Endothelin 3 Induces Skin Pigmentation in a Keratin-Driven ... - Core

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Jul 5, 2007 - Endothelin 3 (Edn3) encodes a ligand important to developing neural crest ... coat color suggesting a previously unreported role for endothelin ...
ORIGINAL ARTICLE

Endothelin 3 Induces Skin Pigmentation in a Keratin-Driven Inducible Mouse Model Roman J. Garcia1, Avner Ittah1, Sheyla Mirabal1, Jessica Figueroa1, Lidice Lopez1, Adam B. Glick2 and Lidia Kos1 Endothelin 3 (Edn3) encodes a ligand important to developing neural crest cells and is allelic to the spontaneous mouse mutation occurring at the lethal spotting (ls) locus. Edn3ls/ls mutants exhibit a spotted phenotype due to reduced numbers of neural crest-derived melanocyte precursors in the skin. In this study, we show that when Edn3 is driven by the keratin 5 promoter and thereby placed proximal to melanocyte lineage cells, adult mice manifest pigmented skin harboring dermal melanocytes. Using a tetracycline inducible system, we show that the postnatal expression of Edn3 is required to maintain these dermal melanocytes, and that early expression of the Edn3 transgene is important to the onset of the hyperpigmentation phenotype. Crosses into Edn3ls/ls mutants demonstrate that the Edn3 transgene expression does not fully compensate for the endogenous expression pattern. Crosses into tyrosine kinase receptor KitWv mutants indicate that Edn3 can partially compensate for Kit’s role in early development. Crosses into Ay mutant mice considerably darkened their yellow coat color suggesting a previously unreported role for endothelin signaling in pigment switching. These results demonstrate that exogenous Edn3 affects both precursors and differentiated melanocytes, leading to a phenotype with characteristics similar to the human skin condition dermal melanocytosis. Journal of Investigative Dermatology (2008) 128, 131–142; doi:10.1038/sj.jid.5700948; published online 5 July 2007

INTRODUCTION Neural crest cells (NCC) are a transient population of epithelial cells situated over the dorsal neural tube in the post-neurula embryo (Schoenwolf and Nichols, 1984). Some NCC in the embryonic trunk undergo an epithelial to mesenchymal transition, and undertake a dorsolateral migratory route between the epidermis and the dermamyotome. These NCC eventually differentiate into the melanocytes found in the skin (Hall, 1999; Le Douarin and Kalcheim, 1999). In the mouse, some of the NCC-derived melanocyte precursors, the melanoblasts, remain in the dermis, while the majority colonize the epidermal basal layer, and subsequently the hair follicles (Mayer, 1973; Erickson and Weston, 1983; Kunisada et al., 1996). Eventually, most of those melanoblasts that remained in the skin are no longer present (Kunisada et al., 2000). Several in vitro studies have implicated a 21 amino-acid peptide, Endothelin 3 (Edn3), with increases in the melanocyte lineage population and its differentiation (Lahav et al., 1996; Reid et al., 1996; Opdecamp et al., 1998; Hou et al., 1

Department of Biological Sciences, Florida International University, Miami, Florida, USA and 2Department of Veterinary Science, Pennsylvania State University, University Park, Pennsylvania, USA Correspondence: Dr Lidia Kos, Department of Biological Sciences, Florida International University, Miami, Florida 33199, USA. E-mail: [email protected] Abbreviations: Dct, dopachrome tautomerase; Dox, doxycycline; Edn3, endothelin 3; Ednrb, endothelin receptor B; NCC, neural crest cells

Received 7 December 2006; revised 21 March 2007; accepted 3 April 2007; published online 5 July 2007

& 2007 The Society for Investigative Dermatology

2004; Trentin et al., 2004). In vivo, Edn3 is expressed in a variety of tissues including the skin (Bloch et al., 1989; Matsumoto et al., 1989; Onda et al., 1990; Reid et al., 1996). In situ hybridization studies suggest that Edn3 is expressed between embryonic day 8.0 (E8.0) and E15.0 in the mesenchymal cells that surround the NCC in all parts of the murine enteric neural and melanocyte pathways (Rice et al., 2000). Edn3 binds a member of the G-protein coupled heptahelical superfamily called Endothelin receptor B (Ednrb) (Arai et al., 1990; Sakurai et al., 1990; Sakamoto et al., 1993; Yanagisawa, 1994). Ednrb is expressed in NCC including melanoblasts (Reid et al., 1996; Opdecamp et al., 1998; Lee et al., 2003). Ednrb and Edn3 are both allelic to the spontaneous mouse mutations occurring at the piebald and lethal spotting (ls) loci, respectively (Baynash et al., 1994; Hosoda et al., 1994). Recessive mutants at either of these loci give rise to similar phenotypes consisting of differing degrees of hypopigmentation and aganglionic megacolon. The hypopigmentation phenotype has been attributed to a decrease in the melanoblast population as early as E10.5 and to abnormal cell migration identified at E11.5 (Pavan and Tilghman, 1994; Lee et al., 2003). The similarity between human genes and those from model organisms has given scientists an opportunity to obtain valuable hints about the genes involved in skin-related pathologies. A homozygous mutation in Edn3 and a nonsense mutation in the Ednrb locus have been linked to Waardenburg syndrome in humans (Hofstra et al., 1996; Syrris et al., 1999). This syndrome comprises sensorineural hearing loss, www.jidonline.org

