Melanocyte fate in neural crest is triggered by Myb proteins
through activation of c-kit
V. Karafiat, M. Dvorakova, P. Pajer, V. Cermak and M. Dvorak*
Department of Molecular Virology, Institute of Molecular Genetics, AS CR v.v.i., V?den ˇsk? 1083, Prague 4,
14220 (Czech Republic), Fax: +420 296443586, e-mail: email@example.com
Received 20 July 2007; received after revision 27 August 2007; accepted 19 September 2007
Online First 20 October 2007
Abstract. The c-myb proto-oncogene and its onco-
genic derivative v-mybAMVencode transcriptional
regulators engaged in the commitment of hemato-
poietic cells. While the c-Myb protein is important for
the formation and differentiation of various progen-
itors, the v-MybAMVoncoprotein induces in chicks a
progression and transformation of the single (mono-
of cell fate-directing abilities of c-Myb and v-MybAMV
proteins in avian neural crest (NC), where both
proteins determine melanocytogenesis. The increased
concentration of c-Myb induces progression into
dendritic melanocytes and differentiation. The v-
myb oncogene converts essentially all NC cells into
melanocytes and causes their transformation. Both
Myb proteins activate in NC cells expression of the c-
kit gene and stem cell factor c-Kit signaling – one of
the essential pathways in melanocyte development.
These observations suggest that the c-myb-c-kit path-
way represents a common regulatory scheme for both
hematopoietic and neural progenitors and establishes
a novel experimental model for studies of melanocy-
togenesis and melanocyte transformation.
Keywords. c-myb proto-oncogene, v-mybAMVoncogene, neural crest, cell fate determination, melanocytes, c-kit
Transcription factors c-Myb and v-Myb were origi-
nally recognized as key regulators in hematopoietic
cells. c-Myb sustains proliferation of all types of
immature hematopoietic cells (reviewed in ). It is
required for the development of definitive erythroid
and myeloid lineages  as well as for the survival and
differentiation of specific stages of T- and B-cells [3,
4]. In addition, c-Myb regulates stem/progenitor cells
in colonic crypt and adultneurogenic zones (reviewed
in ) and in the neuroectoderm of the early chick
embryo . The gene is also expected to function in
vascular smooth muscle cells [7, 8]. Graded intra-
cellular concentrations of c-Myb significantly influ-
ence its biological activity, including the lineage
choice process. Higher c-Myb concentrations in de-
finitive murine hematopoietic progenitors accent the
development of erythroid cells, while low concentra-
tions result in the preferential development of mega-
karyocytes and macrophages . Graded levels of c-
Myb also participate in the formation, survival and
motility of avian neural crest (NC), with higher
concentrations activating the epithelio-mesenchymal
transition in the neuroectoderm and the formation of
migratory NC cells .
* Corresponding author.
Cell.Mol.Life Sci. 64 (2007) 2975–2984
?Birkh?user Verlag, Basel, 2007
Cellular and Molecular Life Sciences
The oncogene of avian myeloblastosis virus, v-my-
bAMV, is the truncated version of the c-myb gene. It
contains only the central part of c-myb coding
sequences with several point mutations, and its con-
stitutive expression is driven by the retroviral pro-
moter. Similarly to c-Myb, the v-Myb oncoprotein
binds specifically to the Myb recognition DNA
element (MRE) and regulates transcription of adja-
cent genes (reviewed in ). In contrast to c-Myb,
however, it distorts the development of common
phage lineage at the expense of other lineages ,
blocks terminal differentiation of immature macro-
phage precursors – monoblasts – and induces their
malignant transformation (reviewed in ). In
cooperation with bFGF, v-Myb can also promote
exclusive erythroid development in primitive pro-
The second myb allele, v-mybE26, composed of fused
gag-myb-ets sequences, transforms myeloid and er-
ythroid cells as well as their multipotent progenitors.
The E26 oncogene also has the potential to affect the
developmental fate of the progeny of multipotent
hematopoietic progenitors . In addition, it has
cultures in cooperation with the exogenous EGF
(epidermal growth factor) receptor .
The NC cells develop in early vertebrate embryos at
the border between the neural plate and the non-
neural ectoderm. These multipotential cells delami-
nate from the dorsal neural tube in a temporary
rostrocaudal wave and migrate throughout the em-
bryo. NC cells derived from the trunk region of the
neural tube differentiate into neurons and glia of the
sensory and sympathetic ganglia, neuroendocrine
cells of the adrenal medulla and melanocytes [15–
tissues [18, 19]. Formation of NC cells and specifica-
tion of their developmental fates are controlled by
complex signalling events that are not yet completely
known (reviewed in [20–25]).
