Human Melanoblasts in Culture: Expression of BRN2 and Synergistic Regulation by Fibroblast Growth Factor-2, Stem Cell Factor, and Endothelin-3

Article (PDF Available)inJournal of Investigative Dermatology 121(5) · December 2003with34 Reads
DOI: 10.1046/j.1523-1747.2003.12562.x · Source: OAI
The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.
Human Melanoblasts in Culture: Expression of BRN2 and
Synergistic Regulation by Fibroblast Growth Factor-2, Stem
Cell Factor, and Endothelin-3
Anthony L. Cook,
w Philippe D. Donatien,zyyy Aaron G. Smith,
Mark Murphy,z Malcolm K. Jones,#
Mee nhard Herlyn,yy Dorothy C. Bennett, z J. Helen Leonard,w and Richard A. Sturm
The Institute for Molecular Bioscience, Center for Functional and Applied Genomics,The University of Queensland, Brisbane, Australia; wQueensland
Radium Institute Research Unit and #Division of Infectious Diseases and Immunology,The Queensland Institute of Medical Research, Brisbane,
Australia; zUniversity College London, Institute of Ophthalmology, London, UK; yDepartment of Basic Medical Sciences, St. Georges Hospital Medical
School, London, UK; yyThe Wistar Institute, Philadelphia, Pennsylvania, U.S.A.; zDepartment of Anatomy and Cell Biology, University of Melbourne,
Melbourne, Australia
The BRN2 transcription factor (POU3F2, N-Oct-3) has
been implicated in development of the melanocytic
lineage and in melanoma. Using a low calcium med-
ium supplemented with stem cell factor, ¢broblast
growth factor-2, endothelin-3 and cholera toxin, we
have established and partially characterised human mel-
anocyte precursor cells, which are unpigmented, contain
immature melanosomes and lack L-dihydroxyphenyla-
lanine reactivity. Melanoblast cultures expressed high
levels of BRN2 compared to melanocytes, which de-
creased to a level similar to that of melanocytes when
cultured in medium that contained phorbol ester but
lacked endothelin-3, stem cell factor and ¢broblast
growth factor-2. This decrease in BRN2 accompanied
a positive L-dihydroxyphenylalanine reaction and in-
duction of melanosome maturation consistent with
melanoblast di¡erentiation seen during development.
Culture of primary melanocytes in low calcium med-
ium supplemented with stem cell factor, ¢broblast
growth factor-2 and endothelin-3 caused an increase in
BRN2 protein levels with a concomitant change to a
melanoblast-like morphology. Synergism between any
two of these growth factors was required for BRN2
protein induction, whereas all three factors were re-
quired to alter melanocyte morphology and for maxi-
mal BRN2 protein expression. These ¢nding implicate
BRN2 as an early marker of melanoblasts that may
contribute to the hierarchy of melanocytic gene con-
trol. Key words: melanoma/POU/melanocyte/tyrosinase. J
Invest Dermatol 121:1150 ^1159, 2003
OU domain DNA-binding transcription factors are
developmental regulators of multiple cell lineages and
represent a conserved family of proteins throughout
metazoan evolution (Ryan and Rosenfeld, 1997; Dailey
and Basilico, 2001). The Class III factor BRN2 (also
known as POU3F2 a nd N-Oct-3) has been implicated in the de-
velopment of several neural and glial cell lineages, including neu-
rons and astrocytes (Fujii and Hamada, 1993; Schreiber et al,1994),
as well as the neural-crest-derived melanocytic lineage, owing to
its expression in murine melanoblasts (Eisen et al,1995).BRN2
also appears to be important for development of malignant mel-
anoma because its expression in human melanoma cell lines is
much higher than in primary melanocytes (Cox et al,1988;
Thomson et al, 1993; Ei sen et al, 1995), and melanoma cell lines
expressing antisense BRN2 lose the ability to form tumors i n
mice (Thomson et al, 1995). Additio nally, BRN2 ablated melano-
ma cells revert to a less mature cell type lacking many markers of
di¡erentiated melanocytes such as tyrosinase (TYR)-related pro -
tein (TYRP) pigmentation ge nes and the microphthalmia asso-
ciated transcription factor (Thomson et al, 1995), implicating
BRN2 in sustai ning the melanocytic phenotype. Interestingly,
expression of BRN2 can be modulated in human melanoma cell
lines, with di¡erentiating agents decreasing expression and
depigmentation agents increasing expression (Sturm et al,1991),
implying a role for BRN2 in mai ntai ning the undi¡erentiated
melanocytic phenotype.
Melanoblasts are the neural-crest-derived precursor of the
melanocytes. Their study is valuable for the analysis of basic
mechanisms in cell di¡erentiation, for comparison with poorly
di¡erentiated cells from melanoma, and for the molecular
analysis of the many known genetic disorders of melanocyte de-
velopment. Melanoblasts have been de¢ned as unpigmented cells
that lack functional TYR, the critical enzyme of melanin synth-
esis (Hirobe, 1992; Sviderskaya et al, 1995) containing only imma-
ture melanosomes (Kawa et al, 20 00). However, others have
de¢ned melanocytes as cells able to synthesize melanosomes
(Jimbow et al, 1999) which mature through a four stage process,
with Stage I^II melanosomes unable to synthesize melanin and
hence considered immature, whereas Stage III^IV contain mela-
nin (Marks and Se abra, 2001).
Reprint requests to: Richard A. Sturm, Institute for Molecular
Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
Abbreviations: DOPA,
L-dihydroxyphenylalanine; EDN3, endothelin-3;
EMSA, electrophoretic mobility gel shift assay; FBS, fetal bovine serum;
FGF2, ¢broblast growth factor-2; IFA, anti-intermediate ¢lament protein;
PBS-T, phosphate-bu¡ered saline/0.05% Tween 20; SCF, stem cell factor;
SILV, silver locus; TPA, 12-O-tetradecanoylphorbol-13-acetate; TYR, tyro-
sinase; TYRP, tyrosinase-related protein.
Manuscript received January 30, 2003; revised May 3, 2003; accepted for
publication May 29, 2003
Copyright r 2003 by The Society for Investigative Dermatology, Inc.
Expression of the melanogenic enzymes TYR, TYRP1, a nd
dopachrome tautomerase is useful in identifying melanoblasts.
Of these, dopachrome tautomerase is the earliest melanoblast
marker in murine embryos, being detected at 10 d postcoitum,
whereas Tyr and Tyrp1 are not expressed until 14.5 d postcoitum
and pigment production is not visible until 16.5 d postcoitum
(Steel et al, 1992). In human embryos, melanoblasts have been
detected by expression of another melanosomal protein SILV
(determined by HMB-45 immunoreactivity) in the epidermis
after 7 wk of gestation, with premelanosomes detectable at 10 wk
(reviewed in Reedy et al,1998).
Recently, Nishimura et al (2002) demonstrated that murine
melanoblasts reside in the lower permanent portion of the hair
follicle and act as stem cells to supply melanocytes that provide
melanin to the growing hair. Additionally, migration of melano-
blasts from one hair follicle through the epidermis to follicles
lacking melanoblasts can occur, where they assume the stem cell
role in the new follicle. In hairless epidermis, melanoblasts di¡er-
entiate to melanocytes which provide the surrounding keratino-
cytes with melanosomes for protection from ultraviolet radiation
(Herlyn et al, 2000; Seiberg, 2001).
Cultures of murine melanoblasts have been described pre-
viously (Sviderskaya et al,1995; Kawaet al, 200 0; Sviderskaya
et al, 2001) and are capable of di¡erentiation in vitro to melano-
cytes. Several peptide growth factors are known to be required
for melanocytic development in mouse a nd for the culture of
both human and mouse melanocytes (Bohm et al, 1995; Moell-
mann and Halaban, 1998; Reedy et al, 1998; Goding, 2000; Hala-
ban, 2000). Conditions for melanoblast culture typically include
these growth factors in the medium. Stem cell factor (SCF) acts
as a chemotactic factor for migration of murine melanoblasts into
hair follicles (Jordan and Jackson, 2000) and can mediate interac-
tions with the environment by altering integrin expression (Scott
et al, 1994). SCF enhances murine melanoblast survival and prolif-
eration in culture a nd can inhibit pigmentation (Re id et al,1995;
Sviderskaya et al, 2001). Endothelin-3 (EDN3) causes the di¡eren-
tiation of chick neural crest cultures to melanocytic and glial pre-
cursor cells (Lahav et al, 1996; Lahav et al, 1998); however,
proli feration of immortal murine melanoblasts is not ected by
removal of EDN3 (Sviderskaya et al, 2001). Interestingly, murine
melanoblast cultures from piebald mice, which carry mutations in
the EDN3 receptor Ed nrb, have impaired growth and di¡erentia-
tion (Sviderskaya et al,1998).