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hypopigmentation of the skin and hair, and pigmentary disturbances of the irides. Mice with spontaneous mutations in the Pax3, Sox10, Mitf, Slug, Edn3, and Ednrb genes at least partially model elements of the four different Waardenburg syndromes (Tachibana et al., 2003). To investigate if higher levels of Edn3 would lead to a novel model of melanocyte-related skin conditions, we created transgenic mice which conditionally overexpress Edn3. In vitro studies have previously indicated that the effects of Edn3 signaling on melanocyte precursors are dosedependent (Lahav et al., 1996; Reid et al., 1996; Opdecamp et al., 1998). Studies conducted on an Edn3 transgenic mouse driven by the Cytokeratin 14 promoter have also been briefly described and show that Edn3 can lead to hyperpigmented skin (Aoki et al., 2005). To clarify the cellular and temporal basis for this phenotype, we used the tetracycline-responsive transgene system. Tetracycline regulatory systems have been previously used in several conditional expression studies in mice (Furth et al., 1994; Chin et al., 1999; Shin et al., 1999; Diamond et al., 2000; Jaisser, 2000). We have used K5-tTA and -rTA mice (Diamond et al., 2000) to conditionally regulate the overexpression of Edn3. K5-tTA or -rTA transgenic mice express tetracycline-sensitive transcriptional activators (tTA or rTA) under the control of the regulatory region of the Bos taurus gene for Keratin 5 (K5). These mice will help us clarify Edn3’s role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. When driven by the K5 promoter, the transgene-mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics developed increased pigmentation on most areas of the skin. Doxycycline (Dox)-based temporal studies showed that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of our Edn3 transgenic mice may offer some insight into the genetics behind human skin dermal pigmentation and offer clues about the time periods important in establishing these conditions. RESULTS A tetracycline inducible Edn3 transgene results in higher levels of embryonic Edn3 transcripts

We have used K5-tTA and -rTA mice (Diamond et al., 2000) to conditionally overexpress Edn3. These mice express tetracycline-sensitive transcriptional activators (tTA or rTA) under the control of the Bos taurus K5 promoter (Figure S1a and b). In the absence of dox, this fusion protein comprised of a tet-repressor-binding domain and a VP16 activation domain (Gossen and Bujard, 1992; Resnitzky et al., 1994; Kistner et al., 1996), binds to the TRE-Edn3-lacZ transgene which contains a tetracycline-responsive tet-operator sequence (tetO). After binding tetO, the tTA protein induces transcription from bidirectional CMV promoters (Figure S1a) and leads to 132

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Edn3 and lacZ expression (Baron et al., 1995). However, if dox is added to the drinking water supply, it binds to tTA and prevents its interaction with tetO. We have also made use of K5-rTA mice that express rTA (Figure S1b), a protein analog of tTA that does not bind tetO unless dox is present (Kistner et al., 1996). Reverse transcriptase-PCR was performed on E12.5 whole embryos (Figure S1c) and E14.5 skin (Figure S1d) to assess the relative levels of Edn3 mRNA transcripts in K5-tTA;TRE-Edn3lacZ and control embryos (wild-type embryos or those with only a single transgene). In the E12.5 embryo, Edn3 mRNA was present at slightly elevated levels in transgenics (0.45 RU (relative units)70.02 vs 0.18 RU70.05; Po0.05). This difference was more pronounced in the E14.5 skin (0.39 RU70.08 vs 0.07 RU70.01; Po0.05). K5-tTA;TRE-Edn3-lacZ mice exhibit a hyperpigmented phenotype

The initial PCR screen of 124 tail biopsies revealed 11 TREEdn3-lacZ founder mice. To obtain K5-tTA;TRE-Edn3-lacZ mice, all TRE-Edn3-lacZ founders were crossed into K5-tTA mice. Resultant K5-tTA;TRE-Edn3-lacZ (F1) offspring were then mated to yield the mice, and embryos used in this study. All K5-tTA;TRE-Edn3-lacZ mice developed a pigmented skin phenotype. Two highly pigmented lines (369, 476) were chosen for detailed analysis with five and 15 transgene copy numbers, respectively. Line 476 was specifically chosen because it did not express lacZ during embryogenesis thereby allowing for crosses into transgenic mice that express lacZ under the control of the Dopachrome tautomerase (Dct) promoter. Over 100 mice from each line were observed to develop a hyperpigmented phenotype. Mice with just the TRE-Edn3lacZ transgene alone never manifested a hyperpigmentation phenotype. As a result of their mixed genetic background (FVB/N and C57BL/6xSJL), K5-tTA;TRE-Edn3-lacZ transgenic mice could have a variety of coat colors (Yokoyama et al., 1990; Brilliant et al., 1994). However, only mice with black and agouti coats were examined in this study. The pigment responsible for the coat color of normal mice resides in the hair follicles and hair shafts, and not in the skin. However, the K5-tTA;TRE-Edn3-lacZ transgenic mice developed prominent skin pigmentation (Figure 1a). Increased pigmentation is evident at E16.5 in K5-tTA;TRE-Edn3lacZ(369) mice around the eye and in the muzzle around vibrissae and within the nostrils (Figure 1b). K5-tTA;TRE-Edn3-lacZ pups from both lines were pigmented at 0 day with varying tones of pigmentation, and in some cases not pigmented. By 2 days (Figure 1a), the dorsal aspect, muzzle, and most of the tail of all transgenic pups in both lines were hyperpigmented. Control wild-type and single transgenic mice (TRE-Edn3-lacZ(369), TRE-Edn3-lacZ(476), or K5-tTA) did not manifest a hyperpigmented phenotype. At 10 days (Figure 1a), control mice manifested a pigmented dorsal aspect. However, K5-tTA;TRE-Edn3-lacZ mice maintained a darker skin tone throughout this time, and they also had darker pigmentation on their tail and muzzle. The dark coloration in control mice has been shown to be due to

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Figure 1. Transgenic mice exhibit a hyperpigmentation phenotype. K5-tTA;TRE-Edn3-lacZ(369) mice (arrowheads). (a) Dorsal skin of 0, 2, 10, and 20 days controls (wild-type) and K5-tTA;TRE-Edn3-lacZ(369) mice (not all 0 day transgenics were born with pigmentation). Hyperpigmented muzzle of (b) E16.5 and (c) 30 days mice. Adult (d) foot pads and (e) tail.