NC-derived melanocyte precursors in chicken em-
bryos emerge relatively late and enter the dorsal path
of migration [26–28]. In addition to Wnt signals ,
their formation also requires signals from stem cell
factor (SCF) and endothelin 3, which activate their
cognate receptors c-Kit and endothelin B – ET (B) –
respectively [30–32]. These signals activate intrinsic
determinants of melanocytic differentiation, includ-
ing Pax3 and several members of the Sox gene family,
and activate synthesis and function of the micro-
which is a central factor of the melanogenic cascade
We found that both Myb proteins support the devel-
opment of melanocyte lineage in chick embryo neural
tube explants. In contrast to c-Myb, the v-Myb
oncoprotein strongly interferes with the differentia-
tion of NC cells, inducing formation of transformed
We show that a significant part of the molecular
mechanism that Myb proteins trigger to determine
melanocyte cell fate is activation of the SCF/c-Kit
Materials and methods
Embryos, cell cultures and viruses. Brown Leghorn
chick blastoderms and neural tube explants were
and cultivated in liquid or semisolid media as descri-
bed elsewhere [6, 11]. The v-myb mutations N118D
, L237A  and L3,4A  were prepared by a
myb genes by replacing the TAG stop codon with the
and the mouse estrogen receptor containing the
G525R mutation . 200 nM 4-hydroxytamoxifen
(Sigma) was used for induction of v-MybER proteins.
In the control Dmyb virus genome the myb gene was
deleted. Proportions of melanocytes in the cultures
the cultures. Images of 10 randomly selected fields of
each culture were captured for cell counting using a
reticle. Data are expressed as a sample mean. Error
bars represent standard deviations.
Antibodies and chemicals. The rabbit polyclonal anti-
Myb and neutralizing anti-SCF antibodies were de-
scribed [6, 38]. Monoclonals 1E8, MelEM and kit2c75
were from DSHB (Iowa City, Iowa, USA) and South-
ernBiotech (Birmingham, AL, USA), respectively.
Affinity-purified F(ab’)2 fragments of goat anti-
mouse FITC (fluorescein isothiocyanate)-conjugated
or anti-rabbit Cy3-conjugated IgGs (immunoglobu-
(West Grove, PA, USA). The AG1296 inhibitor was
purchased from Calbiochem (Darmstadt, Germany).
Immunofluorescence and flow cytometry analyses.
Cells for immunofluorescence analysis or Giemsa
staining were processed as described . Fluores-
cence images were captured using a DMIRB micro-
scope, DFC480 camera and IM500 software (Leica
Microsystems, Wetzlar, Germany) and processed
using Adobe software (Adobe Systems, San Jose,
CA, USA). Growing NC cells and CEFs for flow
2976 V. Karafiat et al.Myb proteins activate melanocytogenesis
kit2c75 and the secondary FITC-conjugated antibod-
ies (1 h each) in the media+0.5% BSA at 37oC.
Unreacted antibodies were removed by 3 washes with
the media+0.5% BSA. Cells were detached by 1%
collagenase (PAA Laboratories, Linz, Austria) for
30–45 min at 37oC, until approximately 90% of cells
were released, collected by low-speed centrifugation
(Becton Dickinson, San Jose, CA, USA). Analysis of
data and histogram overlays were performed with
WinMDI software. All histograms were gated on
propidium iodide-negative cells.
3 timeswith culture
Western blot analysis. Western blot analyses were
were compared on Coomassie-stained gels. Equiva-
lent protein samples were loaded. PageRuler pre-
stained protein ladder (Fermentas) was used as
RT-PCR. Total RNA was isolated from cell cultures
using RNAwiz (Ambion). Carefully quantitated
RNAs (for semiquantitative RT PCR) were reverse-
transcribed to cDNA (complementary DNA) using
M-MLV reverse transcriptase (Promega, Madison,
WI, USA). Semiquantitative PCR reactions con-
tained 200 mM dNTPs, 200 nM primers, 0.7 ml of
cDNA and 0.02 U/ml of Taq polymerase in the
magnesium-containing buffer (Promega) in 20 ml.
