c-Myc is involved in the control of diverse cellular processes and implicated in the maintenance of different tissues including the neural crest. Here, we report that c-Myc is particularly important for pigment cell development and homeostasis. Targeting c-Myc specifically in the melanocyte lineage using the floxed allele of c-Myc and Tyr::Cre transgenic mice results in a congenital gray hair phenotype. The gray coat color is associated with a reduced number of functional melanocytes in the hair bulb and melanocyte stem cells in the hair bulge. Importantly, the gray phenotype does not progress with time, suggesting that maintenance of the melanocyte through the hair cycle does not involve c-Myc function. In embryos, at E13.5, c-Myc-deficient melanocyte precursors are affected in proliferation in concordance with a reduction in numbers, showing that c-Myc is required for the proper melanocyte development. Interestingly, melanocytes from c-Myc-deficient mice display elevated levels of the c-Myc paralog N-Myc. Double deletion of c-Myc and N-Myc results in nearly complete loss of the residual pigmentation, indicating that N-Myc is capable of compensating for c-Myc loss of function in melanocytes.
Deregulation of c-Myc is a common event in cellular transformation and is also found in a variety of primary and metastatic cutaneous melanomas where it has been associated with a poor prognosis. Here, we focused on the role of c-Myc in pigment cell development and homeostasis after birth, and we specifically delete this gene in the melanocyte lineage using Tyr::Cre transgenic mice. Removal of c-Myc during melanocyte development affects the proliferation of melanoblasts in the epidermis, which are then reduced in number. This leads to a gray hair phenotype, which is not progressive, in contrast to human hair graying, or removal of the Notch signaling pathway. These data add c-Myc to the genes critical for the development of the melanocyte lineage and will provide additional arguments for targeting c-Myc expression in melanoma.
Melanocytes are neural crest–derived pigment cells that are responsible for coat color in many species. In mammals, mainly located in the epidermis of the skin, interfollicular and hair follicular melanocytes produce and transfer pigment to the surrounding keratinocytes, thus defining coat color and protecting skin from UV irradiation. Cycles of hair growth involve repeated phases of proliferation, differentiation, and apoptotic events (Paus and Cotsarelis, 1999). At every cycle of hair regeneration, melanocyte stem cells (MSCs) are activated and generate a new population of differentiated cells to fill the hair follicle bulb and ensure the pigmentation of the hair follicle shaft. MSCs have been identified as Dct-positive cells in the bulge region of the hair follicle and have been intensively studied using the Dct::LacZ reporter mouse line (Nishimura et al., 2002).
During embryogenesis, prespecified melanocyte precursors delaminate from the dorsal part of the neural tube with other neural crest derivatives such as neurons and glial cells, cardiac cells, facial cartilage, and bone. In the traditional view, after leaving the neural tube, future pigment cells follow the dorsolateral pathway to reach their final destination at different sites of the mouse body. Recent findings, however, suggest that at least a part of the melanocyte lineage is derived from Schwann cell precursors taking the ventral pathway (Adameyko et al., 2009). Before being engaged in the complete pigmentary unit of the hair follicle, melanoblasts migrate through the dermis and begin to enter the epidermis around day 11 of gestation (Luciani et al., 2011) where they undergo numerous proliferative stimuli and eventually become incorporated into the developing hair follicle.
Many genes have been implicated in specific aspects of melanocyte/melanoblast proliferation/migration/differentiation, and more than 370 loci are identified in the mouse affecting pigmentation (Bennett and Lamoreux, 2003; Lamoreux et al., 2010; Montoliu et al., 2012). The cellular mechanisms underlying the highly patterned neural crest cell migration and differentiation are mainly based on the expression of cell surface receptors (e.g., Kit) and the activity of their respective ligands. Many of the factors involved in the proliferation and differentiation of the melanocyte lineage act synergistically at specific stages of embryonic development. In addition to the genes involved in signal transduction pathways (as Kit, SCF, Ednrb, and Wnt), others are transcription factors (as Mitf, Pax3, Sox10), or genes modulating melanin production (as Mc1r, ASP) (Hou and Pavan, 2008; Robinson and Fisher, 2009; Uong and Zon, 2010). A number of genes were shown to contribute to the maintenance of MSCs within the niche; namely, Bcl2 acts downstream of microphthalmia-associated transcription factor (MITF) as a survival factor for MSCs (Mak et al., 2006; Nishimura et al., 2005) and TGFβ signaling regulates MSC immaturity and quiescence (Nishimura et al., 2010). Finally, Notch signaling appears to be crucial for MSC maintenance and, when inactivated, leads to the progressive loss of MSCs during the hair regeneration cycle (Aydin and Beermann, 2011; Moriyama et al., 2006; Schouwey et al., 2007, 2010). The Wnt/β-catenin signaling pathway has been shown to control the specification and survival of pigment cells (Dorsky et al., 1998; Hari et al., 2002), and is important in establishing the melanocyte lineage by regulating melanoblast proliferation (Luciani et al., 2011). One of the targets of Wnt/β-catenin signaling is MITF, encoding for a member of the Myc supergene family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors, and it is known to play a pivotal role in melanocyte development and homeostasis being implicated in the maintenance and self-renewal of MSCs and in the control of proliferation and differentiation of pigment cells (Carreira et al., 2005, 2006; Lang et al., 2005; Levy et al., 2006; Nishimura et al., 2005).
