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Keywords:

  • alternate transcripts;
  • cutaneous malignant melanoma;
  • hair follicle cycling;
  • melanogenesis;
  • Pax3

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The transcription factor encoded by PAX3 is among the first expressed in the embryo, with a key role in development of the melanocytic lineage. Re-expression of PAX3, consistently observed in cutaneous malignant melanoma (CMM) as compared to normal melanocytes, appears linked to progression of CMM. Previous research has identified PAX3d (encoded by exons 1–9) as the predominant isoform present in CMM, together the with an alternate isoform PAX3c (encoded by exons 1–8). We investigated the expression of Pax3c and Pax3d transcripts during mouse development. The reverse transcription–polymerase chain reaction and immunohistochemistry experiments presented here implicate these transcripts in melanoblast development and demonstrate significant spatial and temporal differences in their expression. Differences in expression were also noted during active hair regrowth in adult skin, which is accompanied by proliferation and migration of melanoblasts into the hair cortex to color new hair. Results indicate that the defined spatial and temporal expression of Pax3d may be linked to either melanoblast proliferation or migration during melanogenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The paired box gene Pax3 is expressed in the neural tube, somite and neural crest during early embryonic development (Goulding et al. 1991; Stoykova & Gruss 1994; Williams & Ordahl 1994; Mansouri et al. 1996; Maroto et al. 1997; Tremblay et al. 1998; Galibert et al. 1999; Heanue et al. 1999; Hornyak et al. 2001). Of particular interest here is the role of Pax3 in the specification, proliferation and migration of neural crest cell derived melanoblasts as they migrate to the skin. Once they reach the skin, they terminally differentiate into melanocytes, with concomitant downregulation of Pax3 (Mayer 1973; Lang et al. 2005). The complex process of melanogenesis is strictly regulated by Pax3 acting in concert with a multitude of factors, in a concentration-dependent manner, to activate or repress melanocyte differentiation (Watanabe et al. 1998; Bondurand et al. 2000; Potterf et al. 2000; Lang et al. 2005). Pax3 is homozygous lethal, whereas the heterozygote (Splotch mouse) exhibits defects in melanocyte proliferation and migration (Henderson et al. 1997), as well as skeletal muscle and central nervous system defects (Epstein et al. 1991; Franz et al. 1993; Chalepakis et al. 1994).

The multiple functions regulated by the encoded Pax3 transcription factor are mediated by its highly conserved paired and homeo DNA-binding domains, as well as by its C-terminal transactivation domain; once Pax3 is bound to the target gene via its DNA-binding domains, the transactivation domain contacts other protein transcription factors to regulate target gene transcription (Chalepakis et al. 1994; Bondurand et al. 2000). Alternate isoforms of Pax3 (a–h), that differ in amino acid sequence within the paired and transactivation domains, have been identified (Barber et al. 1999; Barr et al. 1999; Parker et al. 2004). These alternate isoforms exhibit altered transcriptional specificity and activity (Vogan & Gros 1997).

While differences in function have been implied for alternate Pax3 transcripts (Du et al. 2005), and some differences in spatial and temporal expression patterns have been reported in human, murine and zebrafish tissues (Tsukamoto et al. 1994; Seo et al. 1998; Brown et al. 2005; Cook et al. 2005), the role and expression of individual transcripts during melanocyte development remains unknown. In addition, transcript expression during melanogenesis of mature skin has not been determined. Following catagen, melanocytes of the hair follicle are replaced by melanoblasts arising from a small population of stem cells within the follicular niche. Pax3 is known to be important for the proliferation and migration of these melanocytic stem cells from the hair follicle to a region proximal to the dermal papilla where they differentiate in order to produce melanin for new hair (Nishimura et al. 2002; Lang et al. 2005).

Interestingly, recent studies show that PAX3 transcripts, specifically PAX3c and PAX3d, are present at high levels in cutaneous malignant melanoma (CMM), whereas no PAX3 expression is found in surrounding tissue or in benign nevi (Barr et al. 1999; Scholl et al. 2001). These results indicate an association between PAX3 transcripts and CMM, where PAX3-controlled developmental programs of melanocyte proliferation, migration and differentiation may be reactivated in CMM cells.