Samples of all PCR reactions were checked electro-
phoretically at various numbers of PCR cycles to
ensure that all the reactions are observed in the
exponential phase. Cycling parameters for electro-
phoretograms shown in figures were as follows: 95oC
Tmofagivenpairofprimers)20 s,72oC45 s:28cycles
with CKIT-F (5’-GCAAAGGGCATGAGCTTCC
TGG) and CKIT-R (5’-GGCTCCTCTTCTTCCA-
GATGCCAC), resulting product 624 bp; 30 cycles
GAGTCGCTG) and SCF-R (5’-GCAAACATG-
406 bp (these reactions also contained, in addition to
standard components, 1 M betain); and 22 cycles with
and GAPDH-R (5’-TCCCCACAGCCTTAGCAG),
resulting product 164 bp. The primer pairs were
derived from different exons.
Real-time PCR. For quantitation of c-kit cDNA
CACTGTGAACGCCAGC) and 3KITAE (5’-
the 187-bp product were used in addition to the above
GAPDH-F and GAPDH-R. Reactions were per-
formed in triplicate using DyNAmo SYBR Green
qPCR kit (Finnzymes) on the DNA Engine Opticon2
(MJ Research) according to the manufacturer?s pro-
c-Myb and v-Myb proteins induce accumulation of
pigment cells in chicken blastoderm cultures; v-Myb
causes their transformation. We previously demon-
strated that c-Myb is synthesized in the majority of
cells of the early chicken embryo and is significantly
involved in the formation of the trunk NC . In the
present work we analyzed the influence of c-Myb and
v-Myb proteins on early chick embryo cells, including
First, c-myb or v-myb genes were expressed in
explanted blastoderms of the developmental stage
HH10  using MAV-1-based retroviruses, and the
synthesis of Myb proteins was determined (Fig. 1c).
Infected explants were either grown in liquid cultures
or dispersed and seeded into semisolid media. The
liquid c-myb and v-myb cultures differed after 7 days
of cultivation from cultures infected with the control
(Dmyb) virus by high counts of hematopoietic blasts
and by the morphology of cells attached to dishes
(Fig. 1a). In c-myb cultures, hematopoietic cells were
represented mainly by myeloblasts, which frequently
differentiated into macrophages. Up to 5% of attach-
a rather high content of pigment granules and differ-
entiated morphology (Fig. 1b, c-myb panels). v-myb
cultures contained mainly typical v-myb-transformed
monoblasts and usually over 5% of attached cells
were melanocytes characterized by rather immature
appearance, with a lower content of pigment granula
(Fig. 1b, v-myb panels). In uninfected or control
retrovirus-infected cultures virtually no pigmented
cells were found (Fig. 1a, Dmyb panel).
Only v-myb monoblasts and melanocytes formed
colonies in semisolid media (Fig. 1d-i). Some colonies
of pigmented cells reached a 1-mm diameter (Fig. 1d-
ii) and contained over 105cells (16 generations). Cells
isolated from such colonies continued proliferating in
liquid cultures for an additional 20–30 generations
with a doubling time of 32–36 h. In dense cultures,
pigmented cells detached from the dish surface,
Cell.Mol.Life Sci. Vol.64, 2007
time, these clusters readhered, and pigmented cells
grew out from them and multiplied until they reached
new floating clusters. After a total of 40–50 gener-
ations cells stopped proliferating, turned into adher-
ent dendritic melanocytes and finally decomposed.
Repeated experiments clearly showed that both c-
myb and v-myb genes activate the occurrence of
pigment cells in whole-embryo cultures, and that the
v-myb oncogene transforms these cells and induces
their anchorage-independent growth and long-term
Myb proteins activate formation of NC-derived mel-
anocytes. Two types of pigment cells are easily
detectable in developing embryos – retinal pigment
epithelium and NC-derived skin melanocytes. In
order to confirm that analyzed pigment cells are NC
derivatives, trunk neural tube fragments containing
unfused neural folds were isolated, infected with
concentrated retroviral stocks (c-myb, v-myb or
Dmyb) and placed onto collagen-coated plates. Fol-
lowing 12–16 h pioneer cells started to emigrate from
successfully adhered explants. The emigrated cell
populations were analyzed 7–12 days later. The
results from six independent sets of cultures showed
that while Dmyb virus-infected and non-infected
populations contained at most 20% melanocytes, c-
myb and v-myb cultures invariantly contained about
40 and 95% of pigment cells, respectively, document-
ing the capability of Myb proteins either to activate
melanocyte cell fate in NC cells or to support the
proliferation of committed melanocytes.