c-Myc is another downstream target of Wnt/β-catenin signaling and belongs to the family of bHLH-Zip transcription factors. c-Myc was one of the first oncogenes identified and possesses a very broad spectrum of target genes controlling many physiological processes including cellular growth, proliferation, differentiation, and apoptosis and significantly contributes to tumorigenesis (Gustafson and Weiss, 2010; Meyer and Penn, 2008). Two other members of the Myc family, N-Myc and L-Myc, are closely related to c-Myc and have been implicated in the genesis of specific tumors (Nesbit et al., 1999). Besides the role of c-Myc in oncogenesis, its function during development and normal tissue homeostasis is poorly understood. While L-Myc appeared to be dispensable for mouse development (Hatton et al., 1996), c-Myc-deficient embryos do not survive day 10.5 of gestation owing to placental insufficiency (Charron et al., 1992; Davis et al., 1993; Dubois et al., 2008). N-Myc is highly expressed during embryogenesis, and its homozygous deletion leads to embryonic lethality at mid-gestation. In non-mammalian vertebrates (amphibians and fish), c-Myc was shown to be important for the maintenance of the neural crest. c-Myc is expressed at the neural plate border during Xenopus development, and morpholino knockdown of c-Myc leads to the downregulation of neural crest markers and results in the loss of neural crest derivatives including melanocytes. Consequently, it has been suggested that c-Myc is important for neural crest cell fate specification and might be a key factor to prevent the premature cell differentiation or cell fate decision (Bellmeyer et al., 2003). Expression of c-Myc during zebrafish development is restricted to the dorsal and ventral domains at gastrulation and is equally required for neural crest survival and eye development (Hong et al., 2008). Conditional deletion of murine c-Myc using the Wnt1::Cre transgenic line results in viable mice with different defects linked to neural crest cells, namely coat color, skull frontal bone, and middle ear ossicle development (Wei et al., 2007). Moreover, it was shown that c-Myc is expressed in melanoblasts at E15.5 (Colombo et al., 2012). Therefore, c-Myc appears to play an essential role during embryogenesis including different lineages of the neural crest. Here, we addressed the role of c-Myc in mouse neural crest–derived pigment cells, the melanocytes. For this purpose, a conditional allele of c-Myc (Trumpp et al., 2001) was used in combination with the melanocyte-specific Tyr::Cre transgenic line allowing for c-Myc ablation specifically in the melanocyte lineage from embryonic day 10.5 onwards (Delmas et al., 2003). This results in a gray hair phenotype, which can be seen directly after birth.
Gray hair owing to melanocyte-specific inactivation of c-Myc
Complete loss of c-Myc during embryogenesis results in embryonic death before E10.5 because of multiple developmental defects affecting the embryo size, heart, pericardium, neural tube, and blood cells, with most of these effects being a consequence of placental insufficiency (Charron et al., 1992; Davis et al., 1993; Dubois et al., 2008). To address the role of c-Myc in the melanocyte lineage, we specifically deleted the gene in pigment cells. Mice carrying a conditional allele of c-Myc (c-Mycflox) (Trumpp et al., 2001) were mated to the Tyr::Cre mouse line, enabling recombination specifically in the melanocyte lineage starting from 10.5 days of gestation (Delmas et al., 2003) (Figure 1A). Heterozygous loss of c-Myc in Tyr::Cre; c-Mycflox/+ animals did not reveal any phenotype when compared to wild-type mice or mice carrying only Tyr::Cre (not shown), revealing as expected the recessivity of the mutation. Tyr::Cre; c-Mycflox/+ (hereafter control) mice were used as a control for further experiments. In contrast, homozygous deletion of c-Myc in Tyr::Cre; c-Mycflox/flox (hereafter MyccKO) mice caused a dilution of coat color, which was observed shortly after birth, with the beginning of pigmentation, and led to a gray hair phenotype in adult animals (Figure 1B). The gray color of MyccKO coat is comprised by a mixture of white and different degree of gray hair (Figure S1A–D). Measurement of melanin concentration in the truncal dorsal hair (Figure 1D) showed a significant reduction in melanin content in the MyccKO mice. Moreover, histological examination of the skin revealed that the bulbs of the MyccKO hair follicles are less pigmented or unpigmented (Figures 1E and S1A, C), thus resulting in the dilution of coat color. Most importantly, this phenotype remained constant throughout life, and 1-year-old mice showed a similar degree of graying (not shown). In addition, we estimated the quantity of melanin in dorsal truncal fur at different time points. No significant change in melanin amount was observed through the life of animals (Figure 1F). This confirms that the gray hair phenotype of MyccKO mice does not progress with time, suggesting no additional effect of c-Myc loss after birth.