Fibroblast growth factor-2 (FGF2) is a mitogen for melano-
cytes (Halaban et al, 1988a, b) and like SCF is required for murine
melanoblast proliferation (Sviderskaya et al, 2001). FGF2 also may
be involved in melanoma progression, because adenovirally de-
livered FGF2 conferred melanoma-l ike growth characteristics to
human melanocytes (Nesbit et al, 1999). In combination with
ultraviolet B it also induced lentiginous melanoma in sk in recon-
struction models (Berking et al, 2001). The phorbol ester 12-O-
tetradecanoylphorbol-13-acetate (TPA) is routinely used as a
mitogen in melanocyte cultures to prolong survival (Arita et al,
2000; Halaban, 2000). Importantly, FGF2, SCF, EDN3, and TPA
have been classi¢ed as potent synergistic mitogens for human
melanocyte growth owing to the convergence of signaling in-
itiated by these growth factors on the mitogen-activated protein
kinase pathway and activation of cAMP-responsive element-
binding protein (Bohm et al, 1995; Halaban, 2000).
Conditions for the culture of primary human melanoblasts
have yet to be de scribed. Here, we report the establishment and
partial characterization of primary human melanoblast cultures
and demonstrate molecular and morphologic cha nges in response
to di¡erent media conditions. Furthermore, we show that human
melanoblasts express the BRN2 protein at high levels comparable
to that seen in metastatic melanoma cell lines and in contrast to
the low levels expressed in melanocytes. Reports describing the
exquisite sensitivity of the levels of POU proteins in dimeriza-
tion which subseque ntly determine stem cell fate (Niwa et al,
2000) and adult tissue speci¢c gene expression (Ryan and Rosen-
feld, 1997) implicate the BRN2 molecule as a possible essential
control mechanism in the melanocytic di¡erentiation pathway.
Synergistic regulation of BRN2 levels by peptide growth factors
within the melanocytic lineage may enable cell fate to be deter-
mined by an analogous dimerization mechanism (Smit et al,
2000). For this reason, we have examined the response
of BRN2 to peptide growth factors in human melanoblast and
melanocyte cell cultures.
This project complies with the provisions contained in the Na-
tional Statement on Ethical Conduct in Research Involving Humans and
complies with the regulations govern ing experimentation on
Mammalian cell culture Throughout this article, melanoblasts refers
to melanocytic cells established from neonatal foreskin tissue in
melanoblast growth medium and MB:MC refers to melanoblasts grown
in melanocyte growth medium for 1 wk before assay. Likewise,
melanocytes refers to cells established from foreskin tissue in melanocyte
medium and MC:MB refers to melanocytes grown in melanoblast
medium for 1 wk before assay. Both melanoblasts and melanocytes were
obtained from neonatal foreskin tissue, and melanocytes cultured as
described with 10 ng per mL TPA and 0.6 mg per mL cholera toxin (Smith
et al, 2001) after release of epidermal sheets from the dermis by Dispase II
(Roche, Basel, Switzerland) treatment. For melanocytes, trypsin was
neutralized by addition of RPMI 1640 medium plus 10% fetal bovine
serum (FBS; CSL Biosciences, Victoria, Australia) or for melanoblasts
MCBD 153 (Sigma Chemical Co., St. Louis, MO) medium containing
10% chelated FBS and 2% FBS. Melanoblasts were plated out in MCBD
153 medium containing 10% chelated FBS, 2% FBS, 2 mM glutamine,1.66
mg per L cholera toxin, 10 ng per mL SCF (Sigma), 100 nM EDN3, and 2.5
ng per mL FGF2 (Nesbit et al,1999)at371C in 5% CO2/5%O2 . Chelated
FBS was prepared by mixing 15 g of C helex-100 (Sigma) per 500 mL of
FBS for 1.5 h at 41C with gentle stirring. Culture medium for melanoma
cell lines was 10% FBS in RPMI 1640. The MM96L c8 LC cell line was
established by phenotypically ablating BRN2 expression using BRN2
antisense RNA (Thomson et al, 1995). All culture media contained FBS of
the same batch and contained penicillin (50 U/mL) and streptomycin (50
mg/mL). Primary murine neural crest cell lines were as de scribed in
Murphy et al (1991). Fresh cultures were e stablis hed according to Reid et al
(1996). Cultures that were Kit-negative contained less than 0.1% Kit-
positive cells and were cultured in medium contai ning 10% FBS with no
added growth factors. Cultures that were Kit-positive and unpigmented
were cultured in the presence of SCF (Reid et al, 1995, 1996) and
contained approximately 10% Kit-positive cells. Pigmented cultures that
were Kit-positive contained at least 65% pigmented cells and were
cultured in the presence of SCF, EDN3, and a-MSH (Reid et al,1996).
Fresh neural crest cultures were harvested 2 wk after initiation of culture.
Transmission electron microscopy Cell s were harvested and washed
in PBS, before resuspension of cell pellets in 3% glutaraldehyde in 0.1 M
phosphate bu¡er, pH 7.4. Samples were post¢xed in potassium^
ferricyanide-reduced osmium tetroxide in phosphate bu¡er (1% osmium
and 0.25% ferricyanide) and stained en bloc in 2% uranyl acetate.
Blocks were dehydrated in ascending concentrations of acetone and
subsequently in¢ltrated and embedded in Spurrs resin. Sections were
contrasted with lead citrate and viewed on a Hitachi H600 transmission
electron microscope at 100 kV.
L-Dihydroxyphenylalanine reactivity Cells were grown onto glass
coverslips overnight at 371 C.
L-Dihydroxyphenylalanine (DOPA; Sigma
Chemical Co.) was dissolved at 250 mM in 0.5 M HCl and then added to
the growth medium to a ¢nal concentration of 1, 2, or 5 mM, with the
medium pH after assay of approximately 7.5. Cells were cultured with
DOPA for 3 h, washed 1 in PBS for 2 min, ¢xed in freshly diluted Fast
Green Conce ntrate (Sigma Chemical Co.) for 5 min, washed in PBS, and
mounted inverted onto ethanol-cleaned slides with Crystal/Mount
(Biomeda Corp. Foster City, CA). Cells were photographed using an
Olympus BH2 inverted microscope, and photographs were scanned to
convert to digital images.
Western blotting Western blotting was performed essentially as
described previously after resolution of 10 mg of total protein in SDS^
PAGE gels (Leonard et al, 2002). Membranes were blocked for 1 h at room
temperature in 5% skim milk powder in phosphate-bu¡ered saline/0.05%
Tween 20 (PBS-T), before incubation with primary antibody for 1.5 h at
room temperature. Primary antibodies were anti-BRN2-C-terminal
(anti-BRN2C) a⁄nity-puri¢ed rabbit polyclonal antibody (Smith et al,
1998), anti-MART-1 (Dako Corporation, Carpinteria, CA) diluted 1:500
in 5% skim milk powder in PBS-T, or undiluted anti-TYR 5C13
(McEwan et al, 1988) or anti-TYRP1 B8G3 (Takahashi and Parsons, 1990)
monoclonal supernatant. Secondary antibody was anti-rabbit (for BRN2)
or anti-mouse (for 5C13, B8G3, and MART-1) Ig^HRPO conjugate
diluted 1:500 in 5% skim milk powder in PBS-T for 1.5 h at room
temperature followed by washes and ECL detection (Perkin Elmer Life
Sciences, Wellesley, MA). The membrane was then stripped according to
instructions (Perkin Elmer Life Sciences), washed in a large volume of
PBS-T, and blocked again in 5% skim milk powder in PBS-T before
incubation with primary antibodies to other proteins of interest as
described above, or as a loading control, with anti-intermediate ¢lament
protein (IFA) monoclonal supernatant (Pruss et al, 1981) being applied
overnight at 41C. Following washes, anti-mouse Ig^HRPO diluted 1:50 0
in 5% skim milk powder in PBS-T was applied for 1.5 h, the membrane
was washed, and ECL detection was performed. After each primary
antibody had been detected, the membrane was stripped as described.
Di¡erences in protein expression levels were determined by normalizing
to IFA and subsequently the appropriate control using ImageQuant
(Molecular Dynamics, Sunnyvale, CA).
Immunohistochemistry Cells were plated on to glass coverslips a nd
allowed to attach for 36 h. Subsequently, the medium was removed and
the cell s were ¢xed in two changes of 4% paraformaldehyde in PBS at
room temperature (10 min each). After a rinse in PBS, endogenous
peroxidase activity was blocked by addition of 1% H
in PBS followed
by 3 2-min washes in PBS. The cells were subsequently blocked in 3%
FBS in PBS (melanocytic markers) or 1% goat serum/0.1% Triton X-100
in PBS (for BRN2) for 20 min at room temperature before incubation
with primary a ntibody overnight at 41C. Primary antibodies i ncluded
anti-TYR 5C12, anti-TYR 5C13, and anti-TYRP1 B8G3 undiluted
hybridoma supernatants; MART-1, HMB45 (Bioge nex, San Ramon,
CA), and chromogranin A (Biogenex) a⁄nity-puri¢ed monoclonal
antibodies diluted 1:200 in 3% FBS in PBS; or 1:50 dilution of anti-
BRN2C in 1% goat serum/0.1% Triton X-100 in PBS. Following 3 2-
min washes in PBS, immunohistochemistry was completed essentially as
described (Cook et al, 2001; Leonard et al, 2002), using a broad-spectrum
Histostain-SP Kit (Zymed Laboratories, South San Francisco, CA) using
avidin^biotin ampli¢cation with AEC as the chromogen. The hematoxylin
counterstain was omitted for immunohistochemical analysis of BRN2.