Myb proteins commit NC cells to the melanocyte cell
lineage. To find out whether Myb proteins can
determine melanocyte lineage choice, c-myb and v-
myb genes were introduced into naive neural plates
which contain only prospective NC cells and no
committed melanocyte progenitors. Retroviruses
used in this work efficiently transduce myb genes
. In neural plate explants both myb genes caused
cell delamination and formation of pigmented mela-
, can activate very early steps in the formation of
trunk NC, and both Myb proteins appear to sustain
fragments containing unfused neural folds were used
for further experiments. In the embryo, trunk NC
Figure 1. c-myb and v-myb genes promote accumulation of
immature myeloid cells and melanocytes in blastoderm cultures.
(a) Phase contrast images of cultures infected with the control
Dmyb, c-myb and v-myb retroviruses. Bar, 50 mm. (b) Giemsa-
panels) in cultures infected with c-myb or v-myb retroviruses. Bar,
10 mm. (c) Western blot detection of c-Myb (76 kD) and v-Myb
(48 kD) proteins in the lysates of cultured cells shown in (a)
selected by G418. (d) Transformation of hematopoietic cells and
dark colonies of melanocytes in semisolid media. (ii) The large
melanocyte colony with outgrowing unpigmented melanoblasts.
(iii) Floating clusters of v-myb-transformed melanoblasts in a
liquid culture. Bar, 0.5 mm.
Figure 2. myb genes induce formation of melanocytes (dark
pigmented cells in c-myb and v-myb panels) in naive neural plate
explants. Phase contrast images of representative fields of cultures
infected with the control Dmyb, c-myb and v-myb retroviruses.
2978 V. Karafiat et al. Myb proteins activate melanocytogenesis
cells delaminate from neural folds and invade
surrounding tissues in waves. Cells of the early
wave differentiate mainly into neurons and glial
cells of the peripheral nervous system. Cells of the
second delayed wave differentiate into melanocytes
[27, 40]. This mode of NC emigration/differentiation
is to some extent also recapitulated in tissue culture.
vitro mainly into glial cells. However, as they contain
oligopotent precursors, including bipotential glial-
melanocytic progenitors [40, 41], they have the
potential to develop along the melanocyte cell
lineage when induced, for example, by bFGF or
bFGF+SCF, which both participate in the induction
cells and used them to study the effects of Myb
proteins on cell lineage determination. As expected,
the vast majority of these cells contained the HNK-1
epitope (Fig. 3a), which is typical for undifferenti-
ated migrating NC cells . As shown in Figure 3b,
which summarizes results of four independent ex-
periments, Dmyb virus-infected cells differentiated
into non-pigmented cells (mainly glial), while after
10 dayssuch cultures
bFGF+SCF contained 25 or 80% of pigment cells,
respectively. The early wave cells were then infected
with c-myb and v-myb retroviruses and analyzed 7–
12 days later. Both c-myb and v-myb directed early
wave cells to the melanocyte cell lineage. In c-myb
cultures pigment cells represented about 50% of
cells, while in v-myb cultures typically 90% of the
cells were melanocytes.
The strong melanocyte-promoting activity of v-Myb
was strictly dependent on the integrity of DNA
binding, transactivation and leucine zipper domains.
As shown in Figure 3b, the N118D mutation, which
completely eliminates the specific DNA binding of
Myb proteins , and the L237A mutation, which
inactivates Myb transactivation domain , elimi-
nated melanocyte formation. Similarly, replacement
of crucial leucine residues L325 and L332 within the
zipper domain by alanines, referred to as the L3,4A
mutation, significantly reduced v-Myb ability to
redirect glial development to melanocyte cell lineage.