Because pigmentation was reduced but not absent, we performed further experiments to address the efficiency of recombination. To trace recombined melanocytes/melanoblasts during development, we crossed MyccKO mice to R26R::LacZ reporter mice. In parallel, we mated MyccKO mice to Dct::LacZ mice to follow cells of the melanocyte lineage (Mackenzie et al., 1997). At E14.5, a reduced number of recombined melanoblasts is seen in MyccKO embryos (Figure 2B). Importantly, this decrease is similar to the one observed in the MyccKO, Dct::LacZ embryos (Figure 2A). Altogether, these results suggest that c-Myc is properly defloxed in melanoblasts. In MyccKO; R26R::LacZ newborn mice, immunolabeling for β-Gal and Pax3 revealed that cells of the melanocyte lineage (Pax3 is used as a melanocyte marker) in the bulb and bulge of the hair follicle were LacZ-positive (Figure 2C, D). This suggests that cells of the melanocyte lineage having previously expressed Cre are still present in MyccKO animals. Expression analysis of isolated differentiated melanocytes later confirmed a more than eightfold reduction in c-Myc mRNA levels in cells isolated from c-MyccKO newborn skin (see Figure 6). This therefore suggests that the majority of melanocytes present after birth are c-Myc deficient. We therefore conclude that the deletion of c-Myc in melanocyte precursors/melanoblasts leads to a congenital non-progressive hair graying phenotype after birth.
Melanocytes in gray mice are present but reduced in number
The hair follicles of MyccKO mice show less pigmentation when compared to the control littermates. This could imply several reasons starting with simple reduced number of melanocytes and finishing by the improper function of c-Myc-deficient melanocytes because of defects in melanin production and/or transfer. It appears that MyccKO melanocytes were able to produce pigment and transfer it to the hair cortex because the majority of hairs, even in old animals, are partially pigmented (Figures 1 and S1A, C not shown). Nevertheless, the possibility of impaired pigment machinery in c-Myc-deficient melanocytes cannot be formally excluded. When we analyzed the expression of melanocyte-specific markers (Pax3, Mitf, Tyr, Tyrp1 (not shown), and Dct) in the skin of MyccKO and control mice (Figure 3A–D), these proteins were present in melanocytes of mutant skin, providing another evidence for MyccKO melanocytes to maintain their functional identity. However, the number of positive cells was reduced. The number of differentiated melanocytes in MyccKO animals was also calculated as the number of MITF-positive cells per bulb of hair follicle and appeared to be three times lower than in control skin (Figure S1E). We thus suggest that the major reason for the observed gray hair phenotype of MyccKO is a lower number of differentiated cells residing in the hair follicle.
In human, hair graying is associated with simultaneous reduction in both differentiated cells in the bulb and melanocyte progenitors in the bulge of the hair follicle. We thus analyzed the presence of melanocytes in the bulge area, where stem cells reside (Nishimura et al., 2002). Dct- and Pax3-positive MSCs (Lang et al., 2005; Nishimura et al., 2002) are still present in the bulge of hair follicles, but similar to the differentiated cells, fewer of them could be seen in the MyccKO skin (Figure 3C, D). We then analyzed whether the maintenance of these melanocyte precursors is affected similarly to differentiated melanocytes in the bulb. Using the Dct::LacZ reporter mouse strain (Mackenzie et al., 1997), we further quantified the number of melanocyte precursors in the bulge region in the skin of 8- and 30-day-old mice. At these stages, the hair follicle is in anagen (Muller-Rover et al., 2001) and MSCs have returned to the dormant stage (Nishimura et al., 2002). At P8, the number of LacZ-positive cells in the bulge of the MyccKO skin was threefold reduced as compared to the control (Figure 3E, F). Through the following hair cycle, at P30, the number of MSCs in the MyccKO skin was equally reduced about three times (Figure 3E, F) and thus not different from P8. This indicates that c-Myc deficiency did not affect the maintenance of melanocyte progenitors explaining the stability of the gray hair phenotype. Indeed, we did not observe a hair cycle–associated dilution of the coat pigmentation in MyccKO mice, which is in contrast to the progressive hair graying and loss of stem cells in mice lacking the Notch signaling pathway in melanocytes (Schouwey et al., 2007). We thus conclude that similar to the hair graying in human, the postnatal reduction in differentiated pigment cells in MyccKO mice is attributable to fewer progenitors found in the bulge region of the hair follicle and at least partially accounts for the gray hair phenotype of the MyccKO mouse.
