inflammatory infiltrate compared with control (K5Cre/Rac1wt/wt or

Rac1 deletion phenotype in the absence of 4OHT treatment (Figure 1i–k). Five out of sixteen 6- to 8-month-old K14CreER/Rac1flox/flox mice showed patchy hair loss on the neck, ears, and back. These areas contained thickened interfollicular epidermis (Figure 1i, j), expansion of the sebaceous glands, and infundibulum cysts (Figure 1j), all features of the early and middle phenotypes resulting from 4OHT-induced deletion of Rac1. Two 8-month-old

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Figure 1. Inducible deletion of Rac1 causes irreversible loss of the epidermis. Representative hematoxylin and eosin stained histological sections of the phenotypes observed upon inducible deletion of Rac1. (a) K14CreER (b–d) or K14CreER/Rac1flox/flox mice were treated with 4OHT (3mg/200 ml acetone) 3 days a week during 3 weeks. (b) Corresponds to day 7, (c) to day 11, and (a, d) to day 16 of 4OHT treatment. (e–h) Intraperitoneal administration of Tam (5 mg/100 ml peanut oil). (e) Corresponds to Rac1flox/flox mice collected at day 9 of treatment; (f) correspond to K14CreER/Rac1flox/flox mice collected at day 7 and (g, h) 9 of treatment. (i) Six-month-old control and (j) 6- or (k) 8-month-old K14CreER/ Rac1flox/flox mice that had not been treated with 4OHT. Bar: 100 mm.

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Figure 2. Embryonic deletion of Rac1 induces similar epidermal changes to deletion in adult epidermis. (a–f) Four-week-old K5Cre/Rac1flox/flox mice (b–f) and K5Cre/Rac1flox/wt control littermate (a). (g–j) Tail epidermal whole mount immunofluorescence of Rac1flox/flox (g, i) and K5Cre/Rac1flox/flox littermates (h, j) stained with the antibodies indicated (green). (i, j) Blue fluorescence is 40 ,6-diamidino-2-phenylindole dihydrochloride counterstain; red fluorescence is Nile Red. Bars: (a–f) 100 mm, (g–j) 20 mm.

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SA Benitah and FM Watt Epidermal Deletion of Rac1

Rac1flox/flox) littermates (Figure 2a–c). In 19/21 mice, we found areas of skin with few hair follicles and a thin epidermis consisting largely of cornified cells, resembling the late phenotype of K14CreER/Rac1flox/flox mice treated topically with 4OHT or intraperitoneally with Tam (Figures 2d and 1j). Most of the remaining hair follicles developed infundibulum cysts in K5Cre/ Rac1flox/flox mice (Figure 2b). 2/21 K5Cre/Rac1flox/flox mice remained healthy at 4 months of age, even though they were runted and had lost their hair. Histologically, the skin of these mice showed regions of epidermal hyperproliferation and hair follicle degeneration, but lacked spontaneous wounds and degeneration of the viable epidermal layers (Figure 2e, f). These mice therefore exhibited only the early and middle phases of the Rac1 deletion phenotype. Whole mounts of tail epidermis from the severely affected K5Cre/Rac1flox/flox mice showed changes similar to those described in 4OHT-treated K14CreER/ Rac1flox/flox mice (Benitah et al., 2005; Braun et al., 2003). Most of the hair follicles had a thickened infundibulum, very small sebaceous glands and showed degeneration of the bulb (Figure 2g–j). Expression of the a6 integrin, which is normally highly expressed in the hair follicle bulge, was reduced, indicative of stem cell depletion (Figure 2g, h). There was also a reduction of the level of K14 expression in basal cells in the HF, which was accompanied by degeneration and loss of sebaceous glands (Figure 2i, j). We conclude that loss of Rac1 from the epidermis during embryogenesis

has similar consequences to adult epidermal deletion of Rac1 (Benitah et al., 2005). One possible reason for the differences between our results and those of Chrostek et al. (2006) is that the phenotype is modified by genetic background (Sibilia et al., 1998; Chrostek et al., 2006). A second possibility is incomplete Rac1 deletion, something that could be evaluated by using Rosa26 reporter mice (Lo´pez-Rovira et al., 2005) or by performing immunostaining for Rac1 (Benitah et al., 2005) rather than relying on Western blots of whole skin (Chrostek et al., 2006). Finally, as Chrostek et al. (2006) acknowledge, by flanking exon 3 of the rac1 gene with LoxP sites, it is possible that a truncated protein containing exons 1 and 2 is made. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We are grateful to everyone who provided advice, reagents, and technical assistance. This work was supported by Cancer Research UK and the EuroStemCell FP6 programme. SAB was the recipient of an EMBO long-term fellowship and an EU Marie Curie Fellowship.

Salvador A. Benitah1,2 and Fiona M. Watt1,3 1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, London, UK E-mail: [email protected] 2 Current address: ICREA, Center of Genomic Research, Barcelona, Spain. 3 Current address: CR-UK Cambridge Research Institute, Cambridge, UK.

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