pigment induction in hair follicle melanocytes as opposed to melanocytes in the skin (Mecklenburg et al., 2000). By 20 days (Figure 1a), control mice generally took on a pink skin tone, whereas transgenics retained their black coloration on their dorsal and ventral aspects. Between 20 and 40 days, the hyperpigmentation phenotype was still pronounced in transgenic mice. Relative to littermate controls, K5-tTA;TRE-Edn3-lacZ mice showed increased pigmentation on most areas of the skin such as in the nose and muzzle, foot pads, tail (Figure 1c–e), external genitalia, ears, and the dorsal/ventral aspects of the skin. However, the lining of the oral cavity and paw pads were not pigmented. The K5-tTA;TRE-Edn3-lacZ transgene is expressed in the dorsal aspect of the neural tube and in keratinocyte lineage cells

The lacZ expression pattern of K5-tTA;TRE-Edn3-lacZ(369) embryos corresponded better to that generated by the human Keratin 5 promoter (Byrne et al., 1994) than to the one previously described for the same K5 promoter (Ramirez et al., 1994) used in this study. At E9.5 (Figure 2a), lacZ þ cells were found in the head, in the posterior aspect of the trunk, and tail. High levels of b-galactosidase activity were also detected in the branchial arches. A day later (Figure 2a), the number of lacZ þ cells increased in the branchial arches and in the trunk starting at the level of the forelimb buds as well as in the tail. There was also expression dorsally over the neural tube beginning at a level just rostral of the forelimb. At E10.5, the aberrant expression in TRE-Edn3-lacZ embryos was localized to stained cells in the third and second branchial arch and between the hindlimbs (Figure 2c). At E11.5 (Figure 2a) and E12.5 (Figure 2b), embryos displayed a general increase in lacZ þ cells, and the staining in the neural tube persisted. The aberrant expression in TRE-Edn3-lacZ(369) E12.5 embryos was localized to stained cells in the otocysts, third branchial arch, and between the hindlimbs (Figure 2c). Sections of E12.5 mouse embryos (Figure 2b) revealed that b-galactosidase activity was strongest in the surface ectoderm

and roof plate of the neural tube, with little to no staining in the underlying mesenchyme proximal to the surface ectoderm. In transitional areas, b-galactosidase activity was higher in some regions of the epidermis and sparser in others, as exemplified by the region between the lateral ridge and the roof plate of the neural tube at the level of the forelimb (Figure 2b). By E15.5 (Figure 2a), the last vestiges of unstained areas in the skin disappeared, and promoter activity could be found over the entire surface of the embryo. Wild-type or K5-tTA transgenic controls were negative for b-galactosidase activity at all examined ages. E12.5 K5-tTA;TRE-Edn3-lacZ(476) embryos did not display lacZ staining. In the skin of the K5-tTA double transgenic mice, b-galactosidase activity was observed in the outer root sheath of the hair follicles and in the cornified interfollicular epidermis. K5-tTA;TRE-Edn3-lacZ(369) skin sections from 0 day pups (Figure 2d) showed limited lacZ expression in the epidermis coupled with strong expression in the connective tissue underneath the integument’s striated muscular layer. At 2 days (Figure 2e), the expression in the connective tissue persisted and was accompanied by a more robust and consistent epidermal lacZ expression which included most of the hair follicles. The general localization of this expression pattern was maintained in 20 and 90 days adult skin. LacZ expression was absent from wild-type and K5-tTA mice; however, TRE-Edn3-lacZ(369) mice maintain a faint and aberrant lacZ expression confined to small and interspersed regions of the epidermis (Figure 2f). TRE-Edn3lacZ(476) lacZ expression is absent in 2-day-old mice and staining in K5-tTA;TRE-Edn3-lacZ(476) mice was faint and sparsely localized to the integument’s underlying connective tissue (Figure 2g). Transgenic embryos have increased melanocyte precursor numbers

An important murine melanoblast and melanocyte marker is Dct (Steel et al., 1992; Tsukamoto et al., 1992; Mackenzie www.jidonline.org

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difference in melanoblast numbers and distribution. However, sections of E13.5 K5-tTA;TRE-Edn3-lacZ(476)/Dct-lacZ transgenic embryos and Dct-lacZ littermate controls showed that transgenics had an increased number of both dermal (20.475.8 (SD) (n ¼ 3) vs 10.173.7; Po0.001) and epidermal melanoblasts (24.774.8 vs 8.472.5; Po0.001; Figure 3a and b). At E16.5, melanoblasts in transgenics also outnumbered controls in the dermis (18.974.0 vs 11.07 5.1; Po0.001) as well as in the epidermis (36.178.0 vs 29.575.7; Po0.001). A small but significant increase in the number of Dct þ cells per hair follicle was also observed in E16.5 transgenics (6.171.9 vs 4.670.9; Po0.001; Figure 3a and b). Skin biopsies from 1-day-old K5-tTA;TRE-Edn3lacZ(476)/Dct-lacZ mice and controls were also stained for Dct-lacZ cells (Figure 3c). Not all pigmented melanocytes found in the skin of double transgenic animals were Dct þ . Transgenic mice display an increased number of differentiated melanocytes in the dermis

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A histological analysis of Dopa þ cells was conducted on 0, 2, and 90-day-old K5-tTA;TRE-Edn3-lacZ(369) mice to evaluate the presence and distribution of differentiated melanocytes in the changing skin of juvenile and adult mice. The dorsal aspect of the skin revealed the presence of several melanocytes in the dermis of dark skin transgenic mice since 0 day (Figure S2a). Dopa þ cells localized to the superficial most layer of the dermis and around the hair follicles in the skin of 2 days transgenic mice (Figure S2b). Some melanocytes persist around the hair follicles in 90 days dark skin mice (Figure S2c). Control littermates revealed considerably fewer Dopa þ cells for all ages. Continual transgenic Edn3 expression is required to maintain the hyperpigmentation phenotype