Melanoblast proliferation is affected by c-Myc deletion
Deletion of c-Myc in the melanocyte lineage leads to gray hair directly after birth. Here, we suggested that this phenotype is attributable to a reduced number of pigmented cells in the hair follicle. In addition, when crossed to Rosa26::LacZ mice, MyccKO mice demonstrated diminished melanoblast number at E14.5 (Figure 2B). This indicated that c-Myc is important during melanocyte development. We used the Dct::LacZ reporter line to evaluate the number of melanoblasts at different time points of gestation from E10.5 to E16.5 (Figure 4A–F). A first significant decrease in melanoblast number was detected at E12.5 (P < 0.0001). This did not change until E14.5 when a further reduction (30% of melanoblasts left in MyccKO embryos) was observed. From this time point, the number of cells was not further decreased (Figure 4F) and, accordingly, is reflected by the number of melanocyte progenitors detected at 8 days after birth (Figure 3E, F); notably, the period between E12.5 and E 13.5 coincides with the main melanoblast migration from the dermis to the epidermis (Yoshida et al., 1996). We therefore analyzed the distribution and number of melanoblasts in the dermal and epidermal parts of the skin in E13.5 embryos using the Dct::LacZ reporter mouse (Figure 5A, B, D). In the MyccKO embryos, the number of dermal melanoblasts was not significantly different from the control, whereas the epidermal population of MyccKO cells was 2.5-fold decreased (Figure 5D), suggesting that c-Myc is required to establish a normal population of epidermal melanoblasts at this stage.
c-Myc might influence melanocyte development by different mechanisms. For example, melanoblasts might undergo apoptosis, have a proliferation defect, or do not migrate properly from the dermis toward the epidermis upon c-Myc loss. However, TUNEL assays did not detect any change in the number of apoptotic events in melanoblasts, neither in the epidermis nor in the dermis (not shown). As the number of dermal melanoblasts was not changed and no accumulation of pigment cells was seen in the dermis, we consider it unlikely that partial loss of epidermal melanoblasts is predominantly caused by a migration from the dermis to the epidermis. To assess the proliferation properties of c-Myc-deficient melanoblasts, we then performed BrdU incorporation assays at E13.5. The percentage of BrdU-positive melanoblasts in the dermis was similar in the control and MyccKO embryos (Figure 5C, E). Interestingly, the number of proliferating cells in the control was increased twofold after entering the epidermis (Figure 5E), suggesting the presence of a stimulating signal in the epidermis. In contrast, in MyccKO embryos, melanoblasts in the epidermis showed a ∼50% reduction in BrdU uptake in comparison with the control (Figure 5E). This therefore suggests that the drop in melanoblast number as seen at E14.5 is preceded and caused by a proliferation defect in epidermal melanoblasts. In summary, these results highlight c-Myc to be a crucial factor for melanoblasts and their proliferation upon entry into the epidermis, one of the important steps during melanocyte development.
FACS of differentiated postnatal melanocytes reveals c-Myc deletion and elevated levels of N-Myc
Melanocytes represent a rare population of cells in the skin and thus have to be purified to perform molecular analyses. It has previously been reported that melanocytes can be isolated as CD45−/c-kit+ population from the embryonic skin (Yoshida et al., 1996). Here, we applied a similar approach to analyze the melanocytes in the skin of newborn mice. While unpigmented as newborns, mice develop fully pigmented coat by 4 days after birth (P4) (Figure S2A). Concomitantly, FACS analysis of total skin from P0, P2, and P4 wild-type mice was carried out. Single cell distribution of total dorsal skin showed an increase in the population of highly granular cells, which is characterized by high side scatter (SShigh) (Figure S2B). The same increase can be seen within the CD45−/c-kit+ population (Figure S2D). Knowing that differentiated melanocytes bear mature melanosomes, these cells should be more granular than unpigmented progenitors and thus might be characterized as a subpopulation of granular c-kit-positive cells (CD45−/c-kit+/SShigh). This is supported by another study, where melanocytes were isolated using Dct::Cre × CAT::eGFP transgenic mice (Nishikawa and Osawa, 2007; Nishikawa-Torikai et al., 2011), with c-kit expressing cells having high side scatter. To confirm our assumption, we then used Tyr::DT-A transgenic mice that express an attenuated form of the diphtheria toxin-A gene in melanocytes (Camacho-Hübner and Beermann, 2001). These mice lack pigmentation, as evident already a few days after birth (Figure S4A). Immunostaining against Pax3 and Dct confirms that these mice lack both differentiated melanocytes in the bulb and unpigmented progenitors in the bulge of hair follicles (Figure S4B). SShigh granular cells of Tyr::DT-A newborns were almost absent from the skin (arrow, Figure S4C), with a decrease in the total amount of CD45−/c-kit+ cells (Figure S4D, F). Further analysis of the granular CD45−/c-kit+/SShigh population showed that these cells are lost in Tyr::DT-A mice (Figure S4E, G). The CD45−/c-kit+/SSlow population still remains and probably represents a mix of melanocyte progenitors and other cell types (Figure S4E, G). From this, we concluded that the population of CD45−/c-kit+/SShigh cells belongs to the melanocyte lineage and represents differentiated melanocytes.
We then analyzed melanocytes from MyccKO mice after birth, and it was evident that the differentiated melanocytes in the SShigh population were affected (Figure 6A), and the CD45−c-kit+ and CD45−/c-kit+/SShigh population was reduced in comparison with the control (Figure 6B, C). In order to demonstrate that the remaining melanocytes in the MyccKO skin are c-Myc deficient, we isolated the CD45−/c-kit+/SShigh population from MyccKO mice by FACS. qRT-PCR analysis of cDNA from cells of the CD45−/c-kit+/SShigh population of MyccKO mice revealed a more than eightfold reduction in c-Myc mRNA in comparison with the control (Figure 6D). This provides further evidence that the recombination efficiency is high, and we thus suggest that remaining melanocytes in MyccKO mice are c-Myc deficient.