Electrophoretic mobility shift assay Nuclear extracts were prepared
from two T-75 £asks as described (Schreiber et al, 1994), except that
protease inhibitor tablets (Roche) were added to the extraction bu¡ers
(Smit et al, 2000). DNA-binding probes used were wild-type (H2B,
Baumruker et al, 1988) and divergent (OA25, Bendall et al, 1993) octamer
sequences, a nd the mutant octamer probe dpm8 (Sturm et al, 1988). Probes
were released from plasmids by overnight digestion with EcoRI and
HindIII at 371C, followed by end-labeling with Klenow polymerase (New
England Biolabs, Beverly, MA) using [a-
P]dATP and [a-
Labeled probes were puri¢ed by gel electrophoresis and 5000 cpm used in
subsequent electrophoretic mobility shift assays (EMSA) with 1 mgof
nuclear extract as described (Smit et al, 2000; Leonard et al,2002).For
quantitation, the ratio of BRN2 (N-Oct-3) DNA-binding activity was
normalized to Oct-1 for each lane after exposure of a phosphor screen and
reading on a phosphoimager (Molecular Dynamics). Th is ratio was
subsequently compared to the ratio obtained for primary melanocytes,
with each probe being considered in isolation.
Establishment of primary human melanocyte and
melanoblast cultures
In establishing the conditions for the
growth of human epidermal melanoblasts, a number of di¡erent
growth factors and concentrations were examined. Prel iminary
experiments (Donatien et al,1997
; Donatien and Bennett, 1998
demonstrated that a medium containing a low concentration of
calcium (MCDB 153; 0.03 mM Ca
2 þ
) was better tha n ones
containing higher levels (RPMI 1640; 0.42 mM Ca
2 þ
) as this
inhibited growth of any contaminating ¢broblasts. The
concentration of EDN3 was best at 100 nM (range tested 1^100
nM) although there was not much increase in growth between 10
and 100 nM concentrations. Additions of FGF2 at 2.5 ng per mL
and SCF at 50 to 100 ng per mL gave the best increase in growth,
but culture with acceptable growth rates occurred at 10 ng per
mL SCF and this was used routinely.
Melanoblast morphology and marker expression Primary
human melanoblasts typically had a tr iangular or bipolar
morphology (Fig 1A, pane l 1), similar to that reported for
murine melanoblasts (Sviderskaya et al,1995;Kawa et al,2000).
Like murine melanoblasts, they were unpigmented and
unreactive to DOPA (Fig 1A, panel 1), indicati ng lack of active
TYR and consistent with the cells having predominantly Stage
I and II melanosomes as seen by transmission electron microscopy
(Fig 1 B, pane l 1). When melanoblasts were cultured in melanocyte
medium (MB:MC cells), the cells changed subtly to be
predominantly more bipolar in morphology, typical of primary
melanocytes, a nd became DOPA reactive to a level comparable to
that of melanocyte cultures (Fig 1A, panels 2 and 3). HeLa cells
(Fig 1A, panel 4) are not melanocytic and therefore are DOPA-
negative and were used as a negative control. Consistent with a
positive DOPA reaction, the melanosomes in MB:MC cells had
matured to Stage III and IV melanosome s (Fig 1 B, panel 2)and
resembled those of primary melanocytes (Fi g 1B, pane l 3).
Additionally, melanoblast cells became pigmented after 1 wk in
melanocyte medium, as seen in the cell pellets (Fig 1C).
A number of pigmentation markers were examined by
immunohistochemistry in melanoblasts, MB:MC cells, a nd pri-
mary melanocytes (Fig 2A). It was found that melanoblasts
express TYR, although at lower levels than in melanocytes (Fig
2A, panels 1^3 and 4^6). Localization in melanoblasts was
primarily perinuclear, whereas TYR in MB:MC cells and mela-
nocytes was dispersed throughout the cytoplasm. Immunoblot
analysis showed that melanoblasts and MB:MC cells con-
tained amounts of TYR protein similar to mature melano-
cytes (Fig 2B, lanes 1^3), suggesting that the DOPA reactivity in
MB:MC cells occurred through protein activation or maturation,
rather than de novo synthesis. The human melanoma cell line
MM96L expressed lower amounts of TYR by immunoblot (Fig
2B, lane 4) and MM96L c8 LC cells (Fig 2B, lane 5)servedasa
negative control as it had previously been shown these BRN2
ablated cells express no pigmentation markers (Thomson et al,
19 95).
In a similar manner, the distribution of TYRP1, detected
using the B8G3 hybridoma (Takahashi and Parsons, 1990) in
melanoblasts, was predominantly perinuclear, whereas in melano-
cytes and MB:MC cells, TYRP1 was distributed throughout
the cytoplasm (Fig 2A, panels 7^9). Western blot analysis
revealed that melanoblasts expressed TYPR1 at similar levels to
both MB:MC cells and melanocytes (Fig 2B, lanes 1^3).
MM96L cells expressed lower amounts of TYRP1 by Western
blot analysis, and as expected, TYRP1 was not detected in
MM96L c8 LC cell extracts (Fig 2B, lanes 4 and 5,
The HMB45 monoclonal antibody has been previously us ed to
detect melanocytes in human skin (Holbrook et al,1989).The
antibody recognizes the product of the silver locus, SILV (Adema
et al, 1994; Kawakami et al, 1994), mutation of which causes
progressive graying of hair owi ng to loss of follicular
melanocytes (Kwon et al, 1991, 1995). SILV has been implicated as
having several roles in melanin synthesis, including melanosome
biogenesi s (Berson et al, 2001) a nd melanin stabilization and/
or polymerization (Chakraborty et al, 1996; Lee et al,1996).
Immu nohi stochemical analysis of SILV expression in melano-
blasts showed a perinuclear localization (Fig 2A, panels 10^12),
Donatien PD, Robay D, Bennett DC: Extended culture of normal hu-
man melanoblasts and e¡ect of endothelin-3. Pigment Cell Res 10 :338, 199 7
Donatien PD, Bennett DC: Extended Culture and Di¡erentiation of
Normal Human Melanoblasts. Pigment Cell Res 11:170, 1998 (abstr.)
similar to TYR and STYRP1. In MB:MC cells, however,
localization of the SILV extended throughout the cytoplasm.
This distribution di¡ered from that of melanocytes, where SILV
localization was perinuclear.
Recently another melanocytic protein, MART-1, has been
shown to have a distinct localization from melanocytic enzymes
and to decrease in level upon melanosome maturation (De
Maziere et al, 2002), implying a role in melanosome biogenesis.
In melanoblasts, immunohistochemical analysis of MART-1
(Fig 2A, panels 13^15) revealed a perinuclear localization where
present but not all cells were positive. Nevertheless, in both
melanocytes and MB:MC cells, MART-1 local ization was
highly speci¢c and presented as regularly spaced foci along the
length of the dendrites. MART-1 was detectable by immuno-
blotting (Fig 2B) in melanoblasts and MB:MC cells at simi lar
levels, which was approximately twice that of primary melano-
cytes, whereas MM96L and BRN2 ablated MM96L c8 LC cells
had no detectable MART-1.
Figure 1. Phenotypic analysis of pigmentation in melanoblasts. (A)
DOPA reactivity of melanocytic cells. Melanoblasts (1), MB:MC cells (2),
melanocytes (3), and HeLa cells (4) were cultured in medium supplemented
with 5 mM DOPA, ¢xed, and counterstained. Melanoblasts (panel 1)are
nonreactive to DOPA, but when grown in melanocyte medium (MB:MC
cells, panel 2) become DOPA-positive and acquire a morphology similar to
that of melanocytes (pane l 3). HeLa cells (panel 4) are not melanocytic and
therefore used as a negative control. (B) Transmission electron micrographs
of melanoblasts (1), MB:MC cells (2), and melanocytes (3). Melanoblasts
contain immature, electron-light Stage I^II melanosomes, which mature
in MB:MC cells to resemble the Stage III^IV melanosomes seen in mela-
nocytes. (C) Cell pellets of melanoblasts (MB) a nd MB:MC cells after 1 wk
of growth in melanocyte medium.
Figure 2. Expression of melanocytic markers in melanoblasts. (A)
Immunohistochemical analysis of melanoblasts (left column), MB:MC cells
(middle column), and melanocytes (right column) for 5C12 and 5C13 (TYR,
panels 1^3 and 4^6, respectively), B8G3 (TYRP1, panels 7^9), HMB45
(SILV, panels 10^12), MART-1 (panels 13^15), and chromogranin A (CHMA ,
panels 16^18). Immunohistochemistry was performed using biotinylated
secondary antibodies and streptavidin conjugated horseradish peroxidase
with AEC as the chromogen. (B) Western blot analysis of melanogenic en-
zymes in melanoblasts (lane 1), MB:MC cells (lane 2), melanocytes (lane 3),
MM96L melanoma cells ( lane 4), and BRN2-ablated MM96L c8 LC mel-
anoma cells (lane 5). The antibody used is indicated to the left of the panel
and the sample name above each lane. IFA was used as a loading control.
Chromogranin A (Fig 2A, panels 16^18) is a speci ¢c marker for
Merkel cells in human epidermis (Hartschuh et al, 1989; Leonard
et al, 2002) and was used as a negative control. No immuno-
reactivity was seen in any cell in melanoblast, MB:MC cell, or
melanocyte cultures, indicating that spec i¢c reactivity had occur-
red with other antibodies tested.