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Figure 2. Embryonic and postnatal cells expressing the tetracycline system establish melanocytes in the dermis. (a) At E9.5, b-galactosidase activity is absent along the trunk neural tube (arrows) in K5-tTA;TRE-Edn3-lacZ(369) embryos. By E10.5, b-galactosidase activity begins along the neural tube and persists during early development (E11.5). By E15.5, b-galactosidase activity is found over most areas of the developing skin. (b) E12.5 cryosections reveal b-galactosidase activity on the surface ectoderm (arrowhead indicates lateral ridge) and roof plate of the neural tube (NT; bar ¼ 50 mm). (c) TRE-Edn3lacZ(369) expressing cells in E10.5 and E12.5 embryos. (d and e) lacZ staining in K5-tTA;TRE-Edn3-lacZ(369) skin at (d) 0 day and (e) 2 days localizes to the epidermis, hair follicles, and underlying connective tissue. An ectopic population of dermal melanocytes can be observed in these mice (arrows). (f) TRE-Edn3-lacZ(369) expression in the epidermis of 2 days pups (arrow). (g) 2 days lacZ-stained skin from the K5-tTA;TRE-Edn3-lacZ(476) line reveals sparse to no b-galactosidase activity. Bar ¼ 100 mm.

et al., 1997; Raamsdonk et al., 2004). Since K5-tTA;TREEdn3-lacZ(476) mice do not have embryonic lacZ expression, they were crossed into Dct-lacZ mice to follow melanoblasts during development. Whole mounts of K5-tTA;TRE-Edn3lacZ(476)/Dct-lacZ embryos at E12.5 did not reveal any overt 134

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Pregnant K5-tTA;TRE-Edn3-lacZ(369) dams were treated with dox at E10.5, to identify the ensuing transgene-expressing cells. At E11.5, K5-tTA;TRE-Edn3-lacZ embryos revealed a significant decrease in lacZ-positive cells compared to controls (Figures 2a and 4a). The lacZ-positive cells present in dox-treated litters at E12.5 embryos were restricted to those resulting from the aberrant transgene expression in TRE-Edn3lacZ controls (Figures 2c and 4a). Thus, the repression of transgene expression was effective approximately 2 days after dox administration. The pigmentation phenotype was not induced when the transgene was inactivated in K5-tTA;TRE-Edn3-lacZ(369) or K5-tTA;TRE-Edn3-lacZ(476) mice by dox administration at conception or at E12.5 (Figure 4b). When K5-tTA;TRE-Edn3lacZ(369) litters were treated with dox at 0 day, black transgenic neonates gradually lost their skin color, and by 20 days they manifested the pink skin tone of controls (Figure 4c). To detect differentiated melanocytes and unmelanized melanoblasts, skin biopsies of pink 20-day-old K5-tTA;TREEdn3-lacZ(369) mice (treated with dox at 0 day) were subjected to silver nitrate staining. Stained biopsies revealed a reduced population of dermal melanocyte lineage cells in relation to non-treated K5-tTA;TRE-Edn3-lacZ(369) littermates (Figure 4d).

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Figure 3. Transgenic embryos have large numbers of Dct cells. Dct cells in Dct-lacZ control mice (C) and K5-tTA;TRE-Edn3-lacZ(476)/Dct-lacZ (Tg) mice. (a) E13.5 and E16.5 Dct þ cells in Tg and C (TRE-Edn3-lacZ(476)/Dct-lacZ ) embryos. Sections reveal Dct þ cells scored to the dermis (horizontal arrows), epidermis (vertical arrows), and E16.5 hair follicles (HF). (b) Number of Dct þ cells in representative areas of the dermis and epidermis of E13.5 and E16.5 embryos. E16.5 embryos were also assayed for Dct þ cells in individual hair follicles. Significant increases (asterisks; Po0.001) are seen in dermal, epidermal, and hair follicle Dct þ cell populations. (c) Dct þ cells (arrows) localize to pigmented regions in 1-day Tg mice. Bar ¼ 25 mm.

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Figure 4. Transgene expression is required at all times for maintenance of the hyperpigmentation phenotype. (a) Embryos that receive dox at E10.5 lose b-galactosidase activity over 2 days. (b) K5-tTA;TRE-Edn3-lacZ(369) embryos administered dox at E12.5 never manifest hyperpigmented skin; a 4-day-old litter determined to have two K5-tTA;TRE-Edn3-lacZ(369) pups after PCR analysis is shown. (c) K5-tTA;TRE-Edn3-lacZ(369) mice (arrowheads) repressed with dox at 0 day begin to lose their hyperpigmented skin. (d) Ammoniacal silver nitrate staining indicates melanocyte lineage cells (arrows) in the dark skin of a 20 days K5-tTA;TRE-Edn3-lacZ(369) mouse, and on the pink skin of a 20 days K5-tTA;TRE-Edn3-lacZ(369) littermate treated with dox at 0 day. Bar ¼ 25 mm.

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Figure 5. Activation of transgene expression at E10.5 is required for the generation of the hyperpigmentation phenotype. (a) After dox induction at E7.5, b-galactosidase activity first appears 3 days later (E10.5) and increases by E12.5. (b) Two-day-old K5-rTA;TRE-Edn3-lacZ(369) skin induced at E7.5 shows a decreased lacZ staining pattern in the epidermis and little to no muscular staining relative to the skin of 2 days K5-tTA;TRE-Edn3-lacZ(369) mice. (c) A litter of K5-rTA;TRE-Edn3-lacZ(369) mice treated with dox at E8.5. Three pups from this litter were identified as K5-rTA;TRE-Edn3-lacZ(369); they never developed hyperpigmented skin. (d) K5-rTA;TRE-Edn3-lacZ(369) mice (arrowheads) induced with dox at E7.5. Bar ¼ 100 mm.