TheL3,4A mutationwas previouslyshown tostrongly
reduce the ability of v-Myb to direct the commitment
of common myeloid progenitors to monocyte/macro-
none of the mutations significantly affected the
(Fig. 3b), it is evident that the same v-Myb domains
(functions) necessary for the commitment of hema-
topoietic progenitors into the monocyte/macrophage
lineage and transformation of monoblasts are also
required for the direction of NC progenitors to
treatedwith bFGF or
melanocyte cell lineage and transformation of mela-
Antibodies against specific antigens were used to
prove the identity of the cells studied. Early-wave
progenitors infected with the Dmyb retrovirus differ-
Figure 3. myb genes direct the development of early neural crest
(NC) cells into melanocytes. (a) Adobe Photoshop overlay of the
phase contrast and HNK-1 fluorescence (green) images (the same
(b) The ratios of non-melanocytes (others) and pigmented
melanocytes (melano) in cultures of early wave NC cells infected
with Dmyb and treated with bFGFor bFGF+SCF, or infected with
retroviruses carrying c-myb, wt v-myb, DNA-binding domain
mutant of v-myb (N118Dv-myb), transactivation domain mutant
of v-myb (L237Av-myb) and the leucine zipper mutant of v-myb
(L3,4Av-myb). The data were obtained in four independent
experiments. The abundance of Myb proteins in selected cultures
was determined by Western blot using antiMyb antibody. (c) The
presence of lineage-specific antigens in differentiating early wave
NC cells infected with Dmyb and v-myb viruses. 1E8 and MelEM
antibodies reveal glial cell- and melanocyte-specific antigens,
respectively. The 1E8, antiMyb and bright field images are taken
from the same field. Bars, 10 mm.
Cell.Mol.Life Sci.Vol.64, 2007
entiated into 1E8-positive glial cells (Fig. 3c), just like
non-infected progenitors (not shown). On the other
hand, cells in myb-infected cultures (exemplified by
the v-myb cells in Fig. 3c) did not synthesize the 1E8
antigen in accordance with their melanocyte pheno-
ferase subunit synthesized specifically in NC melano-
blasts committedtoproduce the pigment[44,45].The
control glial cells were negative. In v-myb melano-
blasts the MelEM antigen was distributed essentially
as described . In mid-early cells (7 days after
emigration) the antigen was mostly scattered in the
cytoplasm; later (12 days after emigration), it was
found concentrated around the nuclei (Fig. 3c). In
highly pigmented differentiated melanocytes no im-
munoreactivity with the MelEM was observed (not
shown). It is worth noticing that the melanocytogenic
activity of myb genes did not require additional
exogenous inductors (bFGF, SCF).
The induction of melanocyte formation by c-Myb, v-
Myb or by bFGF+SCF was possible only when early
wave cells were treated by these factors soon after
emigration while they were 1E8-negative. Later
infection or addition of factors resulted in progres-
sively lower amounts of melanocytes in cultures;
almost no melanocytes were obtained when myb
genes or cytokines were introduced to 1-week-old
cultures in which the majority of cells were 1E8-
early wave cell population we used is composed
mainly of uncommitted progenitors, which gradually
lose their ability to differentiate into melanocytes
along with their progression into glial cells. Impor-
(c-myb, v-myb or Dmyb) or treated with cytokines
steadily increased, while the massive changes of the
cell death were observed. This suggests that pigment
cells were generated by the conversion of progenitors
and not by the outgrowth of a few pigment cells that
have spontaneously arisen nor by the death of non-
the melanogenic cascade. We analyzed expression of
selected crucial factors engaged in melanocytogen-
The introduction of c-myb and v-myb genes into early
wave cells correlated with the accumulation of tran-
the progression of melanocyte development .
mRNA (messenger RNA) of the tyrosinase gene –
the target gene of the Mitf factor – was also strongly
upregulated in c-myb and v-myb cells (not shown).
The activity ofthe Mitf protein iscontrolled in several
ways. Among them, Mitf modifications induced by
SCF/c-Kit signaling represent an activating cue im-
portant for the development of melanocyte cell
lineage [48, 49]. Since c-kit expression depends on c-
Myb in immature erythroid cells , we asked
whether there is a link between c-Myb and c-kit
expression in NC cells as well. As documented in
v-myb resulted in the accumulation of c-kit mRNA,
while no striking changes were observed in the levels
of SCF mRNA. To find out whether myb genes also
promote accumulation of the c-Kit protein at the cell
surface, NC cells emigrating in early and late waves
from the HH10 neural tube explants were pooled and
infected with Dmyb, c-myb and v-myb retroviruses
and analyzed by flow cytometry with the kit2c75 anti-
chicken c-Kit antibody. The overlays in Figure 4b
document a detectable level of c-Kit in control NC
cells (present on late wave cells, which contain
committed melanocytes) and its significant increase
overlays also reveal that while there are still some c-
Kit-negative cells in the c-myb-infected population,
almost no such cells can be found in v-myb cultures.