BRN2 expression by primary human melanoblasts
Numerous transcription factors have been implicated in melano-
cyte development; nevertheless, given the absence of melanocyte-
speci¢c proteins in BRN2 ablated melanoma cell lines
(Thomson et al, 1995), we focused on the BRN2 tra nscription
factor. Expression was analyzed by immunoblotting using anti-
BRN2C polyclonal antibody (Smith et al, 1998). BRN2 was
detected in melanoblast extracts at a level similar to that of the
metastatic melanoma cell line MM96L (Fig 3A, compare lanes 1
and 9), but not in primary ¢broblasts and HeLa cells (lanes 7 and
8). Nevertheless, in MB:MC cells, expression of BRN2 decreased
to a level more similar to that of primary melanocytes (Fig 3A,
compare lanes 2 and 4), whereas melanoblasts maintained in
melanoblast medium sustained high levels of BRN2 (Fig 3A,
lane 3).
Because POU domain transcription factors bind both wild-
type and divergent octamer sequences and are able to form
homodimers on some s equences (Rhe e et al, 1998; Smit et al,
2000), EMSA were performed using probes containing BRN2-
binding sites (Fig 3B). The position of N-Oct-3 and N-Oct-5
(BRN2 products) DNA-binding activities were ide nti¢ed in
MM96L extracts (Fig 3B, lanes 13^15) compared to the BRN2
ablated MM96L c8 LC (Fig 3B, lanes 16^18). All extracts
contained the ubiquitous Oct-1-binding activity, and no
binding to the mutant octamer probe dpm8 was seen, consistent
with previous results (Thomson et al, 1995). Results showed that
melanoblasts had a typical melanocytic octamer DNA-binding
pro¢le, having Oct-1 as well as N-Oct-3 and trace amounts of
N-Oct-5-binding activities (Fig 3B, lanes 1^3) to H2B wild-
type and OA25 divergent octamer sequences, with no speci¢c
binding to dpm8. Relative to melanocytes (Fig 3B, lanes 7^9),
melanoblasts had more N-Oct-3-binding activity to the H2B
and OA25 probes. For MB:MC cells (Fig 3B, lanes 4^6),
however, no clear di¡ere nce in the N-Oct-3 activity from
melanocytes was seen for either probe.
R e gula ti on of BRN2 expr essi on in primary human melanocyt e
To determine the e¡ects of medium, melanocytes
were cultured in melanoblast medium (MC:MB cells).
After 1 wk, western blot a nalysis showed an increase in BRN2
to levels similar to that of melanoblasts (Fig 3A, compare lanes 6
and 1), whereas continued melanocyte cultures retained lower
levels of BRN2 (Fig 3A, lane 5). Furthermore, MC:MB cells
acquired a morphology more akin to that of melanoblast
concomitant with a reduced DOPA reaction (Fig 3C) and had a
reduced ability to synthesize pigment as seen by a less-pigmented
cell pellet after centrifugation (Fig 3D). When primary
melanocytes were cultured in MCDB 153 (melanoblast) medium
lacking SCF, FGF2, a nd EDN3, but supplemented with TPA,
the cells remained bipolar but did not continue to grow
(unpublished data). Taken together, these results imply BRN2
Figure 3. Correlation of BRN2 expression with pigmentation and morphology in melanocytic cells. (A) Western blot analysis for BRN2. Mel-
anoblasts (lane 1), MB:MC cells (lane 2), melanoblasts retained on melanoblast medium (lane 3), melanocytes (lane 4), melanocyte s retai ned on melanocyte
medium (lane 5), MC:MB cells (lane 6), neonatal foreskin ¢broblasts (lane 7), HeLa cells (lane 8), and MM96L metastatic melanoma cells (lane 9). IFA was used
as a loading control. (B) Melanoblasts, MB:MC cells, melanocytes, MC:MB cells, MM96L melanoma cells, a nd the BRN2-ablated MM96L c8 LC cells
were examined for octamer DNA-binding activity by EMSA using wild-type (H2B), divergent (OA25), and mutant (dpm8) octamer probes. Lanes 19^2 1
contain no nuclear extract. For each lane, BRN2 (N-Oct-3) DNA-binding activity was compared to Oct-1, and then this ratio was normalized to melano-
cytes (lanes 7^9), with each probe considered in isolation. FP, free probe. (C) DOPA reactivity for melanocytes (panel 1, lane 4 of A) and MC:MB cel ls (pane l
2, lane 6 of A). (D) Cell pellets of melanocytes (MC) and MC:MB cells after 1 wk of growth in melanoblast medium. (E) Immunohistochemical analysis of
BRN2 localization in melanoblasts, MB:MC cells, melanocytes, MC:MB cells, MM96L melanoma cells, and MM96L c8 LC BRN2-ablated melanoma
cells (panels 1^6, respectively). BRN2 immunohistochemistry was performed us ing biotinylated secondary antibodies and streptavidin-conjugated horse-
radish peroxidase with AEC as the chromogen, with the hematoxylin counterstain omitted.
upregulation by growth factor(s) in the melanoblast medium or
repression by factor(s) in the melanocyte medium. Interestingly,
despite dramatically increased BRN2 protein levels in MC:MB
cells to a level similar to that of melanoblasts (Fig 3A), there
was only a slight increase in N-Oct-3-binding activity relative
to melanocytes (Fig 3B, lanes 10^12). To determine whether
protein localization was responsible for the slight i ncrease in N-
Oct-3 DNA-binding activity in MC:MB cells, immunohisto-
chemical analysis of BRN2 was performed (Fig 3E).
Melanoblast, MC:MB cells, and MM96L cultures (Fig 3E, panels
1, 4 , and 5, respectively) all showed strong nuclear reactivity for
BRN2, whereas in MB:MC cells and melanocytes (Fig 3E,
panels 2 and 3), BRN2 was undetectable by immuno-
histochemistry and showed only background staining, similar to
BRN2 ablated MM96L c8 LC cel ls (Fig 3E, panel 6). He nce the
ability of BRN2 to bind DNA in vitro is not as e⁄cient in
MC:MB cells compared to melanoblasts.
To determine which growth factor(s) were responsible for
increased BRN2 in MC:MB cells, melanocytes were cultured in
MCDB 153 supplemented with SCF, FGF2, or EDN3 separately,
or in combination, either with or without TPA for 3 d and then
harvested for western blot analysis (Fig 4A). Compared to
routine melanocyte cultures, melanocytes grown for 3 d in
MCDB 153 supplemented with SCF, EDN3, and FGF2 had a
dramatically increased level of BRN2 (3.5-fold, Fig 4A, lane 6),
with further upregulation when TPA was added (5.6-fold, Fig
4A, lane 7). Concomitant with th is was a morphologic change to
a polydendritic phenotype (Fi g 4B, compare pane l 1 (melanocyte
medium) to panel 6 (melanoblast medium) and panel 7 (melano-
blast medium with TPA)), consistent with that seen previously
(Fig 3). Increased BRN2 protein levels and polyde ndritic
morphology were also observed when RPMI 1640 was
supplemented with SCF, EDN3, and FGF2 (Fig 4A, lanes 4 and
5; Fig 4B, pane l 4), although not to the sam e extent as with
Figure 4. Regulation of BRN2 in mel-
anocytic cells. (A) Western blot analysis
of melanocytes cultured in RPMI 1640 or
MCDB 153 supplemented with FGF2,
SCF, EDN3, or TPA as indicated. For each
lane, the level of BRN2 was compared to
IFA expression levels and the BRN2:IFA
ratio was normalized to melanocytes cul-
tured in melanocyte growth medium
(lane 1). (B) Phase contrast photomicro -
graphs for comparison melanocyte mor-
phology (panel 1) to cell morphology of
selected examples of melanocytes grow n
with varying growth factor supplements
as indicated in A; inset, lane number from
A.(C) EMSA analysis of selected exam-
ples from A. The level of BRN2 (N-Oct-
3) DNA-binding activity was compared to
Oct-1 for each lane, with this ratio being
normalized to that of melanocytes (lanes
1^3 ). FP, free probe.
MCDB 153. When melanocytes were grown in MCDB 153
supplemented with only one growth factor, BRN2 levels did
not increase and actually decreased in some cases (Fig 4A, lanes
8^13 ), irrespective of TPA. Interestingly, when RPMI 1640 was
supplemented with FGF2 alone, BRN2 levels increased in the
presence , but not absence of TPA (Fig 4A, compare lanes 2 and
3). For melanocytes grown in MCDB 153, addition of two
growth factors (SCF and EDN3, FGF2 and EDN3, or FGF2 and
SCF) resulted in increased BRN2 (Fig 4A, lanes 14^19; ratios 1.8^
6.1), again regardless of TPA supplementation. Maximal increase
was observed for melanocytes cultured with SCF, EDN3, and
TPA (4.9- and 6.1-fold, Fig 4A, lanes 14 and 15), whereas
melanocytes cultured with FGF2 and EDN3 ( Fig 4A, lanes 16
and 17 ) or FGF2 and SCF (Fig 4A, lanes 18 and 19) expressed
lower levels of BRN2, similar to each other. It was noted that
irrespective of the growth factor combination used, addition
of TPA to the culture medium increased BRN2 for each
combination with the exception of FGF2 and SCF. Increased
BRN2 levels were also usually seen on EDN3 addition.