The early embryonic expression of Edn3 is important to the onset of the hyperpigmented phenotype

The use of K5-tTA;TRE-Edn3-lacZ(369) mice does not allow us to establish the latest time at which transgenic Edn3 expression can begin and still generate a hyperpigmented phenotype. Therefore, K5-rTA;TRE-Edn3-lacZ(369) mice were used to identify this time. To find the time when lacZpositive cells were first seen, pregnant K5-rTA;TRE-Edn3lacZ(369) dams were treated with dox at E7.5 and embryos were harvested at E9.5, E10.5, and E12.5; skin biopsies were also taken from 2-day-old mice. While E9.5 K5-rTA;TREEdn3-lacZ(369) embryos did not reveal any lacZ-stained cells (data not shown), E10.5 embryos showed b-galactosidase activity over a large region of the embryo (Figure 5a) that increased considerably by E12.5 (Figure 5a). Sections of 136

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E10.5 mouse embryos at the forelimb reveals a pattern of strong b-galactosidase activity along the surface ectoderm and roof plate of the neural tube (Figure 5a). Thus, the onset of transgene expression occurs approximately 3 days after dox treatment. The 2 days transgene expression pattern in the skin of dox-treated K5-rTA;TRE-Edn3-lacZ(369) mice is less pronounced in the epidermis and shows little to no muscular staining relative to K5-tTA;TRE-Edn3-lacZ(369) transgenics (Figures 2e and 5b). To determine when transgene expression was required for the generation of the hyperpigmentation phenotype, K5-rTA;TRE-Edn3-lacZ(369) mice were administered dox since conception, at E7.5, E8.5, and E12.5. When K5-rTA; TRE-Edn3-lacZ mice were administered dox since conception (n ¼ 15) or at E7.5 (n ¼ 12), they revealed a phenotype similar

RJ Garcia et al. Hyperpigmentation in End3 transgenic mice

to K5-tTA;TRE-Edn3-lacZ mice that were born pink or with a slightly darker dorsal skin and ears (Figure 5d). By 2 days (Figure 5d), these mice were substantially darker than littermate controls, although so less than K5-tTA;TRE-Edn3lacZ pups. By 20 days, these mice had darkly pigmented skin (Figure 5d) similar to K5-tTA;TRE-Edn3-lacZ mice. However, K5-rTA;TRE-Edn3-lacZ(369) mice that were induced with dox at E8.5 (n ¼ 11; Figure 5c) or E12.5 (n ¼ 9; data not shown), did not generate hyperpigmented skin at any time.

of melanocytes, Edn3 expression was induced in K5-rTA;TREEdn3-lacZ(476)/Edn3ls/ls mice since E13.5 (n ¼ 3) (Figure 6d), a time when melanoblasts begin to colonize the epidermis. Neither the white areas nor the coat pigmentation in general were altered. An analysis of K5-tTA;TRE-Edn3-lacZ(476) mice with either KitWv/ þ or KitWv/Wv alleles allowed us to investigate if Edn3 could compensate for Kit’s role during melanocyte development. Heterozygous animals display a dilute coat color with a white belly spot (Hanks et al., 1988). Homozygous animals have an almost complete lack of coat pigmentation. K5-tTA;TRE-Edn3-lacZ(476)/KitWv/ þ mice (n ¼ 5) showed a normal coat indicating a rescue of the ventral spot phenotype seen in KitWv/ þ mice (Figure 6e). K5-tTA;TRE-Edn3-lacZ(476)/KitWv/Wv mice (n ¼ 6) developed darkly pigmented skin in the rostral and caudal aspects with a completely white coat (Figure 6f and g). Agouti K5-tTA;TRE-Edn3-lacZ mice displayed an overtly darkened coat phenotype relative to littermate controls. This was due to the augmented pigmentation in both pheomelanin and eumelanin bands in the hair shafts of transgenic mice (Figure 6h). Ay mice have a systemic overexpression of agouti-like transcripts that lead to a completely yellow coat

Transgenic Edn3 alters the Edn3ls, KitWv, and Ay mutant phenotypes

K5-tTA;TRE-Edn3-lacZ(476) mice were crossed into spontaneous Edn3ls, KitWv, and Ay mutants to evaluate resulting changes in skin and coat color phenotypes. K5-tTA;TREEdn3-lacZ(476)/Edn3ls/ls (n ¼ 4) mice displayed white regions in both the dorsal and ventral aspects of the coat (Figure 6a and c). A partial correction of the Edn3ls/ls phenotype was predominantly restricted to the rostral areas in transgenics (Figure 6a and b). The skin underlying the white areas of the coat did not display any pigmentation (Figure 6b). In an attempt to separate the effects of the transgene on coat pigmentation and the maintenance of the dermal population 10d

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Figure 6. Transgene-mediated Edn3 expression alters the phenotypes of Edn3ls/ls, KitWv/Wv, and Ay mice. (a–c) K5-tTA;TRE-Edn3-lacZ(476)/Edn3ls/ls mice (arrowheads) are shown with Edn3ls/ls littermates. (a and b) White dorsal patches in the coats (a) of 10 days K5-tTA;TRE-Edn3-lacZ(476)/Edn3ls/ls mice correlate to (b) depigmented regions of the skin underlying these same areas in the coat. A partial correction to the spotting phenotype is seen in the rostral areas of transgenic mice (arrows) (a and b). (c) The ventral belly spot of Edn3ls/ls mice is not rescued in 30 days K5-tTA;TRE-Edn3-lacZ(476)/Edn3ls/ls transgenics. (d) The coat of 20 days K5-rTA;TRE-Edn3-lacZ(476)/Edn3ls/ls mice (arrowhead) induced with dox at E13.5 resembles its Edn3ls/ls littermate. (e) Rescued ventral coat phenotype of K5-tTA;TRE-Edn3-lacZ(476)/KitWv/ þ mice (arrowhead) shown with a hypopigmented KitWv/ þ littermate. (f and g) K5-tTA;TRE-Edn3-lacZ(476)/ KitWv/Wv mice (arrowheads) manifest rostral and caudal areas of dark skin (arrows) at (f) 7 days; however, by (g) 15 days, these areas give rise to unpigmented white hair follicles (arrows). (h) The dorsal agouti coat of K5-tTA;TRE-Edn3-lacZ mice (arrowhead) is darkened relative to littermate controls. Magnified individual hair shafts are shown for each coat. (i) K5-tTA;TRE-Edn3-lacZ(476)/Ay mice (arrowhead) display a darkened coat color relative to Ay littermates. (j) The depilated skin of K5-tTA;TRE-Edn3-lacZ(476)/Ay mice (arrowhead) develop pigmented skin in contrast to their Ay littermates. Bar ¼ 15 mm.