In this investigation we sought to delineate Pax3c and Pax3d transcript expression during melanogenesis in developing and mature skin. To do this we have used reverse transcription–polymerase chain reaction (RT–PCR) and immunohistochemistry to assess the spatial distribution of these transcripts at developmental stages coinciding with melanoblast proliferation, migration and differentiation. Similarly, we have assessed the spatial and temporal distribution of these transcripts in mature murine skin during depilation-induced melanogenesis. Investigation of Pax3 expression profiles at key stages of melanogenesis may clarify functional roles for specific Pax3 transcripts and provide information regarding their putative role in CMM.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Collection of murine embryonic samples

Pregnant female mice and embryos were euthanized by CO2 overdose. Staged embryos were surgically removed at embryonic day 11 (E11), E12.5, E15 and E20. For mRNA analyses, embryonic tissue was frozen in liquid nitrogen following excision and kept at −80°C until use. Tissues isolated from embryos included skin, skeletal muscle (trunk) and brain. Skin was segregated to examine Pax3 transcripts, in particular at E15, during melanoblast migration into the developing hair placode, and at E20, during differentiation of the melanoblast into a melanin producing melanocyte. Ethical clearance for use of the animals was acquired through the Animal Ethics Committee of Edith Cowan University (project no. 03-A8) and conformed to the provisions of the Declaration of Helsinki, 1995 and Edinburgh, 2000.

Collection of mature murine skin samples

To assess transcript-specific expression of Pax3 in mature skin during normal hair follicle re-pigmentation, active synchronized hair regrowth (anagen) was induced by depilation; such experimental methods are commonly used to assess gene expression during murine hair regrowth (Paus et al. 1994).

Wild-type C57BL/6 mice were sedated with midazolam (Roche, Basel, Switzerland) via intramuscular injection at a dosage of 0.1 mg/kg. A 15 mm × 15 mm area of skin was depilated at the cervical region of the back using a wax/rosin mixture warmed to 38°C. At 12, 24, 36, 48 and 60 h post-depilation, mice were euthanized by CO2 overdose and the depilated skin was surgically removed, cleansed with 100% ethanol, frozen in liquid nitrogen and kept at −80°C until use. The depilation experiments and use of the animals conformed to guidelines laid down by the National Health and Medical Research Council of Australia and ethical clearance for the experiments was acquired through the Animal Ethics Committee of Edith Cowan University (project no. 03-A8).

Human tissue collection

Five metastatic CMM samples surgically excised from regional lymph nodes were kindly donated by Clinical Professor Dominic Spagnolo of PathCentre (Perth, WA, Australia). Samples were frozen in liquid nitrogen immediately after surgical excision and stored at −80°C until use. Ethical clearance for use of the human tissues was acquired through the Human Ethics Committee of Edith Cowan University (project no. 02-68). Anonymity of donors was preserved.

Isolation of total RNA: Murine embryos, mature murine skin and cutaneous malignant melanoma

Murine embryo tissues and CMM samples were homogenized in a glass-col homogenizer using 1 mL of Trizol (Invitrogen, San Diego, CA, USA) per 50–100 mg of tissue. Skin samples from mature mice were frozen in liquid nitrogen and crushed in a mortar and pestle prior to homogenization in Trizol. Homogenized samples were passed through a 19 gauge needle several times prior to addition of 10 µL proteinase K solution (10 mg/mL ddH2O) followed by incubation at room temperature for 10 min. Total RNA was isolated as previously described (Ziman et al. 2001). Integrity of total RNA was assessed by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining under ultraviolet (UV) light.

Poly(A)+ mRNA purification

mRNA was isolated from total RNA using an Oligotex mRNA kit (QIAGEN, Tokyo, Japan) according to the manufacturer's instructions and stored at −80°C until further use. mRNA was isolated from total RNA, as amplification of Pax3c by RT–PCR used a reverse primer that corresponds to a retained intronic sequence. To ensure genomic DNA was not amplified from tissue samples, negative controls for each sample included elimination of the reverse transcription portion of the RT–PCR reaction.