The morphology of selected cultures assayed for total BRN2
protein levels are shown in Fig 4B. When melanocytes (Fig 4B,
panel 1) were cultured in MCDB 153 supplemented with one or
two of SCF, EDN3, or FGF2 (Fig 4B, p a n el s 8, 10, 12, 14, 16, and
18), the cells remained bipolar. When all three growth factors
were present in RPMI 1640 (Fig 4B, pane l 4) or in MCDB 153,
with or without TPA (Fig 4B, panels 6 and 7, respectively) an
increase in the proportion of polydendritic cells in the culture
was consistently observed. Hence, despite the pres ence of two
growth factors i n MCDB 153 resulting in a similar BRN2
increase similar to that obtai ned with all three factors, the
change to a polydendritic morphology required SCF, FGF2, and
Additionally, primary melanocytes were cultured for 3 d in
melanoblast medium supplemented with two of FGF2, SCF, and
EDN3, or all three factors, and then harvested for EMSA analysis
(Fig 4C) to support the ¢ndings by western blot analysis. When
cultured with FGF2, SCF, and EDN3, there was an increase in
BRN2 DNA binding to both the H2B and OA25 probes (Fig
4C,comparelanes 4 and 5 with lanes 1 and 2). Interestingly, this
increase is greater than the BRN2 increase obser ved after 7 d of
culture (MC:MB cells, Fig 3B).When melanocytes were cultured
in melanoblast medium supplemented with any two of FGF2,
SCF, or EDN3 (Fig 3C, lanes 7^15), there was a slight increase in
N-Oct-3-binding activity to wild-type and divergent octamer
sequences compared to melanocytes, with no binding to mutant
octamer sequences.
Because MCDB 153 and RPMI 1640 di¡er greatly in the
concentration of Ca
2 þ
, CaCl
was added to MCDB 153 such
that the concentration in MCDB 153 and RPMI 1640 was
the same. Subsequently, melanoblasts were cultured in CaCl
supplemented MCDB 153 with TPA alone or SCF, EDN3, and
FGF2 and then assayed for BRN2 by western blot analysis.
Results showed that in CaCl
-supplemented MCDB 153, the
addition of SCF, EDN3, and FGF2 maintained high levels of
BRN2 and the polydendritic melanoblast morphology, whereas
addition of TPA (and absence of peptide growth factors)
resulted in decreased BRN2 protein levels and a bipolar morpho-
logy (unpublished data) similar to MB:MC cells. Additionally,
supplementation of RPMI 1640 with SCF, EDN3, and FGF2
also increased BRN2 protein levels in melanocytes (Fig 4A,
lanes 4 and 5). Hence, the presence of peptide growth factors and
not the di¡erences in Ca
2 þ
between the media is responsible for
the di¡ering BRN2 levels.
N-Oct-3 activity in murine neural crest To determine
whether primary neural crest cells express BRN2, retrovirally
transformed murine cell lines established from isolated trunk
neural tubes of 9 d postcoitum embryos (Murphy et al,1991) as
wel l as mouse brain a nd MM96E melanoma cell nuclear extracts
were assayed by EMSA for octamer DNA-binding activity. Oct-1
DNA-binding activity was detected in all extracts examined (Fig
5A). Strong N-Oct-3 and N-Oct-5 activity was seen in the
MM96E melanoma cell line extract (Fig 5A, lane 3), whereas
mouse brain had lower N-Oct-3 activity (Fig 5A, lane 1). Of the
neural crest cultures examined, NC 14.3.3G, NC 14.4.9D, NC
14.4.8, and NC 14.4.6E had detectable N-Oct-3 -binding activity
Figure 5. Expression of N-Oct-3 in murine neural crest. (A) Cell lines derived from murine neural crest were assayed for BRN2 (N-Oct-3) DNA-
binding activity using H2B (odd-numbered lanes) and dpm8 (even-numbered lanes) octamer probes. BRN2 (N-Oct-3) DNA-binding activity was detected in
MM96E melanoma cell extract and in mouse brain, as well as in cultures derived from neural crest. The positions of the Oct-1, N-Oct-3, and N-Oct-5
complexes as well as unbound probe (FP) i s indicated. (B) Octamer expression in mouse neural crest cells cultured in the presence or absence of SCF, EDN3,
and a-MSH to generate cultures enriched i n Kit-negative cells (lanes 4^6), Kit-positive unpigmented (lanes 7^9), or Kit-positive pigme nted cells (lanes 10^12).
A2058 (lanes 1^3) and mouse brain (lanes 13^15) octamer pro¢les to the OA25 (lanes 2 and 3, 5 and 6, 8 and 9, 1 1 and 12, and 14 and 15) and dpm8 (lanes 1, 4, 7, 10,
and 13) were included for comparison purpose s. Positions of the Oct-1, BRN2 (N-Oct-3) a nd BRN2 (N-Oct-5) complexes are indicated. Inclusion of anti-
BRN2 POU domain a ntibody was used for con¢rmation of N-Oct-3 and N-Oct-5 complexes (supershift indicated by arrowhead). FP, free probe.
(Fig 5A, lanes 5, 11, 13, and 15, respectively). Two of these cell lines,
NC 14.4.9D and NC 14.4.6E, have been classi¢ed as dif-
ferentiated cell lines, whereas NC 14.4.8 are considered partial ly
di¡erentiating cells and NC 14.3.3G as an undi¡erentiated
neural crest-like cell type (Murphy et al,1991).
Further to this, Reid et al (1995) demonstrated that Kit-positive
cells in neural crest cultures were precursors of melanocytes and
that addition of TPA resulted in cell pigmentation. Hence, we
examined primary neural crest cultures of Kit-negative, Kit-
positive, unpigmented and Kit-positive, pigme nted cells using
mouse brain and A2058 melanoma cell extracts as controls for
N-Oct-3 DNA-binding activity. The A2058 melanoma cells
(Fig 5B, lanes 1^3) contained the Oct-1, N-Oct-3, and N-Oct-5
DNA-binding activities. The N-Oct-5 complex was detected in
all three neural crest cultures, whereas N-Oct-3 was not
apparent (Fig 5B, lanes 4^12). The N-Oct-5 protein has been
speculated to arise from proteolytic clipping of the N-Oct-3
protein (Atanasoski et al, 1997; Smith et al, 1998) and its presence
in less di¡erentiated primary melanoma cell lines prompted
speculation that it may play a role in the di¡erentiation process
during melanocyte development. Inclusion of an anti-BRN2C
antibody resulted i n a supershift of the N-Oct-5 complex,
indicating that the protein complex detected contained a BRN2
gene product.
Melanoblasts are a neural-crest-derived cell type de¢ned as spe-
ci¢ed melanocyte precursors which are unable to synthesize
melanin (Hirobe, 1992; Sviderskaya et al, 1995). Here, we have
described conditions for culturing human melanoblasts. We have
shown that they are unpigmented cells containing immature mel-
anosomes that are unreactive to DOPA (Fig 1). This is despite hu-
man melanoblasts expressing TYR and TYRP1 protein, unlike
murine melanoblasts in which Tyr was detected in only a mi nor-
ity of cells by immunohistochemistry (Sviderskaya et al,1995;
Kawa et al, 200 0). Examination of the POU domain transcription
factor BRN2, implicated in di¡erentiation of the melanocytic
lineage (Eisen et al, 1995; Thomson et al, 1995), demonstrated that
melanoblasts expressed high levels of BRN2 protein and N-Oct-
3 DNA-binding activity, similar to metastatic melanoma cell
lines and at a much higher level than melanocytes (Fig 3). Pre -
sumably, the lower relative fold increase in N-Oct-3 activity
compared to the increase in total BRN2 protein is due to a num-
ber of factors i ncluding normalization to di¡erent proteins and
inherent di¡erences between western blot analysis and DNA-
binding assays. We have shown that BRN2 localization is not
the re ason for these di¡erences (Fig 3E). Nevertheless, in all ex-
periments, changes in BRN2 protein levels followed the same
qualitative trends as N-Oct-3 DNA-binding activity.
Upon change of growth factor supplementation, we were able
to induce primary human melanoblasts to become DOPA-posi-
tive and furthermore acquire the ability to synthesize melanin
owing to melanosome maturation as determined by transmission
electron microscopy (MB:MC cells, Fig 1). Concomitant with
this was a change in cell morphology to one resembling that of
melanocytes (Fig 1A) and a decrease in BRN2 to levels similar to
those of melanocytes (Fig 3A). Given the similarities between
MB:MC cells and melanocytes, and the de¢nition of a melano-
blast (Sviderskaya et al, 1995), we consider MB:MC cells to be
melanocytes and that melanoblasts have been induced to di¡er-
entiate in vitro by the change in culture conditions.
Further to this, culture of melanocytes in melanoblast medium
(MC:MB cells) produced a decrease in DOPA reactivity and mel-
anin synthesis, a change in morphology, and an increase in
BRN2 protein and DNA-binding activities (Fig 3). Hence,
MC:MB cells are very similar to melanoblasts, and we inter-
preted this as a dedi¡erentiation of melanocytes in response to
medium changes. It is interesting to note that avian melanocytes
cultured with EDN3 have been reported to revert to a bipotent
progenitor cell capable of redi¡erentiation to melanocytes (Dupin
et al , 2000). Furthermore, avian Schwann cells cultured with
EDN3 revert to a bipotent glial-melanocyte precursor cell popu-
lation able to di ¡erentiate into melanocytes (Dupin et al,2003).It
was not reported, however, whether the dedi¡erentiation of avian
melanocytes and glial cells seen with EDN3 resulted in an in-
crease in BRN2, similar to the dedi¡erentiation of melanocytes
seen here.