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color (Michaud et al., 1994). K5-tTA;TRE-Edn3-lacZ(476)/Ay mice (n ¼ 3) (Figure 6i) were also darker than Ay littermate controls. Their hair follicles contain eumelanin deposits in contrast to their Ay littermates, which are pigmented only with pheomelanin. The depilated skin of K5-tTA;TRE-Edn3lacZ(476)/Ay mice (Figure 6j) was hyperpigmented, but less so than K5-tTA;TRE-Edn3-lacZ mice (Figure 1a). DISCUSSION In wild-type mice, only a few regions devoid of coat hair follicles, such as the tail, pinna, and snout, actually carry an extensive melanocyte population in the skin. Skin regions that are covered with hair have most of their melanocytes within hair follicles and, as a result, these areas of skin are unpigmented (Billingham and Silvers, 1960; Hirobe, 1995). K5-tTA;TRE-Edn3-lacZ transgenic mice developed prominent skin pigmentation from melanocytes distributed throughout the dermis. Transgenic mice showed increased pigmentation on most areas of the skin, except for those areas where melanocytes are normally not seen, such as the oral cavity and paw pads. However, these areas were found to be pigmented in hyperpigmented transgenic mice in which the Kit-ligand was driven by the Cytokeratin 14 promoter (Kunisada et al., 1998). These findings suggest that, unlike the Kit-ligand, Edn3 does not act as a secreted chemotactic signal. The extra Edn3 produced by the transgene and the localization of the transgenic Edn3 signal appears to be important to the phenotype of dark-skinned transgenics. However, the early-development Edn3 transgene expression pattern does not correspond to the previously described endogenous mesenchymal Edn3 expression (Leibl et al., 1999; Rice et al., 2000). This difference in localization is the likely explanation for the non-rescue of the hypopigmented coat of Edn3ls/ls mice. Previous work with a tetracycline inducible system found that Ednrb only needs to be transcribed in vivo between E10–E12.5 for normal melanocyte development (Shin et al., 1999). In these mice, the coat hair follicles are normally pigmented. Here, we show that transgenic Edn3 needs to be present at E10.5, for the generation of the dark skin phenotype. Together these results indicate that the early embryonic events important to the establishment of a fully pigmented coat are also important to the establishment of a hyperpigmented skin. Although the critical requirement for endothelin signaling occurs at an early embryonic age (Shin et al., 1999), Edn3 can have an effect on differentiated melanocytes. Edn3, most likely, affects their survival, as postnatal transgenic Edn3 signals from the skin were required to maintain an extensive population of dermal melanocyte lineage cells. Transgenic Edn3 signaling may also lead to a hyperpigmented coat by increasing the number of hair follicle melanocytes. The increased pigment deposits onto the hair shaft may also suggest an increase in the pigmentproducing ability of individual hair follicle melanocytes. This is supported by the finding that Ednrb signaling is required for the expression of tyrosinase (Hou et al., 2004). The synthesis of eumelanin from the hair follicle melanocytes of K5-tTA;TRE-Edn3-lacZ(476)/Ay mice indicates that 138

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the Ednrb/Edn3 signaling pathway partially rescues the inhibited Mc1-R/a-MSH pathway in the hair bulb melanocytes of Ay mice (Kobayashi et al., 1995; Barsh et al., 2000; Hearing, 2000; Lassalle et al., 2003). This is additional evidence that Edn3 signaling can influence melanocytes past the critical time requirement, not only by affecting their survival but also by regulating pigment switching. The lighter pigmented skin of K5-tTA;TRE-Edn3-lacZ(476)/Ay mice relative to K5-tTA;TRE-Edn3-lacZ mice also suggests that the Ay protein has a similar inhibitory function on the Mc1-R/a-MSH pathway in skin melanocytes. This is further supported by findings that show that epidermal melanocytes respond to the agouti protein (Hirobe et al., 2004). K5-tTA;TRE-Edn3-lacZ(476)/KitWv/Wv mice developed darkly pigmented skin in the head and tail regions. Similarly, when KitWv/Wv mice are crossed into those that have gain-offunction mutations in G-proteins associated with Ednrb, the resultant mice develop darkly pigmented ears (Raamsdonk et al., 2004). During development, these areas contain the highest density of melanoblasts (Pavan and Tilghman, 1994). Adult KitWv/Wv or Ednrb null mutants exhibit some pigmentation in these same locations. Thus, as long as either the Kit or Ednrb-mediated pathway is active, melanoblasts in areas of high cell density can survive and differentiate. The increased pigmentation in K5-tTA;TRE-Edn3-lacZ/ KitWv/Wv and K5-tTA;TRE-Edn3-lacZ/KitWv/ þ mice suggests that Edn3 signaling is able to partially compensate for the dearth of melanoblasts in KitWv/Wv mutants (Mackenzie et al., 1997). This result is similar to the white spot reduction seen in KitW57J/W57J mutant mice with a Cytokeratin 14 promoterdriven Edn3 transgene (Aoki et al., 2005). However, the completely white coat found in K5-tTA;TRE-Edn3-lacZ/KitWv/ Wv mice, despite underlying areas of pigmented skin, strongly suggests that Edn3 signaling does not compensate for one or all of the ensuing Kit-dependent events: locating melanoblasts for their entry and proliferation in the epidermis and the onset of hair follicle pigmentation during anagen (Yoshida et al., 1996). Owing to the genetic homology between mice and humans, the study of our Edn3 transgenic mice may offer some insight into the genetics behind different skin-related conditions such as benign dermal pigmentation. Endothelins, in particular, are known to exhibit a high level of zoological conservation (Baynash et al., 1994). Clinically, the presence of melanocytes in the dermis is termed dermal melanocytosis (Nordlund et al., 1998). There are several morphologic forms of dermal melanocytosis, such as the blue nevus, the Mongolian spot, the nevus of Ota, and the nevus of Ito (Velez et al., 1992). Most dermal melanocytoses are congenital such as the Mongolian spot, which typically fades and disappears in childhood, while other cases persist for life (Stanford and Georgouras, 1996). The genesis of dermal melanocytosis is thought to be heterogeneous, reflecting multiple factors or genes that regulate the development of melanocytes (Hori and Takayama, 1988). The transgenic mice described here suggest that Ednrb signaling may be involved in cases of acquired dermal melanocytosis. Acquired dermal melanocytosis appears to be