Reverse transcription–polymerase chain reaction

Poly(A)+ mRNA was converted to cDNA using a One-Step RT–PCR Kit (QIAGEN) according to the manufacturer's instructions. Primers specific for human or murine PAX3/Pax3 sequences were as follows: PAX3c (forward), 5′-GCAATTTCTCCTGGAAGGGA-3′; PAX3c (reverse), 5′-ATTGATACCGGCATGTGTGG-3′; PAX3d (forward), 5′-TGGGCAGTATGGACAAAGTG-3′; PAX3d (reverse), 5′-GGCTGCGAAGACCAGAAAC-3′; Pax3c (forward), 5′-GGGGGTAGTTCCTCCTGG-3′; Pax3c (reverse), 5′-CAATCAGCTGTCTTTGCCAC-3′; Pax3d (forward), 5′-TGGGCAGTATGGACAAAGTG-3′; Pax3d (reverse), 5′-GTGGAGGCCGGAAACAGG-3′.

Cycling conditions for RT–PCR were 30 min at 50°C, 15 min at 95°C, followed by 40 cycles of 30 s at 94°C, 15 s at 56°C and 15 s at 72°C, with a final extension of 10 min at 72°C. Negative controls lacked template mRNA. RT–PCR products were analyzed by electrophoresis on 2% agarose gels and visualized with ethidium bromide staining under UV light. PCR products were sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster City, CA, USA) and an ABI Prism 3730 48 capillary sequencer. Sequences were aligned with known sequences in GenBank using the multiAlign tool in Angis, available on GenBank.

An RT–PCR analysis of Pax3c and Pax3d from each tissue provides a relative expression profile and therefore an internal control for each transcript. This is sufficient for determination of the presence or absence of transcripts, and reactions relative to a standard control gene are therefore not necessary.

Immunohistochemistry

Following surgical excision, embryos were immediately, frozen in thawing isopentane and stored at −80°C until cryosectioned in the sagittal plane at 6 µm onto Superfrost Plus slides. Sections were air-dried prior to immersion in acetone at 4°C. Slides were rehydrated in Tris-buffered saline and incubated in phosphate-buffered saline (PBS) and Triton-X100 (0.2%) for 10 min. Sections were then incubated in PBS containing 3% H2O2 for 10 min, rinsed and blocked in PBS containing 10% fetal calf serum for 30 min. Samples were incubated overnight at 4°C with antibodies to Pax3c or Pax3d (1:10, rabbit polyclonal), kindly donated by Dr Tom Barber, Johns Hopkins University, USA. These antibodies have been assessed as specific for each isoform and therefore are able to clearly detail spatial differences in expression between the two isoforms (Barber et al. 1999). Amino acid sequences from the unique C-termini of Pax3c (KPWTF) and Pax3d (AFHYLKPDIA) were used for generation of the antibodies (Barber et al. 1999).

After 12 h, sections were washed twice in PBS and incubated with biotinylated antirabbit IgG (LSAB2 System, DAKO, Carpinteria, CA, USA) for 10 min at room temperature. Sections were then washed in PBS prior to application of horseradish peroxidase (HRP)-linked streptavidin (LSAB2 System, DAKO) for 10 min at room temperature. Following a wash in PBS, immunohistochemical staining was visualized using 3,3-diaminobenzidine (DAB, Sigma Chemicals, St Louis, MO, USA) as chromogen, and mounted in DePeX (BDH Laboratory Supplies, Poole, UK). Negative controls on the same slide were processed at the same time but contained no primary antibody.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PAX3c and PAX3d transcripts in cutaneous malignant melanoma

Expression of PAX3c and PAX3d is clearly evident in all CMM biopsies tested (5/5; cf. Figure 2). PAX3c/Pax3c mRNA is transcribed by exons 1–8 of the PAX3/Pax3 gene. The 3′ end of PAX3c/Pax3c (beginning at exon 8; human sequence shown in Fig. 1) is generated using the stop codon and polyadenylation signals located within intron 8 (Barber et al. 1999; Barr et al. 1999). PAX3d/Pax3d mRNA is transcribed from exons 1–9; the 5′ splice site for intron 8 is located 11 nucleotides upstream of the PAX3c/Pax3c stop codon with intron 8 branchpoint, polypyrimidine tract and acceptor site located downstream. In generation of the PAX3d/Pax3d transcript, the first stop codon and polyadenylation signal, used to generate PAX3c/Pax3c, are ignored. Instead transcription continues until the second stop codon and polyadenylation signal are reached, located in exon 9. The intron 8 5′ splice site, used to produce the PAX3d/Pax3d transcript, is considered unfavorable due to mismatching of the consensus sequence (Nelson & Green 1988; Zamore et al. 1992), and a polypyrimidine tract that extends only seven nucleotides (Smith et al. 1993). It was thought curious that in CMM, PAX3d mRNA transcripts predominate, although produced from the less favorable splice signals.