N-Myc is another member of the Myc family and is the closest structural and functional homolog of c-Myc. Previously, it has been shown that c-Myc and N-Myc can be co-regulated during embryogenesis and that the level of c-Myc mRNA was elevated in N-Myc-deficient embryos (Stanton et al., 1992). Analysis of N-Myc transcript level in FACS-sorted c-Myc-deficient melanocytes (CD45−/c-kit+/SShigh) revealed a two- to threefold upregulation (Figure 6E). This suggests that N-Myc might be involved in the maintenance of embryonic melanocyte numbers and hence explain the residual coat color of MyccKO animals.
N-Myc upregulation partially compensates for c-Myc deficiency in pigment cells
In order to clarify the possible role of N-Myc in pigment cells, we generated melanocyte-specific conditional knockout mice for the N-Myc gene, N-MyccKO, using the N-Mycflox mouse (Knoepfler et al., 2002) and the melanocyte-specific Tyr::Cre transgenic line (Delmas et al., 2003). Homozygous deletion of N-Myc had no obvious effect on the coat pigmentation (not shown), most probably due to the contribution of c-Myc function. On the other hand, the elevated level of N-Myc in c-Myc-deficient melanocytes might represent a compensatory mechanism. To address whether N-Myc is indeed able to substitute for c-Myc function in melanocytes, we generated double conditional knockout mice (c-MyccKO/N-MyccKO, hereafter dKO). The pigmentation phenotype of these mice (dKO) appeared to be more severe exhibiting coat color as very light gray (Figure 7A). Histological examination of the skin demonstrates the absence of nearly all pigment cells in the bulb of hair follicles (Figure 7C). Melanin content in the dorsal hair of dKO mice was close to albino mice (Tyrc) (Figure 7B), indicating that melanocytes are efficiently eliminated from the dorsal skin of dKO (c-MyccKO/N-MyccKO) mice. Using the Dct::LacZ reporter mouse line, we confirmed a further reduction in the number of melanoblasts at E14.5 in dKO, which is significantly lower than in embryos lacking only c-Myc in melanoblasts (cKO) (Figure 7D, E). This suggests that N-Myc plays an essential role in MyccKO melanocytes already during embryogenesis. In addition, this might explain the maintenance of a certain number of melanoblasts during development, and thus the remaining pigmentation, in MyccKO mice, which might be at least partially due to compensation by N-Myc.
In cells and organs, c-Myc activity is required for multiple functions including growth, proliferation, apoptosis, and differentiation (Gustafson and Weiss, 2010; Meyer and Penn, 2008). c-Myc was shown to be implicated in the maintenance of neural crest lineages in mice (Wei et al., 2007), zebrafish (Hong et al., 2008), and Xenopus (Bellmeyer et al., 2003), indicating its relevance to the melanocyte lineage. Recently, it has been shown that c-Myc is expressed in melanoblasts at E15.5 (Colombo et al., 2012). Here, we specifically addressed the effect of this gene on melanocytes, using a conditional allele of c-Myc in combination with Tyr::Cre mice (Delmas et al., 2003; Trumpp et al., 2001). Mice lacking c-Myc in melanocytes display diluted coat color, with variable degree of gray hair ranging from white to dark gray. Remarkably, this phenotype is non-progressive and thus in contrast to hair graying models. For instance, removal of Notch signaling using the same Tyr::Cre mouse strain leads to a progressive hair graying with a complete white coat after a few months (Schouwey et al., 2007). In such conditional Notch knockout mice, histological sections had revealed a loss of bulge melanocytes, assumed to be MSCs. Histological analysis on the MyccKO mice revealed a reduced number of melanocytes in both bulge and bulb of hair follicles, but no complete or progressive loss of these populations was observed. The deletion efficiency of the c-Myc flox allele appears to be high enough (Figure 6) to suggest that remaining coat pigmentation in cKO is maintained by c-Myc null melanocytes. Similar to human hair graying, reduced number of MSCs in the hair follicle bulge is associated with fewer differentiated cells in the bulb of hair follicle expressing melanocyte-specific markers (Pax3, Mitf, Tyr, Tyrp1, Dct –Figure 3 and not shown). These c-Myc-deficient pigment cells are still capable of melanin production and transfer to the growing hair. We assume that the transfer of pigment granules is rather not affected in MyccKO mice because no clusters of aggregated melanin can be observed in follicular melanocytes similar to those observed in ashen and dilute mutants (Wilson et al., 2000). Nevertheless, whether the pigment machinery of MyccKO melanocytes works with the same efficiency as in wild-type cells remains an open question. To address this question, different experiments might have to be carried out including quantitative analysis of melanogenic gene expression and measurement of tyrosinase activity on single cell level. On the other hand, our preliminary data have shown that c-Myc downregulation by siRNA leads to slight overexpression of MITF in melanocyte cultures (not shown) offering an explication for a compensatory mechanism. These two proteins belong to the same family of transcription factors and might have overlapping target genes. This would also explain how melanocytes are able to survive and normally function after birth without c-Myc – one of the key molecules regulating almost, if not, all cellular processes.