This corresponds well to the proportion of melano-
cytes in c-myb and v-myb cultures.
been already documented , no such data are
available for v-myb. To find out whether the increase
of c-kit mRNA depends on v-Myb and is not solely
caused by altered composition of cells in a differ-
for 32 h to retroviruses transducing the 4-hydroxyta-
moxifen (4-OHT)-inducible v-mybER and N118Dv-
mybER (unable to bind the Myb recognition se-
24 h. cDNAs were assayed for the relative concen-
trations of c-kit sequences by semiquantitative and
quantitative PCR (Fig. 5a) and protein lysates (pre-
pared from cells immediately prior to 4-OHT treat-
ment) for the presence of Myb fusion proteins
(Fig. 5b). At the time of analysis not all the cells
were productively infected, and the amounts of
synthesized fusion proteins were only similar to that
of the endogenous c-Myb (Fig. 5b). Nevertheless, it
was critical to analyze cells well before any signs of
differentiation became apparent and any significant
changes in culture composition could take place. The
results of the real-time PCR (columns) as well as the
semiquantitative PCR (electrophoretograms) shown
in Figure 5a document the increase in c-kit mRNA
concentration already 7 h after 4-OHT induction,
which became more pronounced after 24 h of 4-OHT
treatment. It is apparent that v-MybER displays some
2980 V. Karafiat et al.Myb proteins activate melanocytogenesis
basal activity and increases c-kit mRNA even in the
absence of 4-OHT, as indicated by comparison of v-
mybER unstimulated cells with control cells (infected
with Dmyb virus). Importantly, both the basal and 4-
OHT-inducible effects of v-MybER were completely
binding domain. This supports the concept of direct
involvement of v-Myb in the activation of c-kit
As early NC cells produce SCF , the increased
likely to result in enhanced c-Kit signaling.This might
represent at least some of the mechanism through
Myb proteins activateSCF/c-Kit signaling in NC cells.
In order to learn whether the melanocyte-promoting
activity of Myb proteins is mediated by the SCF/c-Kit
signal, early wave cells were infected with c-myb, v-
myb and Dmyb retroviruses and treated with inhib-
itors of SCF/c-Kit signaling, namely the neutralizing
anti-SCFantibody  and the AG1296 inhibitor of c-
cultures 12 h after retroviral infections. The average
cell counts obtained 10 days after infection in two
independent experiments are plotted in Fig. 6. Both
reduced the number of melanocytes in c-myb and v-
myb cultures to approximately 50–30% of values
obtained in the absence of these compounds. The
melanocytogenic activity of c-Myb was slightly more
sensitive to c-Kit signal inhibitors than the activity of
v-Myb. Concomitant with the decrease in melano-
cytes, the number of other, mainly glial cells in these
cultures increased. We conclude that at least some of
the melanocytogenic activities of c-Myb and v-Myb
proteins are dependent on the SCF/c-Kit signal. This
signal is likely activated by the Myb-dependent
increase in the cell surface concentration of the c-Kit
cell fate determination not only in hematopoietic
progenitors  but also in NC cells.
We identified c-Myb and v-MybAMVtranscription
factors as powerful inducersof the melanocytogenesis
in avian neural tube explants. Both factors efficiently
promoted melanocyte development in explanted
neural folds, where the formation of NC cells has
already been activated and where the early stages of
lineage specification programs have been launched.
Furthermore, Myb proteins induced melanocytogen-
esis in neural plate epithelium, where these programs
have not been initiated. These results confirm our
recent observations that c-Myb is involved in early
inductive events in the neural tube leading to NC
formation  and reveal similar activity of the v-Myb
oncoprotein. Moreover, they document the capability
of both Myb proteins to specify development of
melanocyte cell lineage. At least a part of the
molecular mechanism(s) underlying these Myb activ-
ities could be activation of c-kit transcription or c-kit
mRNA stability, leading to accumulation of c-kit
It can be expected that higher c-Kit levels in NC cells
result in activation of the SCF-c-Kit melanocytogenic
of the c-kit gene has been long considered a candidate
Myb target promoter [53, 54]. However, there is no
evidence that Myb proteins directly regulate c-kit
Figure 4. Upregulation of mela-
nocyte-specific factors in pooled
early and late wave NC cells. (a)
Semiquantitative RT-PCR deter-
mination of relative concentra-
tions of scf, c-kit, mitf and gapdh
transcripts in cells infected with
Dmyb, v-myb or c-myb retrovi-
ruses. (b) Flow cytometry quan-
tification of the cell surface c-Kit
protein. Chick embryo fibro-
blasts (CEF) were used as the
Cell.Mol.Life Sci.Vol.64, 2007
to prove their interaction both in hematopoietic and
NC cells. The promoter of the SCF gene could also be
directly regulated by c-Myb, as suggested by analyses
of SCF production in fetal liver stroma in c-myb–/–
mice . However, in our experiments with NC cells
no changes in SCF mRNAwere observed in response
to elevated c-Myb or v-Myb proteins.