image

Figure 2. Detection of Pax3c, Pax3d transcripts in murine embryos and human cutaneous malignant melanoma (CMM). Murine embryos at embryonic day 11 (E11), E12.5, E15 and E20: reverse transcription–polymerase chain reaction (RT–PCR) products generated from mRNA of mouse embryos, using Pax3c and Pax3d primers, visualized on 2% ethidium bromide-stained agarose gel. Pax3c, amplified Pax3c transcript (expected product length, 117 bp); Pax3d, amplified Pax3d transcript (expected product length, 97 bp); -co, negative control reaction without template mRNA; pUC, DNA ladder (pUC19 digested with HpaII); Hd, head (brain); bd, body (trunk skeletal muscle); sk, skin. At E11 and E12.5, mRNA was isolated from whole embryos and bodies, respectively, since the skin was extremely thin and fragile and could not be separated. At these stages, RT–PCR products may therefore be indicative of expression in neural, skeletal muscle and/or melanocyte cells. Detection of PAX3c, PAX3d transcripts in human skin biopsies. RT–PCR products generated from mRNA of human CMM biopsies, using PAX3c and PAX3d primers, visualized on 2% ethidium bromide-stained agarose gels. PAX3c, amplified PAX3c transcript (expected product length, 89 bp); PAX3d, amplified PAX3d transcript (expected product length, 104 bp); 1–5, CMM samples; -co, negative control reaction without template mRNA.

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image

Figure 1. Generation of PAX3c and PAX3d transcripts by alternate splicing. Shown is the genomic sequence of PAX3, from exon 8 to exon 9. The exons are denoted by upper case letters while the intronic region is denoted by lower case letters. In the generation of PAX3c, the splice site at the 3′ end of exon 8 is not recognized and transcription continues until the stop codon and adenylation site, located near the 5′ end of intron 8 (indicated in red) are reached. In the generation of PAX3d, intron 8 is spliced out at the 5′ and 3′ consensus donor/acceptor sites (indicated in blue), utilizing the branchpoint sequence and polypyrimidine tract (indicated in green and orange, respectively). Thus the first stop site and polyadenylation signal are not used and transcription continues until the second stop signal and adenylation site located in exon 9 (indicated in pink) are reached.

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To assess whether the alternative PAX3d transcript is aberrantly produced only in melanoma cells or is a functional transcript used in human melanoblast cell development, GenBank was searched for PAX3 transcripts with alternative 3′ ends. One expression sequence tag was found in a human melanocyte cDNA library (GenBank H82467). Moreover, an identical C-terminal sequence to that of PAX3d is found in Pax3 from quail (GenBank AF000673) and mouse (skeletal muscle, Barber et al. 1999), suggesting that PAX3d is a conserved isoform. The central aim of this research then was to determine Pax3c and Pax3d expression profiles during normal murine melanocytic development.

Pax3 transcripts in murine embryos

Murine embryonic tissues including skin, brain and skeletal muscle (trunk and limb) were analyzed by RT–PCR for mRNA expression and by immunohistochemistry for locality of Pax3c and Pax3d at key developmental stages (Figs 2–4). More than twenty murine embryonic samples were assayed with between six and nine embryos assayed at each stage, except at E11 when only one embryo was assayed.

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Figure 3. Immunohistochemical staining of Pax3-positive cells in embryonic day 12.5 (E12.5), E15 and E20 skin. Immunohistochemical staining using Pax3c (A,C,E,G) and Pax3d (B,D,F,H) primary antibodies, and a biotin–streptavidin–peroxidase system visualized with diaminobenzidine (DAB) chromogen. At E12.5 (A–D), Pax3-positive cells, such as those indicated by arrows, are seen scattered throughout the dermis (Pax3c, A,C) and epidermis (Pax3d, B,D) in close proximity to the dermal/epidermal border, in skin of the trunk (A,B) and the fold of nare (C,D). Bar, 100 µm. At E15, Pax3c-positive cells (E) and Pax3d-positive cells (F) are seen in the hair follicles (HF) and epidermis, respectively, as well as in panniculus carnosus muscle (PC) as indicated by arrows. At E20, Pax3c-positive cells (G) are seen above the oval shaped dermal papilla (DP) and in the midst of the distal hair follicle epithelium (located closest to the epidermis) as indicated by arrows. At E20, no positive Pax3d-positive cells are observed in the skin (H). D, dermis; DP, dermal papilla; HF, hair follicles; PC, panniculus carnosus muscle; SC, subcutis.