To elucidate which growth factors were responsible for the in-
crease in BRN2 protein, melanocytes were grown with several
combinations of growth factors and then ass ayed for BRN2 pro-
tein. This revealed a possible synergistic mechanism controlling
BRN2, requiring at least two of SCF, FGF2, and EDN3 to in-
crease BRN2 protein levels in MCDB 153, irrespective of the
presence of TPA. Endotheli n-1 and SCF can act synergistically
to enhance proliferation of human melanocyte cultures via trans-
activation of the Kit receptor leading to activation of mitogen-
activated protein kinase (Imokawa et al, 2000), with Kit-activated
mitogen-activated protein kinase increasing the transcriptional
activity of microphthalmia associated transcription factor (Heme-
sath et al, 1998) by speci¢cally recruiting p300 (Price et al,1998).
Because BRN2 is also capable of i nteracting with p300 (Smit
et al, 2000), it is possible that EDN3 and SCF can interact in a
similar manner to endothelin-1 and SCF, with the outcome
being i ncreased production and/or transcriptional activity of
BRN2. Synergism between SCF and EDN3 has been previously
reported to e nhance murine melanocyte precursor proliferation
and survival in culture (Reid et al, 1996). Furthermore, disruption
of either signaling mechanism in vivo causes severe loss of mela-
noblasts in mouse (Steel et al, 1992; Pavan and Tilghman, 1994;
Wehrle-Haller and Weston, 1995) underlying the importance of
BRN2 in melanocytic cells.
Other POU domain transcription factors are downregulated
upon di¡erentiation of speci¢c cell types in vivo and in vitro. For
instance, the Class III factor Oct-6/Tst-1/SCIP (also POU3F1) is
expressed in glial cell progenitors of both the central and the per-
ipheral nervous system (Collarini et al, 1992; Jaegle et al,1996)and
the embryonic POU domain factor Oct-3/4 (POU5F1) is ex-
pressed by ES cells (Niwa et al, 2000; Reubino¡ et al ,2000),but
are downregulated as the cells di¡erentiate. Similar to BRN2 in
melanoma, Oct-3/4 is upregulated again in cancer cell lines
(Monk and Holding, 2001). Interestingly, the neural-crest-derived
peripheral nervous system glial cell, the Schwann cell, expresses
the cell-type-speci¢c transcription factors SOX10 and PAX3 (also
expressed by melanocytes) in addition to BRN2 and grows in
vitro as a dendritic cell similar to melanocytes (reviewed in Jessen
and Mirsky, 1998), consistent with the existence of a melanocyte^
glial bipotent progenitor cell (Le Douarin and Kalcheim, 1999).
Notably, Schwann cell demyelination owing to nerve injury causes
an upregulation of BRN2, which remains until the nerve remye-
linates (Sim et al , 2002), suggesting that BRN2 is upregulated ow-
ing to cellular stress or Schwann cell proliferation.
BRN2/N-Oct-3 DNA-binding activity was also detected in
murine neural crest cultures (Fig 5). The NC 14.3.3G cell line
had high levels of N-Oct-3 and has been classi ¢ed as an undif-
ferentiated neural crest-like cell type, because it expresses no
markers speci¢c for terminally di¡erentiated neurons or glia
(Murphy et al, 1991), suggesting that BRN2 is expressed early in
neural crest cell segregation. Interestingly, the NC 14.4.9D cell
line which was classi¢ed as a di¡erentiated cell type, but was
considered bipotent owing to expression of both Schwann cell
and neuronal markers (Murphy et al, 1991), has N-Oct-3 activity
(Fig 5). Additionally, NC 14.4.9D has a strong mitogenic response
to FGF2, and a dendritic morphology similar to melanocytes and
Schwann cells (Murphy et al, 1991), and hence NC 14.4.9D may
represent a bipotent glia^melanocyte progenitor cell (Le Douarin
and Kalcheim, 1999). N-Oct-3 activity has also been detected in
neural-crest-derived carcinoma cell lines, such as small cell lung
carcinomas, Merkel cell carcinomas, astrocytomas, glioblastomas,
and Ewings sarcomas (Schreiber et al, 1992; Schreiber et al,1994;
Thomson et al, 1994; Leonard et al, 2002) suggesting BRN2 invol-
vement in the development of multiple neural crest lineages.
In conclusion, we have shown that human cultured melano-
blasts express BRN2 at high levels comparable to melanoma cell
lines and that this level decreased on di¡erentiation to melano-
cytes. Thi s supports the i nvolvement of BRN2 in maintenance
of the melanoblast phenotype and a reciprocal impairment of
melanocyte maturation. The £exible DNA recognition and homo-
dimerization properties of BRN2 in melanocytic cells (Rhe e
et al, 1998; Smit et al , 2000) make the levels of this protein criti-
cal and may allow it to act as both an activator and a repressor
of melanocytic gene expression patterns mediating cell fate
A.L.C. was supported by a University of Queensland Postgraduate Scholarship, and
P.D.D., by a European Commission Marie Curie Training Grant. This work was
supported in part byThe Royal Brisbane Hospital Research into CancerTrust Fund
and NIH Grants CA80999 and CA25874. We thank Paul Alewood for the gift of
EDN3; Peter Parsons for 5C12, 5C13 and B8G3 monoclonal hybridoma superna-
tants; and Greg Detrich for help with photography. The Institute for Molecular
Bioscience is a Special Research Center of the Australian Research Council.
Adema GJ, de Boer AJ, Vogel AM, Loenen WA, Figdor CG: Molecular characteriza-
tion of the melanocyte lineage-speci¢c antigen gp100. J Biol Chem 269:
20126^20133, 1994
AritaY, Santiago-Schwarz F, Coppock DL.: Survival mechanisms induced by: 12-O-
tetradecanoylphorbol-13 -acetate in normal human melanocytes include inhi-
bition of apoptosis and increased Bcl-2 expression. Melanoma Res 10:412^420,
Atanasoski S, Schreiber E, Fontana A, Herr W: N-Oct 5 is generated by in vitro
proteolysis of the neural POU-domain protein N-Oct 3. Oncogene 14:
1287^1294, 1997
Baumruker T, Sturm R, Herr W: OBP100 binds remarkably degenerate octamer
motifs through speci¢c interactions with £anking sequences. Genes Dev 2:
1400^1413, 1988
Bendall AJ, Sturm RA, Danoy PA, Molloy PL: Broad binding-site speci¢city and
a⁄nity properties of octamer 1 and brain octamer-binding proteins. Eur J Bio-
chem 217:799^811, 1993
Berking C, Takemoto R, Satyamoorthy K, Elenitsas R, Herlyn M: Basic ¢broblast
grow th factor and ultraviolet B transform melanocytes in human skin. Am
J Pathol 158:943^953, 2001
Berson JF, Harper DC, Tenza D, Raposo G, Marks MS: Pmel17 initiates premelano-
some morphogenesis within multivesicular bodies. Mol Biol Cell 12:3451^3464,
Bohm M, Moel lmann G, Cheng E, Alvarez-Franco M, Wagner S, Sassone-Corsi P,
Halaban R: Identi¢cation of p90RSK as the probable CREB-Ser133 kinase in
human melanocytes. Cell Growth Di¡er 6:291^302, 1995
Chakraborty AK, Platt JT, Kim KK, Kwon BS, Bennett DC, Pawelek JM: Polymer-
ization of 5,6-di hydroxyindole-2-carboxylic acid to melanin by the pmel 17/
silver locus protein. Eur J Biochem 236:180^188, 1996
Collarini EJ, Kuhn R, Marshall CJ, Monuki ES, Lemke G, Richardson WD: Down-
regulation of the POU transcription factor SCIP is an early event in oligoden-
drocyte di¡erentiation in vitro. Development 116:193^200, 1992
Cook AL, Pollock PM, Welch J, et al: CDKN2A is not the principal target of dele-
tions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) car-
cinoma of the skin. Int J Cancer 93:361^367, 2001
Cox PM, Temperley SM, Kumar H, Goding CR: A distinct octamer-binding pro-
tein present in malignant melanoma cells. Nucleic Acids Res 16:11047^11056, 1988
Dailey L, Basilico C: Coevolution of HMG domains and homeodomains and the
generation of transcriptional regulation by Sox/POU complexes. JCellPhysiol
186:315^328, 2001
De Maziere AM, Muehlethaler K, Van Dons elaar E, et al: The melanocytic protein
melan-A/MART-1 has a subcellular localization distinct from typical melano -
somal proteins.Tr a c 3:678^693, 2002
Dupin E, Glavieux C,Vaigot P, Le Douarin NM: Endothelin 3 induces the reversion
of melanocytes to glia through a neural crest-derived glial-melanocytic pro-
genitor. Proc Natl Acad Sci USA 97:7882^7887, 2000
Dupin E, Real C, Glavieux-Pardanaud C, Vaigot P, Le Douarin NM: Reversal of
developmental restrictions in neural crest lineages: Transition from Schwann
cells to glial-melanocytic precursors in vitro. Proc Natl Acad Sci USA
100:5229^5233, 2003
Eisen T, Easty DJ, Bennett DC, Goding CR: The POU domain transcription factor
Brn-2. elevated express ion in malignant melanoma and regulation of melano-
cyte-speci¢c gene expression. Oncogene 11:2157^2164, 1995
Fujii H, Hamada H: A CNS-speci¢c POU transcription factor, Brn-2, is required for
establishing mammalian neural cell lineages. Neuron 11:1197^1206, 1993
Goding CR: Mitf from neural crest to melanoma. Signal transduction and transcrip -
tion in the melanocyte lineage. Genes Dev 14:1712^1728, 20 00
Halaban R: The regulation of normal melanocyte proliferation. Pigment Cell Res 13:
4^14, 2000
Halaban R, Langdon R, Birchall N, et al: Basic ¢broblast growth factor from human
keratinocytes is a natural mitogen for melanocytes. JCellBiol107:1611^ 1619,
1988 a
Halaban R, Langdon R, Birchall N, et al: Paracrine stimulation of melanocytes by
keratinocytes through basic ¢broblast growth factor. Ann N Y Acad Sci
548:180^190, 1988b
Hartschuh W, Weihe E, Egner U: Chromogranin A in the mammalian Merkel cell.