Activation of the c-Kit signal per se is probably not
sufficient to induce mitf transcription. However, Myb
proteins display a rather complex integration with the
signaling network of progenitors. We observed that
introduction of c-Myb into neural tube epithelium
treated with BMP4, SCF or bFGF leads to differing
results. For example, elevated concentrations of c-
Myb combined with bFGF result in massive delami-
nation and outgrowth of melanocyte precursors and
pigmented melanocytes, while in cooperation with
BMP4, rather undifferentiated progenitors accumu-
late in culture [V. Karafiat, unpublished observation].
Thus, other pathways, in addition to the c-Kit signal,
are likely modulated by Myb proteins. One of them
might be the EGF receptor signal .
In neural tube epithelium the v-Myb oncoprotein
induces development of cells of the melanocytic
lineage-restricted precursors it causes a partial block
of their terminal differentiation, activation of their
Myb and v-Myb activate similar events – commitment
of progenitors to the same cell lineage and activation
of a set of genes responsible for melanogenesis.This is
for the experiments and to the inferential spectrum of
surrounding regulatory factors. We suggest that in
cells that naturally use c-Myb for regulation, v-Myb
can operate in the same pathways, although with
Figure 5. Increase in c-kit mRNA depends on v-Myb. (a) Early
wave NC cells were infected with v-mybER-, N118Dv-mybER- or
Dmyb retroviruses; cultures were split 32 h post-infection and half
of them treated with 200 nM 4-OHT. In two independent experi-
ments RNAs were isolated either 7 or 24 h later, and cDNAs were
summarizes results of real-time PCR (RT qPCR). The electro-
phoretograms show results of semiquantitative PCR (RT PCR)
(b) Western blot analysis of first wave cells infected for 32 h with
Figure 6. Inhibition of c-myb and v-myb melanocytogenesis by
compounds interfering with SCF/c-Kit signaling. Early wave NC
cells infected with c-myb or v-myb retroviruses were treated with
preimmune rabbit serum (PRS), neutralizing anti-SCF rabbit
antibody or with AG1296 inhibitor of c-Kit phosphorylation. The
amounts of pigmented melanocytes and other cells in individual
cultures were determined as in Figure 3. The datawere obtainedin
two independent experiments.
2982 V. Karafiat et al. Myb proteins activate melanocytogenesis
eventually different phenotypical outcome caused by
the deregulated nature of the oncoprotein.
The complex effect of both myb genes on NC
formation and melanocyte development recalls their
effectson hematopoieticcells.Theresults ofthiswork
support the idea of overlapping genetic programs and
similar cell fate specification regulatory pathways in
both cell compartments [57, 58]. We hypothesize that
at least in birds, c-Myb is involved in molecular
mechanisms controlling proliferation, survival and
fate determination programs related in both hema-
topoietic and NC progenitors. One of these mecha-
nisms might be activation of c-Kit signaling, as
suggested by recent experiments in mice  and
data in the work described here. If activation of c-Kit
signaling is a common c-Myb property, then other cell
types dependent on the SCF/c-Kit signal may also
require c-Myb as a part of this pathway.
Acknowledgements. We thank Milos ˇ Grim and Petr Bartu ˚ne ˇk for
critical reading of the manuscript and helpful comments. We
and Lenka Pichl?kov? and the assistance of Sˇ?rka Tak?c ˇov? in
preparing the manuscript. This work is supported by project
AV0Z50520514 awardedby the Academy of Sciences of the Czech
and Sports of the Czech Republic and by grant 204/06/1728 from
the Grant Agency of the Czech Republic.
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2984 V. Karafiat et al. Myb proteins activate melanocytogenesis