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Figure 4. Immunhistochemical staining of Pax3-positive cells in embryonic day 20 (E20) midbrain and skeletal muscle. Immunohistochemical staining of E20 sections using Pax3 primary antibodies and a biotin–streptavidin–peroxidase system visualized with diaminobenzidine. Pax3c-positive (A) and Pax3d-positive (B) cells are seen in the dorsal superior colliculus. Pax3c-positive nuclei are seen at the periphery of skeletal muscle cells (C) and in panniculus carnosus muscle (D) as indicated by arrows.

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Analysis using RT–PCR revealed that as early as E11 and E12.5, when neural crest cells proliferate and migrate to the dermis and epidermis (Mayer 1973; Yoshida 1996), both Pax3c and Pax3d transcripts are expressed in murine embryos (Fig. 2). Similarly, Pax3c and Pax3d transcripts are expressed specifically in skin, brain and skeletal muscle at E15, confirming a role for Pax3 transcripts in the specification of cells of these tissues (Barber et al. 1999; Fig. 2). At E20 however, while Pax3c remains expressed in these tissues, Pax3d expression is downregulated in skin but continues to be expressed in brain and skeletal muscle (Fig. 2). Results were confirmed in nine E15 and E20 embryos. RT–PCR negative controls showed no product at all stages examined (Fig. 2).

Spatial distribution of Pax3c and Pax3d during melanocyte development

Yoshida (1996) demonstrated that between E12.5 and E13.5, melanoblasts enter the epidermis synchronously where they proliferate extensively. Positive staining of Pax3c and Pax3d proteins is seen throughout the dermis at E12.5, and expression appears to be localized within melanoblasts (Fig. 3; adjacent hematoxylin–eosin (HE) sections not shown); however, co-localization with melanoblast markers was not performed. Staining confirms RT–PCR results demonstrating Pax3c and Pax3d expression in early embryonic tissues.

At E15, melanoblasts migrate toward the developing hair germs where they localize in hair follicle pigmentary units (Hirobe 1984). At this stage, most Pax3c-positive cells are seen within developing hair follicles, and based on their location within the follicle these cells are likely to be melanoblasts (adjacent HE sections not shown; Müller-Röver et al. 2001). By contrast, few Pax3d-positive cells are observed in follicular cells; most Pax3d-positive cells remain distributed profusely throughout the epidermis where there are few Pax3c-positive cells (Fig. 3).

Murine epidermal melanoblasts begin to terminally differentiate at around E14. At E20, epidermal and hair follicular melanoblasts are characterized by melanin formation indicating an advanced stage of melanocytic differentiation (Hirobe 1984). In E20 skin, positive Pax3c staining is primarily seen within the hair follicle above the dermal papilla and among the distal hair follicle epithelium (Fig. 3). No Pax3d-positive staining is observed in the skin at E20 (Fig. 3), confirming the RT–PCR results.

Pax3c and Pax3d in brain and skeletal muscle during embryonic development

Pax3c-positive and Pax3d-positive cells are present in the midbrain of both E15 and E20 embryos (Fig. 4): the first evidence of a role for the Pax3d transcription factor within the developing brain. Positive staining is also seen for both Pax3c and Pax3d in trunk skeletal muscle at E15 (Fig. 4), confirming the RT–PCR results (Fig. 2). At E20 Pax3c-positive staining can be clearly seen in the nuclei of trunk skeletal muscle cells (Fig. 4), shown clearly for panniculus carnosus muscle (Fig. 4) while Pax3d is not observed in these cells (results not shown).

In summary, RT–PCR and immunohistochemistry reveal that while both Pax3c and Pax3d are expressed during normal murine development, they appear to have distinct spatial and temporal expression patterns at specific stages of melanogenesis.