The primary effect of c-Myc was evident during embryonic development, with a reduction in melanoblast numbers first observed at around E12.5 and remaining at about one-third of the controls throughout development. One can also propose that c-Myc acts in early development of pigment cells when Tyr::Cre is not yet active. Nevertheless, it has been reported that conditional deletion of c-Myc using the Wnt1::Cre transgenic line results in a viable mouse with different defects linked to the neural crest cells, namely coat color, skull frontal bone, and middle ear ossicle development (Wei et al., 2007). This suggests that c-Myc is indeed expressed early in the neural crest cells. The illustrated coat color phenotype of the c-Mycflox/flox, Wnt1::Cre mouse was similar to the one we described here in the c-Mycflox/flox, Tyr::Cre mouse. Therefore, it is feasible that early deletion of c-Myc does not affect the melanocyte generation and that c-Myc is rather important for melanocyte maintenance later during embryogenesis. On the other hand, morpholino knockdown of c-Myc in Xenopus embryos leads to the downregulation of neural crest markers and results in the loss of different neural crest derivatives including melanocytes. It thus appeared that c-Myc is important for the neural crest cell fate specification and might be a key factor to prevent the premature cell differentiation or cell fate decision (Bellmeyer et al., 2003). However, the Tyr::Cre transgene acts on committed melanocyte precursors, at a stage when the cells have already specified and emigrated from the neural crest. Furthermore, tracing of recombined cells with the Rosa26R reporter strain did not reveal any abnormal distribution of LacZ-stained cells, suggesting that c-Myc has not affected the specification of this neural crest–derived lineage.
Notably, c-Myc appears to be important to maintain melanoblast proliferation in the epidermal compartment of the skin. As we observed, wild-type melanoblasts present in the epidermis at E13.5 show higher proliferation rate in contrast to c-Myc-deficient pigment cells (Figure 5E). This leads to fewer cells to be found in MyccKO embryos at E14.5. The interactions of melanoblasts with the surrounding cells in the dermis and epidermis are different because of different molecular and cellular environments. Such differences may explain various proliferative behaviors. In the dermis, melanoblasts interact mainly with fibroblasts by various proteins including N-cadherin (Jouneau et al., 2000), while in the epidermis, melanoblasts interact mainly with keratinocytes using E- and P-cadherin. It was shown that the level of cadherins in the cells is important to regulate the proliferation (Haass et al., 2005). Interestingly, it has been shown that c-Myc regulates the level of E-cadherin but not N-cadherin (Batsche et al., 1998; Liu et al., 2009).
However, melanoblasts lacking c-Myc are still able to proliferate and contribute to the remaining pigmentation of c-MyccKO mice. We showed that c-Myc-deficient melanocytes upregulate N-Myc demonstrating compensatory mechanisms. Both c-Myc and N-Myc are essential, and a deficiency in c-Myc or N-Myc leads to embryonic lethality. In contrast, L-Myc appears to be dispensable for mouse development and L-Myc-null mice are viable and not different from control littermates including the coat color phenotype (Charron et al., 1992; Hatton et al., 1996; Stanton et al., 1992; Trumpp et al., 2001). c-Myc levels are elevated in N-Myc null embryos, indicating coordinate regulation of Myc genes during mouse embryogenesis (Stanton et al., 1992). Similarly, c-Myc-deficient embryos show upregulation of N-Myc mRNA (Trumpp et al., 2001), and N-Myc might compensate the lack of c-Myc during cerebellar development (Wey et al., 2010; Zindy et al., 2006). Lack of functional developmental compensation for the other Myc family gene, as seen in the respective knockout mice, was partially explained by the distinct although overlapping expression pattern of N-Myc and c-Myc during embryogenesis (Downs et al., 1989). Indeed, replacement studies, where endogenous c-Myc was substituted with N-Myc coding sequence, demonstrated that N-Myc is able to rescue the c-Myc function during embryogenesis and is sufficient to generate viable mice (Malynn et al., 2000). Thus, embryonic abnormalities occurring as a result of c-Myc or N-Myc loss cannot be entirely compensated by each other probably due to differences in transcriptional regulation yielding distinct expression patterns (Downs et al., 1989; Malynn et al., 2000). From these different points, it might be envisaged that N-Myc (or even L-Myc) might compensate to a certain degree for the lack of c-Myc and thus permit the presence of c-Myc-deficient melanocytes, which then equally account for the presence of gray hair. Here, we have shown that, in the presence of c-Myc, N-Myc appeared to be dispensable for melanocyte development and homeostasis in adult. Interestingly, c-Myc-deficient pigment cells demonstrated an increase in N-Myc mRNA levels (Figure 6F), indicating the presence of a compensatory