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caused by sunlight or endocrine and paracrine factors acting on immature melanocytes in the dermis. For example, ultraviolet irradiation is known to cause keratinocytes to continuously secrete Endothelin 1, which along with Edn3 can bind to Ednrb (Imokawa et al., 1992; Yanagisawa, 1994; Ahn et al., 1998). This signaling pathway is known to affect melanocytes and accelerate melanogenesis (Mizoguchi et al., 1997). Similarly, the continuous keratinocyte-driven expression of our Edn3 tetracycline transgenic system is required to sustain the population of pigment-producing melanocytes in the dermis of our transgenic mice. MATERIALS AND METHODS TRE-Edn3-lacZ vector construction and identification of founder transgenic mice A 1.4 kb BamH1 fragment containing a 1.068 kb murine Edn3 cDNA (GenBank Accession no. U32330) was obtained after a BamH1 digestion of the DbH-edn3 plasmid (Rice et al., 2000). The BamH1 fragment was cloned into plasmid pBI-3 (Baron et al., 1995). The resulting plasmid, pTRE-Edn3-lacZ, was digested with SapI and AclI, yielding a 7.5 kb transgene (TRE-Edn3-lacZ) that was microinjected into fertilized (C57BL/6XSJL)F2 eggs at the University of Michigan Transgenic Animal Model Core (Ann Arbor, MI). The screening of founder mice was done by PCR with primers that yield a 463 bp product and bind to the 30 end of the Edn3 cDNA (50 -AGGCCTGTGCACACTTCTGT-30 ) and to non-coding DNA (50 -TCCTTGTGAAACTGGAGCCT-30 ) found in the original DbHedn3 plasmid. Transgene copy numbers were determined by Southern blot hybridization on tail biopsy DNA. The probe, TREEdn3-lacZ-P, was generated with [32P]dATP and [32P]dCTP-labeled nucleotides from a 932 bp fragment (resulting from the SalI and Bsu36I digestion of pTRE-Edn3-lacZ).

Mice Mice carrying the TRE-Edn3-lacZ transgene (TRE-Edn3-lacZ) were crossed into K5-tTA or K5-rTA mice (Diamond et al., 2000) generated on an FVB/N genetic background. K5-tTA, TRE-Edn3-lacZ double transgenics (K5-tTA;TRE-Edn3-lacZ) were crossed to Dct-lacZ mice provided by Dr Paul Overbeek (Baylor College of Medicine, Houston) created on an FVB/N background (Zhao and Overbeek, 1999), Edn3ls (strain LS/LeJ), KitWv, and Ay mutants maintained on a C57BL/6J background (obtained from The Jackson Laboratory, Bar Harbor, ME). Noon of the day, a vaginal plug was found defined as E0.5. Harvested embryos were subsequently staged according to the standard references (Kaufman, 1995). All mice used in this study were housed in the Animal Care Facility at Florida International University (Miami, FL). Animal work was performed according to the institutional guidelines established by the NIH.

Genotyping Identification of transgenic animals and embryos was conducted by PCR of tail biopsies or yolk sac DNA. Dct-lacZ mice (Zhao and Overbeek, 1999) were recognized with primers that yield an 850 bp amplicon. One primer 50 -ACAAGGAAGACTGGCGAGAA-30 binds within the Dct promoter, while the other 50 -GCCGAGTTAACGCC ATCAAAAATA-30 adheres to the lacZ gene. K5-tTA or K5-rTA mice were genotyped with primers 50 -CCAGG TGGAGTCACAGGATT-30 and 50 -ACAGAGACTGTGGACCACCC-30

that amplify a 214 bp PCR product from the regulatory region of the Bos taurus Keratin 5 gene – GenBank accession no. Z32746 (Casatorres et al., 1994). The restriction enzyme digest, and primers used to genotype Edn3ls (Rice et al., 2000) and KitWv mutants (Cable et al., 1995) were described previously.

Documentation of transgene expression by reverse transcriptase-PCR K5-tTA;TRE-Edn3-lacZ (369 and 476) and control whole embryos or embryonic skin were triturated, and RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) and purified with DNAse treatment to prevent genomic DNA contamination according to the manufacturer’s instructions. Total RNA was used for cDNA synthesis using the SIII reverse transcriptase (SIII-RT; Invitrogen). PCR reactions were run for 31 cycles. Edn3 was amplified from previously described primers (Leibl et al., 1999) to yield a 244 bp product. Levels of cDNA were normalized with HPRT using primers (50 -GGAGCGGTAGC ACCTCCT-30 and 50 -AATCCAGCAGGTCAGCAAAG-30 ) that yield a 311 bp size product. Each experiment was represented by two dox-responsive embryos and littermate controls (without the TREEdn3-lacZ transgene). RU values were obtained from quantified gel images (IQ Mac v1.2). Values were obtained by dividing Edn3 mRNA levels by HPRT mRNA levels. Differences in RU values were judged as significant if the P-value was o0.05, as determined by the two-sample t-test (assuming equal variances).