Cellular and subcellular distribution. JInvestDermatol93:641^648, 1989
Hemesath TJ, Price ER,Takemoto C, Badalian T, Fisher DE: MAP kinase links the
transcription factor microphthalmia to c-Kit signalling i n melanocytes. Nature
391:298^301, 1998
Herlyn M, Berking C, Li G, Satyamoorthy K: Lessons from melanocyte develop-
ment for understanding the biological events in naevus and melanoma forma-
tion. Melanoma Res 10:303^312, 2000
Hirobe T: Basic ¢broblast growth factor stimulates the sustained proliferation of
mouse epidermal melanoblasts in a serum-free medium in the presence of di-
butyryl cyclic AMP and keratinocytes. Development 114:435^445 , 1992
Holbrook KA, Underwood RA, Vogel AM, Gown AM, Kimball H: The appear-
ance, density and distribution of melanocytes in human embryonic and fetal
skin revealed by the anti-melanoma monoclonal antibody, HMB-45. Anat Em-
bryol 180:443^455, 1989
Imokawa G, Kobayasi T, Miyagishi M: Intracellular signaling mechani sms leading to
synergistic e¡ects of endothelin-1 and stem cell factor on proliferation of cul-
tured human melanocytes: Cross-talk via trans-activation of the tyrosine ki-
nase c-kit receptor. JBiolChem275:33321^33328, 2000
Jaegle M, Mandemakers W, Broos L, et al: The POU factor Oct-6 and Schwann cell
di¡erentiation. Science 273:507^510, 1996
Jessen KR, Mirsky R: O rigin and early development of Schwann cells. Microsc Res
Te c h 41:393^402, 1998
Jimbow K, Quevedo WC Jr, Prota G, Fitzpatrick TB: Biology of melanocytes. In:
Freedberg IM, Eisen AZ,Wol¡ K, Austen KF, Goldsmith LA, Katz SI, Fitzpa-
trick TB (eds). Fitzpatricks Dermatology in General Medicine. New York:
McGraw^Hill Health Professions Division, 1999
Jordan SA, Jackson IJ: MGF (KIT ligand) is a chemokinetic factor for melanoblast
migration into hair follicles. Dev Biol 225:424^436, 2000
KawaY, Ito M, Ono H, et al: Stem cell factor and/or endothelin-3 dependent immor-
tal melanoblast and melanocyte populations derived from mouse neural crest
cell s. Pigment Cell Res 13:73^80, 2000
Kawakami Y, Eliyahu S, Delgado CH, et al: Identi¢cation of a human melanoma
antigen recognized by tumor-in¢ltrating lymphocytes associated with in vivo
tumor rejection. Proc Natl Acad Sci USA 91:6458^6462, 1994
Kwon BS, Chintamaneni C, Kozak CA, et al: A melanocyte-speci¢c gene, Pmel 17,
maps near the silver coat color locus on mouse chromosome 10 and is in a
syntenic region on human chromosome 12. Proc Natl Acad Sci USA 88:
9228^9232, 1991
Kwon BS, Halaban R, Ponnazhagan S, Kim K, Chintamaneni C, Bennett D, Pick-
ard RT: Mouse silver mutation is caused by a single base insertion in the pu-
tative cytoplasmic domain of Pmel 17. Nucleic Acids Res 23:154^158, 1995
Lahav R, Dupin E, Lecoin L, Glavieux C, Champeval D, Ziller C, Le Douarin NM:
Endothelin 3 selectively promotes survival and proliferation of neural crest-de-
rived glial and melanocytic precursors in vitro. Proc Natl Acad Sci USA 95:14214^
14 219, 19 9 8
Lahav R, Ziller C, Dupin E, Le Douarin NM: Endothelin 3 promotes neural crest
cell proliferation and mediates a vast increase in melanocyte number in culture.
Proc Natl Acad Sci USA 93:3892^3897, 1996
Le Douarin N, Kalcheim C:The Neural Crest, (2nd ed). Cambridge/New York: Cam-
bridge University Press, 1999
Lee ZH, Hou L, Moellmann G, et al: Characterization and subcellular localization of
human Pmel 17/silver, a 110-kDa (pre) melanosomal membrane protein asso-
ciated with 5,6,-dihydroxyindole-2-carboxylic acid (DHICA) converting ac-
tivity. JInvestDermatol10 6:6 05 ^ 610, 1996
Leonard JH, Cook AL, Van Gele M, Boyle GM, Inglis KJ, Speleman F, Sturm RA:
Proneural and proneuroendocrine transcription factor expression in cutaneous
mechanoreceptor (Merkel) cells and Merkel cell carcinoma. Int J Cancer 101:
103 ^110, 2 0 02
Marks MS, Seabra MC: The melanosome: Membrane dynamics in black and white.
Nat Rev Mol Cell Biol 2:738^748, 2001
McEwan M, Parsons PG, Moss DJ: Monoclonal antibody against human tyrosinase
and reactive with melanotic and amelanotic melanoma cells. J Invest Dermatol
90:515^519, 1988
Moellmann G, Halaban R: Growth-factors and s ignal transduction regulating the
proliferation and di¡erentiation of melanocytes. In: Norlund J, Boissy R,
Hearing V, King R, Ortonne JP (eds). The Pigmentary System Physiology and
Pathophysiology. New York: Oxford University Press, 1998; p 135^149
Monk M, Holding C: Human embryonic genes re-express ed in cancer cells. Onco-
gene 20:8085^8091, 2001
Murphy M, Bernard O, Reid K, Bartlett PF: Cell lines derived from mouse neural
crest are representative of cells at various stages of di¡erentiation. J Neurobiol
22:522^535, 1991
Nesbit M, Nesbit HK, Bennett J, et al: Basic ¢broblast growth factor induces a trans-
formed phenotype in normal human melanocytes. Oncogene 18:6469^6476,
Nishimura EK, Jordan SA, Oshima H, et al: Dominant role of the niche in melano-
cyte stem-cell fate determination. Nature 416:854^860, 2002
Niwa H, Miyazaki J, Smith AG: Quantitative expression of Oct-3/4 de¢nes di¡eren-
tiation, dedi¡erentiation or self-renewal of ES cells. Nat Genet 24:372^376, 2000
Pavan WJ, Tilghman SM: Piebald lethal (sl) acts early to disrupt the development of
neural crest-derived melanocytes. Proc Natl Acad Sci USA 91:7159^7163, 1994
Price ER, Ding HF, Badalian T, et al: Lineage-speci¢c signaling in melanocytes.
C-kit stimulation recruits p300/CBP to microphthalmia. JBiolChem273:
17983^17986, 1998
Pruss RM, Mirsky R, Ra¡ MC, Thorpe R, Dowding AJ, Anderton BH: All classes
of intermediate ¢laments share a common antigenic determinant de¢ned by a
monoclonal antibody. Cell 27:419^428, 1981
Reedy MV, Parichy DM, Erickson CA, Mason KA, Frost-Mason SK: Regulation of
melanoblast migration and di¡erentiation. In: Norlund J, Boissy R, Hearing V,
King R, Ortonne JP (eds). The Pigmentary System Physiology and Pathophysiology.