effect. This was further confirmed by the deletion of both c-Myc and N-Myc in melanocytes of double conditional knockout mice (dKO: c-Mycflox/flox, N-Mycflox/flox, Tyr::Cre, Figure 7). The dorsal coat color of these double knockout mice was almost white in comparison with MyccKO (c-Mycflox/flox, Tyr::Cre) mice, indicating that N-Myc is indeed involved in the partial rescue of remaining pigmentation in the gray MyccKO mice. This result might suggest that c-Myc has a prevalent function in the melanocyte lineage, because N-Myc can only partially replace c-Myc. This difference in c-Myc and N-Myc functions could thus be due to unequal levels of expression or can be explained by the distinct quantitative activity of their transactivation domains (Barrett et al., 1992; Laurenti et al., 2008). Moreover, N-Myc is ubiquitinylated to a greater extent and subsequently less stable in comparison with c-Myc (Laurenti et al., 2008; Zhao et al., 2008). Therefore, to maintain the same expression pattern mediated by one member of the Myc family, it seems likely that any cell needs more functional N-Myc protein than c-Myc. A balance between c-Myc and N-Myc is exemplified in the hematopoietic system. Here, immature hematopoietic stem cells (HSCs) express both c-Myc and N-Myc at equal levels. During differentiation, HSCs downregulate N-Myc but maintain c-Myc transcription (Laurenti et al., 2008). Deletion of c-Myc affects HSC differentiation and an accumulation of immature stem cells. In contrast to the melanocyte lineage, no compensatory increases in N-Myc mRNA levels were observed (Laurenti et al., 2008), suggesting that the c-Myc/N-Myc ratio and functional requirements are different for pigment cells.
In conclusion, we have shown here that c-Myc is required for proper maintenance of the melanocyte lineage and its loss leads to a congenital non-progressive gray hair phenotype in mice. Importantly, even fewer c-Myc-deficient cells are still able to function and supply pigmentation to hair follicles. Investigation of the possible role of MITF deregulation and other melanogenic genes will shed light on how the pigmentary system is established in melanocytes when c-Myc is deleted. The diminished number of pigment cells is primarily caused by a reduction in melanoblast numbers during embryonic development because of a defect in proliferation. Importantly, N-Myc appeared to play a compensatory role for c-Myc-deficient melanoblasts, and when both c- and N-Myc are deleted, almost no pigment cells can be found in skin at birth. Further analysis of N-Myc function in c-Myc-deficient melanoblast might clarify the role of this gene in pigment cell development and possibly its early role when melanoblasts are specified from neural crest lineage.
Dorsal skin was isolated and fixed with 4% paraformaldehyde (PFA) solution (pH7.2 in PBS) on ice for 2 h. After washing, samples were dehydrated and embedded in paraffin; 4-μm sections were counterstained with nuclear fast red. For Fontana-Masson staining, highlighting argentaffin granules and melanin, sections were deparaffinized and hydrated to distilled water. Slides were placed in 2.5% silver nitrate working solution and left in a 56°C oven for 2 h. This step was followed by incubation in 0.1% gold chloride solution for 10 min, washing and subsequent incubation in 5% sodium thiosulfate solution for 1–5 min. Slides were counterstained with nuclear fast red.
For immunostaining, sections were boiled in Tris-EDTA (pH9) for 20 min. Sections were blocked in 1% goat serum, 2% BSA, 0.05% Tween in PBS at RT for 30–60 min. This was followed by overnight incubation at +4°C with primary antibodies in blocking solution. Secondary antibodies were applied for 1 h, at RT. Nuclei were stained with DAPI, and sections were mounted with DABCO. More than 10 sections of at least two animals per genotype were analyzed. Following primary antibodies were used: Rabbit anti-Tyrosinase (1:1000), anti-TRP1 (1:1000), anti-Dct (1:1000) (provided by V. Hearing, NIH, Bethesda), anti-LacZ (1:1500) (Chemicon, Merck Millipore, Billerica, MA, USA), Rat anti-BrdU (1:200) (MorphoSys UK t/a AbD Serotec, Oxford, UK), mouse anti-Pax3 (1:200) (DSHB, Iowa City, IA, USA), anti-MITF (1:1000) (provided by Simon Saule, Institut Curie, Orsay).
For BrdU labeling of embryos, pregnant mice were injected intraperitoneally with 5-bromodeoxyuridine (BrdU, B-5002; Sigma-Aldrich, Buchs, Switzerland) at 100 μg/g of mouse weight. After 2 h, embryos were dissected, fixed in 4% PFA for 2 h, and incubated at +4°C O/N in 30% sucrose. The next day, embryos were transferred to 30% sucrose with OCT (1:1) and kept for 4 h at +4°C. They were then immersed in OCT and frozen on dry ice. Embryos were sectioned transversely at 8 μm. Prior analysis, slides were dried at RT and washed with PBS. BrdU incorporation was then detected by immunohistochemistry using between 25 and 40 sections per embryo. TUNEL staining was performed on paraffin sections of embryos according to the manufacturer’s protocol (In Situ Cell Death Detection Kit, Fluorescein, Roche, Basel, Switzerland, Cat. No. 11 684 795 910).