Pax3 mRNA in follicular regrowth

After birth most murine epidermal melanocytes, except those of hairless areas such as the ears and tail, undergo apoptosis (Hirobe 1984), leaving only melanocytes of the hair follicles to produce pigmentation of the coat. As the mature hair follicle cycles through stages of growth, regression, resting and shedding, pigment cells or melanoblasts also cycle through periods of proliferation, migration, differentiation and apoptosis (Tobin et al. 1998; Müller-Röver et al. 2001; Nishimura et al. 2002). Histologic and ultrastructural studies have demonstrated that when the hair follicle undergoes active growth (anagen), melanoblasts migrate out of a stem cell niche, proliferate, localize in the hair matrix before further proliferation and differentiation into mature melanocytes (Nishimura et al. 2002). Pax3 is required for maintenance of melanoblasts as undifferentiated stem cells in the follicular niche (Lang et al. 2005).

Here we induced spontaneous anagen by depilation of C57BL/6 pigmented mice (Paus et al. 1994), and Pax3 transcripts were analyzed in skin samples every 12 h up to 60 h post-depilation, at stages corresponding to proliferation and migration of melanoblasts into the regenerating hair placode. At 96 h post-depilation, melanin granules appear above the dermal papilla, indicating melanocyte differentiation, therefore it was presumed that Pax3 would not be expressed at this stage (Müller-Röver et al. 2001). Two separate depilation experiments were performed and at each stage, skin samples were analyzed for Pax3c and Pax3d expression (a total of 10 animals were used). HE staining was performed on adjacent sections of depilated skin to ensure complete removal of hair sheath and associated melanocytes of the inner root sheath, and to ensure that no skeletal muscle tissue was present in the samples (Fig. 5).

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Figure 5. Detection of Pax3c, Pax3d transcripts in mature murine skin after depilation. (A) A representation of one experiment showing the reverse transcription–polymerase chain reaction products generated from depilated skin mRNA samples, using Pax3c and Pax3d primers, visualized on 2% ethidium bromide-stained agarose gels. Pax3c, amplified Pax3c transcript (expected product length, 117 bp); Pax3d, amplified Pax3d transcript (expected product length, 97 bp); 12, 24, 36, 48, 60 indicate time intervals after depilation; -co, negative control reaction without template mRNA; pUC, DNA ladder (pUC 19 digested with HpaII). This experiment was repeated on a separate group of animals and at each time point separate animals were analyzed. (B) Hematoxylin–eosin staining was performed on adjacent sections of depilated skin to ensure complete removal of the hair sheath and associated melanocytes of the inner root sheath and to ensure that no skeletal muscle tissue was present in the samples.

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In both depilation experiments, Pax3c expression was observed at 24, 36, 48 and 60 h post-depilation. By contrast, Pax3d expression was observed as early as 12 h and then only at 48 h post-depilation (Fig. 5). These results indicate that Pax3c and Pax3d are expressed at early stages of anagen, indicative of a role in adult melanogenesis during proliferation, migration and/or differentiation of melanoblasts. Moreover, stage specific differential expression of these transcripts implies functional differences during these processes.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PAX3c and PAX3d transcripts are highly expressed in cutaneous malignant melanoma samples, yet their expression is not found in normal skin (Scholl et al. 2001; Blake & Ziman 2003; Blake, unpubl. obs., 2004). Previous research has identified these transcripts as being among a large number of alternate Pax3 transcripts that encode isoforms with varied binding and transactivation domains (Tsukamoto et al. 1994; Underhill & Gros 1997; Fortin et al. 1998; Seo et al. 1998). Here we have sought to analyze Pax3c and Pax3d transcript expression, specifically during normal murine development, and at stages of melanogenesis in adult murine hair cycling, with the aim of gaining insight into their melanogenic function and their contributing roles in the tumorigenicity of CMM.

Experiments performed here demonstrate that Pax3c and Pax3d transcripts are in fact expressed from early developmental stages: at E11 as neural crest cells migrate from the neural tube, at E12 when melanoblasts migrate from the dermis into the epidermis and proliferate in the epidermis/dermis, at E15 during migration of melanoblasts into the hair follicle and at E20 when melanoblasts remain as quiescent stem cells in the niche of hair follicles.