NewYork: Oxford University Press, 1998; p 75^95
Reid K, Nishikawa S, Bartlett PF, Murphy M: Steel factor directs melanocyte de-
velopment in vitro through selective regulation of the number of c-kit þ pro-
genitors. Dev Biol 169:568^579, 1995
Reid K,TurnleyAM, Maxwell GD, KuriharaY, Kurihara H, Bartlett PF, Murphy M:
Multiple roles for endothelin in melanocyte development. Regulation of pro-
genitor number and stimulation of di¡erentiation. Development 122:3911^3919,
19 96
Reubi no¡ BE, Pera MF, Fong CY, Trounson A, Bongso A: Embryonic stem cell
lines from human blastocysts: Somatic di¡erentiation in vitro. Nat Biotechnol
18:399^404, 2000
Rhee JM, Gruber CA, Brodie TB, Trieu M,Turner EE: Highly cooperative homodi-
merization is a conserved property of neural POU proteins. JBiolChem
273:34196^34205, 1998
Ryan AK, Rosenfeld MG: POU domain family values: Flexibility, partnerships, and
developmental codes. Genes Dev 11:1207^1225, 1997
Schreiber E, Himmelmann A, Malipiero U, Tobler A, Stahel R, Fontana A: Human
small cell lung cancer expresses the octamer DNA-binding and nervous sys-
tem-speci¢c transcription factor N-Oct 3 (brain-2). Cancer Res 52:6 121^6124,
Schreiber E, Merchant RE,Wiestler OD, Fontana A: Primary brain tumors di¡er in
their expression of octamer deoxyribonucleic acid-binding transcription fac-
tors from long-term cultured glioma cell lines. Neurosurgery 34:129^135, 1994
Scott G, Ewing J, Ryan D, Abboud C: Stem cell factor regulates human melanocyte^
matrix interactions. Pigment Cell Res 7:44^51, 1994
Seiberg M: Keratinocyte^melanocyte interactions during melanosome transfer. Pig-
ment Cell Res 14:236^242, 2001
Sim F, Zhao C, Li W, Lakatos A, Franklin R: Expression of the POU-domain tran-
scription factors SCIP/Oct-6 and Brn-2 is associated with Schwann Cell but
not oligodendrocyte remyelination of the CNS. Mol Cell Neurosci 20:669^682,
Smit DJ, Smith AG, Parsons PG, Muscat GE, Sturm RA: Domains of Brn-2 that
mediate homodimerization and interaction with general and melanocytic tran-
scription factors. Eur J Biochem 267:6413^6422, 2000
Smith AG, Box NF, Marks LH, et al: The human melanocortin-1 receptor locus:
Analysis of transcription unit, locus polymorphism and haplotype evolution.
Gene 281:81^94, 20 01
Smith AG, Brightwell G, Smit SE, Parsons PG, Sturm RA: Redox regulation of
Brn-2/N-Oct-3 POU domain DNA binding activity and proteolytic forma-
tion of N-Oct-5 during melanoma cell nuclear extraction. Melanoma Res 8:
2^10, 1998
Steel KP, Davidson DR, Jackson IJ: TRP-2/DT, a new early melanoblast marker,
shows that steel growth factor (c-k it ligand) is a survival factor. Development
115:1111^ 1119, 199 2
Sturm RA, Bisshop F, Takahashi H, Parsons PG: A melanoma octamer binding
protein is responsive to di¡erentiating agents. Cell Growth Di¡er 2:519^524,
19 91
Sturm RA, Das G, Herr W: The ubiquitous octamer-binding protein Oct-1 contains
a POU domain with a homeo box subdomain. Genes Dev 2:1582^1599, 1988
Sviderskaya EV, Easty DJ, Bennett DC: Impaired growth and di¡erentiation of di-
ploid but not immortal melanoblasts from endothelin receptor B mutant (pie-
bald) mice. Dev Dyn 213:452^463, 1 998
Sviderskaya EV, Hill SP, Balachandar D, Barsh GS, Bennett DC: Agouti signaling
protein and other factors modulating di¡erentiation a nd proliferation of im-
mortal melanoblasts. Dev Dyn 221:373^379, 2001
Sviderskaya EV,Wakeling WF, Bennett DC: A cloned, immortal line of murine mel-
anoblasts inducible to di¡erentiate to melanocytes. Development 121: 1 547^1557 ,
19 95
Takahashi H, Parsons PG: In vitro phenotypic alteration of human melanoma cells
induced by di¡erentiati ng agents: Heterogeneous e¡ects on cellular growth
and morphology, enzymatic activity, and antigenic expression. Pigment Cell
Res 3:223^232, 1990
Thomson JA, Leonard JH, McGregor K, Sturm RA, Parsons PG: A nonconsensus
octamer-recognition sequence (TAATGARAT-motif) identi¢es a novel DNA
binding protein in human Merkel cell carcinoma cell lines. Int J Cancer 58:285^
290, 1994
Thomson JA, Murphy K, Baker E, Sutherland GR, Parsons PG, Sturm RA: The
brn-2 gene regulates the melanocytic phenotype and tumorigenic potential
of human melanoma cells. Oncogene 11:691^700, 1995
Thomson JA, Parsons PG, Sturm RA: In vivo and in vitro expression of octamer bind-
ing proteins in human melanoma metastases, brain tissue, and ¢broblasts. Pig-
ment Cell Res 6:13^22, 1993
Wehrle-Haller B, Weston JA: Soluble and cell-bound forms of steel factor activity
play distinct roles in melanocyte precursor dispersal and survival on the lateral
neural crest migration pathway. Development 121:731^742, 1995
    • "Written informed consent was obtained from each donor or donor's parents prior to sample collection. The published method by Cook et al. (Cook, et al. 2003) was used to culture and purify melanocytes. We used the melanin analytical method by Cook et al. (Cook, et al. 2009) to calculate the total amount of melanin and protein of melanocytes in each cell culture dish. "
    [Show abstract] [Hide abstract] ABSTRACT: Skin lightening among Eurasians is thought to have been a convergence occurring independently in Europe and East Asia as an adaptation to high latitude environments. Among Europeans, several genes responsible for such lightening have been found, but the information available for East Asians is much more limited. Here, a genome-wide comparison between dark-skinned Africans and Austro-Asiatic speaking aborigines and light-skinned northern Han Chinese identified the pigmentation gene OCA2, showing unusually deep allelic divergence between these groups. An amino acid substitution (His615Arg) of OCA2 prevalent in most East Asian populations—but absent in Africans and Europeans—was significantly associated with skin lightening among northern Han Chinese. Further transgenic and targeted gene modification analyses of zebrafish and mouse both exhibited the phenotypic effect of the OCA2 variant manifesting decreased melanin production. These results indicate that OCA2 plays an important role in the convergent skin lightening of East Asians during recent human evolution.
    Full-text · Article · Jan 2016
    • "Since hPSC culture can be easily scaled up, hPSCs represent a valuable source to generate a large number of human McSCs and melanocytes for research and potential clinical applications. Previously, techniques to isolate and culture melanoblasts from mouse and human skin were established6970717273. Researchers also attempted to isolate and culture McSCs from hair follicles. "
    [Show abstract] [Hide abstract] ABSTRACT: Melanocytes in the skin play an indispensable role in the pigmentation of skin and its appendages. It is well known that the embryonic origin of melanocytes is neural crest cells. In adult skin, functional melanocytes are continuously repopulated by the differentiation of melanocyte stem cells (McSCs) residing in the epidermis of the skin. Many preceding studies have led to significant discoveries regarding the cellular and molecular characteristics of this unique stem cell population. The alteration of McSCs has been also implicated in several skin abnormalities and disease conditions. To date, our knowledge of McSCs largely comes from studying the stem cell niche of mouse hair follicles. Suggested by several anatomical differences between mouse and human skin, there could be distinct features associated with mouse and human McSCs as well as their niches in the skin. Recent advances in human pluripotent stem cell (hPSC) research have provided us with useful tools to potentially acquire a substantial amount of human McSCs and functional melanocytes for research and regenerative medicine applications. This review highlights recent studies and progress involved in understanding the development of cutaneous melanocytes and the regulation of McSCs.
    Full-text · Article · Dec 2015
    • "Second, we analyzed the expression of the transcription factors SOX9 and SOX10 in pigmentDESS melanocytes. Lately, it was observed that SOX9 drives the transition of undifferentiated melanoblasts to differentiated melanocytes in postnatal skin eventually resulting in melanogenesis , i.e., the production of melanin252627. SOX10 is a widely described transcription factor and one of the most important regulators of embryonic melanocyte development and of maintenance of melanocyte stem cells in hair follicle bulge in postnatal skin [28, 29] . "
    [Show abstract] [Hide abstract] ABSTRACT: PURPOSE Transplantation of pigmented tissue-engineered human autologous skin substitutes represents a promising procedure to cover skin defects. We have already demonstrated that we can restore the patient's native light or dark skin color by adding melanocytes to our dermo-epidermal skin analogs. In this long-term study, we investigated if melanocytes in our skin substitutes continue to express markers as BCL2, SOX9, and MITF, known to be involved in survival, differentiation, and function of melanocytes. METHODS Human epidermal melanocytes and keratinocytes, as well as dermal fibroblasts from light- and dark-pigmented skin biopsies were isolated and cultured. Bovine collagen hydrogels containing fibroblasts were prepared, and melanocytes and keratinocytes were seeded in a 1:5 ratio onto the gels. Pigmented dermo-epidermal skin substitutes were transplanted onto full-thickness wounds of immuno-incompetent rats and analyzed for the expression of melanocyte markers after 15 weeks. RESULTS Employing immunofluorescence staining techniques, we observed that our light and dark dermo-epidermal skin substitutes expressed the same typical melanocyte markers including BCL2, SOX9, and MITF 15 weeks after transplantation as normal human light and dark skin. CONCLUSIONS These data suggest that, even in the long run, our light and dark dermo-epidermal tissue-engineered skin substitutes contain melanocytes that display a characteristic expression pattern as seen in normal pigmented human skin. These findings have crucial clinical implications as such grafts transplanted onto patients should warrant physiological numbers, distribution, and function of melanocytes.
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