Hair was collected from the dorsal region of a mouse, and melanin was extracted using alkali (1 M NaOH) treatment; 1 g/ml of hair was incubated at 85°C for 4 h. Relative melanin content was measured by spectrophotometry at 475 nm. A hair sample from an albino (Tyrc) mouse was used as reference.
Whole-mount embryo LacZ staining
Embryonic day (E)0.5 was determined as noon of the day of detection of a vaginal plug. LacZ staining was essentially carried out as described (Porret et al., 2006; Schmidt et al., 1998). Embryos were dissected free of extraembryonic tissues, washed in PBS, and fixed in 4% paraformaldehyde for 1 h on ice. After two washes with PBS, the embryos were incubated in permeabilization solution (0.1 M phosphate buffer pH 7.3, 2 mM MgCl2, 0.01% deoxycholate, 0.02% NP40) twice for 1 h at RT. They were then incubated in staining solution (3.33 mM potassium ferricyanide, 3.33 mM ferrocyanide, 20 mM Tris–HCl pH7.4, 0.66 mg/ml X-Gal in permeabilization solution) for 4–6 h at 37°C. After two washes in PBS, the embryos were post-fixed for 4–8 h in 4% paraformaldehyde at 4°C. For E16.5 embryos, skin from the dorsal part of the embryo was removed, fixed for 1 h on ice (skin was stretched on a piece of paper to avoid shrinking), and stained as described above. β-Galactosidase staining of the dorsal skin from 8- and 30-day-old mice was performed using the same protocol, with incubation in staining solution at room temperature for 48 h. For counting of embryonic melanoblast numbers, embryos were photographed and defined regions were counted for LacZ-positive cells. To count dermal and epidermal melanoblasts at E13.5, embryos were dehydrated, embedded in paraffin, and sectioned at 6 μm from caudal to rostral limbs. Every 10th section (n > 20 sections/embryo) was counterstained with nuclear fast red, and numbers of epidermal and dermal cells per section were counted using 20–34 sections per embryo.
The protocol for the isolation of single cells from the mouse skin was adopted from a publication (Nishimura et al., 1999). The dorsal skin of newborn mice (≤P4) was isolated and washed with PBS. To separate the dermis from the epidermis, skin was incubated for 1–2 h in 0.25% trypsin at 37°C. After splitting, the dermis and epidermis were washed in PBS and treated separately. Epidermis was minced and incubated in the dissociation buffer (Gibco Life Technologies, Zug, Switzerland) at RT for 10–15 min. Then, two volumes of 5% RPMI/0.002% DNAseI (Roche) were added and kept on ice. Dermis was minced in the Collagenase/Dispase solution [0.1% Collagenase I (Sigma), 0.1% Dispase (Roche), 0.002% DNAseI in 5% RPMI (Gibco)] and incubated at 37°C until fully digested. This was followed by washing with 0.002% DNAseI solution in 5% RPMI. At this stage, cells from dermis and epidermis were combined and counted.
For FACS analysis, 1–3 × 106 cells were used per staining. Staining was performed in 50 μl for 20 min on ice in the dark. 7-AAD was used to exclude dead cells. Along with the experimental staining, corresponding single-antibody staining controls were made. Samples were run on the CyAn ADP analyzer (Dako, Baar, Switzerland). Following antibodies (eBioscience, Vienna, Austria; unless notified) and reagents were used: CD117-PE, CD45-FITC, CD45-PE-Cy5; SAV (streptavidin)-PB, SAV (streptavidin)-PE. Gating strategy for the FACS analysis is shown in Figure S3. For FACS sorting, a single cell suspension was prepared from the skin of newborn mice as described above. Cells were stained with biotinylated anti-CD117 antibody (eBioscience, clone 2B8), further incubated with anti-biotin magnetic beads, and enriched for CD117+ population by MACS separating system. This was followed by staining with CD45-FITC antibody. Cells were sorted directly to Trizol (Invitrogen, Zug, Switzerland) as CD45-CD117+SShigh and CD45-CD117+SSlow populations. Dead cells were discriminated by 7-AAD staining.
Quantitative RT-PCR analysis
Quantitative RT-PCR was performed using cDNA obtained from the reverse transcription of total RNA derived from FACS-sorted melanocytes (SuperScript II and random hexamer primers; Invitrogen). Gene expression was analyzed using the Light Cycler Carousel PCR machine (Roche Diagnostics) following the manufacturer’s instructions. Relative levels of gene expression were normalized to GAPDH gene expression using the 2-ΔΔCT method. Gene-specific primer sequences are available upon request.
Thanks are due to Ian J. Jackson for providing Dct::LacZ mice, to Simon Saule and Vince Hearing for antibodies, and to Michelle Blom for help with mouse breeding. Work in the laboratory of FB was supported by grants from Oncosuisse, Novartis, Emma Muschamps, and The Swiss National Science Foundation. Work in the laboratory of LL was supported by LNCC (labelization) and INCa. RNE was supported by NIH/NCI grant RO1CA20525.