Observed spatial and temporal differences in expression of Pax3c and Pax3d may be related to functional differences. For example, Pax3c-positive cells are primarily localized in the developing hair follicles, while Pax3d-positive cells remain in the epidermis. This may indicate that cells which successfully migrate to the developing hair placode express Pax3c, whereas Pax3d-positive cells either do not migrate, or express Pax3d prior to migration. These differences in Pax3c and Pax3d expression support the notion that they may assist in the complex regulation of melanocytic development by differential regulation of target genes.

Since Pax3c and Pax3d appeared relevant to melanoblast development, their expression during melanogenesis of mature skin was also investigated. Following successful depilation, the only melanocytic cells in murine skin should be those of the follicular stem cell niche; therefore, analysing Pax3 transcript expression during anagen indicates their association with proliferating and migrating follicular melanoblasts.

As during skin development, Pax3c and Pax3d exhibit differential expression patterns during hair cycling of murine skin, further highlighting their proposed functional differences. Using the time-scale of Müller-Röver et al. (2001), follicular transition from resting state (telogen) to active growth (anagen) occurs around 24 h following depilation. Therefore, expression of Pax3d within 12 h post-depilation, prior to anagen induction, may indicate a role for this transcript in proliferation of follicular stem cells (Nishimura et al. 2002). It should be noted that depilation induces a short healing response; however, neither wounded nor healing murine skin expressed Pax3d (Blake, unpubl. obs., 2005). Furthermore, Pax3d expression at 48 h post-depilation, coinciding with anagen, may be related to a secondary wave of melanoblast proliferation. This proliferation occurs as progeny cells of the stem cell population undergo further mitosis proximal to the dermal papilla prior to differentiation into pigmented melanocytes (Nishimura et al. 2002).

Alternately, Pax3d expression may be linked to migration of melanoblasts, initially in the 12 h post-depilation period, as stem cells migrate from the niche, and then again between 36 and 48 h post-depilation, as cells migrate from the bulge area toward the dermal papilla. During murine embryogenesis, Pax3d expression was demonstrated at all times of melanoblast migration, supporting this hypothesis.

Conversely, Pax3c expression is not observed in the 12 h post-depilation period, and it may not have a role in the events prior to anagen induction. Moreover, Pax3c expression directly correlates to the onset of anagen at 24 h and continues through until the onset of melanin production associated with melanocyte differentiation. This is not unexpected, as Pax3 is known to regulate genes involved in melanin synthesis, such as Mitf-m and tyrosinase-related protein-1. Conversely, Pax3 also silences these genes in order to maintain an undifferentiated quiescent stem cell in mature skin (Galibert et al. 1999; Lang et al. 2005). The alternate transcripts Pax3c and Pax3d may differentially regulate these genes to promote melanocyte differentiation or quiescent stem cell maintenance. Interestingly, in our experiments, Pax3c but not Pax3d is present in cells of the hair follicle during development. Thus Pax3d may regulate proliferation and/or migration rather than quiescence.

Finally, it should be noted that in the 36–48 h period post-depilation, both Pax3c and Pax3d transcripts are concurrently expressed within the skin. Further studies are required to differentiate Pax3c and Pax3d expression in different populations of quiescent, proliferating and migrating cells in order to gain insight into their individual and specific roles. Such experiments would include co-localization experiments using isoform specific antibodies and cell stage markers, as well as chromosomal hybridization immunoprecipitation assays to determine transcript-specific target gene sequences.

In conclusion, our results clearly demonstrate a conserved role for the Pax3c and Pax3d transcription factors in early development of the melanoblast, prior to differentiation and melanin production. Our future studies aim to investigate the role of Pax3 isoforms in cell proliferation and migration. As we continue to uncover the cellular events that take place during the transformation of the undifferentiated stem cell to the terminally differentiated adult cell, we may discover key players responsible for the propagation of neoplastic cell properties.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to take this opportunity to gratefully acknowledge and thank the following persons without whom this research would not have been possible: Dr Tom Barber, Johns Hopkins University, for Pax3 antibodies; Clinical Professor Dominic Spagnolo, for human CMM biopsies; and Dr Adrian Charles, Dr Lawrence Yu, Dr Fiona Wood and Dr Peter Heenan for consultations.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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  • Barr, F. G., Fitzgerald, J. C., Ginsberg, J. P., Vanella, M. L., Davis, R. J. & Bennicelli, J. L. 1999. Predominant expression of PAX3 and PAX7 forms in myogenic and neural tumor cell lines. Cancer Res. 59, 54435448.
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