Dox treatment Dox (3 mg/ml; Sigma, St Louis, MO) and sucrose (5%) were added to the drinking water of the mice subjected to transgene induction or repression studies on the morning of the indicated experiment. Based on the assumption that mice drink 5 ml of water every 24 hours, treated mice received 15 mg of dox daily. Each timed dox experiment was represented by at least three litters and three dox-responsive embryos or mice and littermate controls.

Postnatal histology Murine skin was harvested from the medial and dorsal aspect of the trunk and sectioned to obtain longitudinal cryosections (14 mm) through the hair follicles (Paus et al., 1999). Mice were depilated with Nair (Church and Dwight Co. Inc., Princeton, NJ) to expose the integument in hairy regions. Skin samples were stained with the Dopa reaction, lacZ stain, or for ammoniacal silver nitrate activity and counterstained with eosin as indicated. Biopsies were embedded in freezing medium (Triangle Biomedical Sciences, Durham, NC) and snap-frozen in liquid nitrogen. Each age group was represented by at least three dox-responsive transgenic mice and three littermate controls. Every sixth section, for a total of 30 sections (14 mm), was sampled from each mouse. Sections were photographed with a Leica DC500 digital camera mounted on a Leitz DMRB microscope.

Dopa, silver nitrate, and b-galactosidase staining Postnatal skin was subjected to the Dopa reaction to stain differentiated melanocytes with tyrosinase activity according to previously described protocols (Hirobe, 1984; Hirobe et al., 2002). To stain melanocyte lineage cells in postnatal skin, cryosections were rinsed in 0.05 M PBS and treated with a 10% ammoniacal silver nitrate solution (Sigma) for 5 minutes at room temperature. This stain www.jidonline.org

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permits the light microscopy detection of differentiated melanocytes and unmelanized melanoblasts (Hirobe, 1995). Whole embryos or skin biopsies were stained for b-galactosidase according to the standard protocols (Furth et al., 1994). Each lacZ experiment was represented by at least three dox-responsive transgenic mice and littermate controls. For the localization of embryonic lacZ þ transgene-expressing cells, 30 serial sections (14 mm) were taken from three sites in E12.5 K5-tTA;TRE-Edn3-lacZ embryos (the forelimb, hindlimb, and tail). E10.5 K5-rTA;TRE-Edn3lacZ embryos were assessed in a similar fashion at the forelimb bud.

Numerical analysis of embryonic cells

Baron U, Freundlieb S, Gossen M, Bujard H (1995) Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 23:3605–6 Barsh G, Gunn T, He L, Schlossman S, Duke-Cohan J (2000) Biochemical and genetic studies of pigment-type switching. Pigment Cell Res 13(Suppl 8):48–53 Baynash AG, Hosoda K, Giaid A, Richardson JA, Emoto N, Hammer RE et al. (1994) Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79: 1277–85 Billingham RE, Silvers WK (1960) The melanocytes of mammals. Q Rev Biol 35:1–40

The number of epidermal, dermal, and hair follicle Dct þ cells was quantified in cryosections obtained from K5-tTA;TRE-Edn3-lacZ/DctlacZ embryos. Dct þ cells at E13.5 were determined by counting the dorsolateral population of cells lying between the apical dorsal aspect of the embryo to the end of the forelimb; at E16.5, the density of Dct þ cells underlying the dorsal skin was determined by counting those cells underneath a curve of length 1.8 mm, as measured along the epidermis with Spot Advanced Software (Version 3.5, Diagnostic Instruments Inc., Sterling Heights, MI). Seventy hair follicles were analyzed per embryo. Thirty transverse serial sections (14 mm) were made from each embryo, with the first section containing the superior aspect of the forelimb and the others proceeding caudally. Each age group was represented by three K5-tTA;TRE-Edn3-lacZ/ Dct-lacZ mice and three littermate controls. Means and SD were calculated from pooled data and indicated on bar graphs. Differences were judged as significant if the P-value was o0.001 as determined by the two-sample t-test (assuming equal variances).

Byrne C, Tainsky M, Fuchs E (1994) Programming gene expression in developing epidermis. Development 120:2369–83

CONFLICT OF INTEREST

Diamond I, Owolabi T, Marco M, Lam C, Glick A (2000) Conditional gene expression in the epidermis of transgenic mice using the tetracyclineregulated transactivators tTA and rTA linked to the keratin 5 promoter. J Invest Dermatol 115:788–94

The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Dr Raj Kapur (University of Washington, Seattle) for the pDbHedn3 construct; Dr Hermann Bujard (Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg) for the pBI-3 construct; Dr Paul Overbeek (Baylor College of Medicine, Houston) for the Dct-lacZ mice; Sabrina Gerzenstein, Ronald Yglesias, Erasmo Perrera, Sean Mandat, and other members of Dr Lidia Kos’ laboratory (Florida International University, Miami) for their helpful discussions and technical assistance. We also thank Dr Kalai Mathee (Florida International University, Miami) for her immense generosity and Dr Kermit Carraway (University of Miami Medical School) for editing the manuscript. This work was partially supported by the College of Arts and Sciences (Florida International University, Miami). R.G. and J.F. were supported by Grant no. NIH/NIGMS R25 GM061347. L.L. was supported by Grant no.NIH/NIGMS T34 GM008771.

SUPPLEMENTARY MATERIAL Figure S1. Tetracycline sensitive transcriptional activators mediate Edn3 and lacZ transgene expression. Figure S2. Melanocytes in the skin of transgenic mice are found mostly in the dermis.

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