1 Genetic Disease Research Branch, National Human Genome Research Institute,
National Institutes of Health, Bethesda, MD 20892-4472, USA
2 Cell and Developmental Biology Program, Fox Chase Cancer Center, Philadelphia,
PA 19111, USA
3 Laboratory of Developmental Neurogenetics, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892,
USA
* Author for correspondence (e-mail: lhou{at}mail.nih.gov)
Accepted 30 March 2004
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SUMMARY |
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Key words: G-protein-coupled receptor, Endothelin, Kit ligand, Mitf, Phorbol ester, Neurocristopathy, Neural crest cell culture, Tyrosinase, Melanogenesis
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Introduction |
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It is well known that Ednrb is expressed in mouse melanoblasts
(Lee et al., 2003;
Opdecamp et al., 1998
) and
hence thought to act cell-autonomously
(Hosoda et al., 1994
), in
contrast to its ligand EDN3 which is secreted and clearly works in a paracrine
fashion during melanocyte development. Nevertheless, a study in chimeric mice
composed of wild-type and Ednrb null mutant cells
(Ednrbs-l, piebald-lethal) has suggested that
Ednrb can act cell non-autonomously during enteric neuroblast
development (Kapur et al.,
1995
). Because of a lack of suitable markers for mutant
melanoblasts, however, this chimeric study did not allow conclusions as to the
fate of the mutant pigment lineage in the wild type environment, and so the
question of whether melanocyte development is likewise subject to cell
non-autonomous actions of Ednrb remained unanswered.
In order to be able to track Ednrb-expressing pigment cells even
when they are deficient in functional Ednrb, we here used a novel
null allele, EdnrblacZ, that was obtained by targeted
insertion of the bacterial lacZ marker gene into the first exon of
mouse Ednrb (Lee et al.,
2003). Although chimeric mice composed of wild-type and mutant
cells, as mentioned above, usually provide a straightforward approach to test
for cell non-autonomous gene actions, they may offer limited possibilities to
further characterize the cell types and growth factors involved. This
limitation prompted us to perform tissue recombination experiments in vitro,
using neural crest cell cultures established from explanted mid-gestation
neural tubes (NTs).
The experiments show that in the absence of functional Ednrb, the
development of the melanocyte lineage is initiated but no tyrosinase-positive
or mature melanocytes are being generated. The mutant cells can be rescued,
however, by a two-step procedure whereby a first step, involving cell
non-autonomous EDNRB signaling in cells other than melanoblasts, leads to
tyrosinase expression but not pigmentation, and a second step, mimicking cell
autonomous EDNRB signaling in melanoblasts, leads to pigmentation. The results
imply that Ednrb plays a significant role in melanocyte
differentiation at time points beyond the narrow developmental period between
E10.5 and E12.5 through which it has been shown to be required in vivo
(Shin et al., 1999), and they
suggest that this later role is mediated by both cell-autonomous and cell
non-autonomous signaling pathways.
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Materials and methods |
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Noon on the day a vaginal plug was found was defined as embryonic day 0.5
(E0.5). Embryos were harvested at E9.5. The polymerase chain reaction (PCR)
was used to genotype embryos, using the head portion as source of genomic DNA.
Three oligonucleotides were used: Ednrb1,
5'-CCAGACTGAAAACAGCAGAGCGGC-3' (forward); Ednrb2,
5'-GGTCTCCCAGAGCCAGACTGGCGATA-3' (reverse); and
Ednrb-lacZ, 5'-CTGTTGGGAAGGGCGATCGGTGC-3' (reverse).
Because of a deletion associated with the targeted insertion,
EdnrblacZ/EdnrblacZ embryos could be detected
by the presence of a 270 bp band and the absence of a 495 bp band, wild-type
embryos by the presence of the 495 bp band and the absence of the 270 bp band,
and heterozygotes by the presence of both bands. To genotype embryos
heterozygous or homozygous for KitlSl, the
oligonucleotides used were those described previously
(Ono et al., 1998). For
genotyping, genomic DNA was denatured (94°C for 3 minutes) in the presence
of primers, and subjected to PCR in a 50 µl reaction for 30 cycles
(94°C for 40 seconds, 55°C for 1 minute and 72°C for 1 minute)
using Taq polymerase (Promega) and the manufacturer's buffer in the presence
of 1.5 mM MgCl2.
Neural tube explant cultures, in vitro reconstitutions and growth factor treatments
Embryos at E9.5 were obtained from the matings described above. Pure NT
explants containing neural crest cells were isolated and cultured as described
previously (Ito and Takeuchi,
1984). For most experiments, culture medium consisted of 90% DMEM,
1 mM L-glutamine, 1 mM penicillin-streptomycin, 10% FBS and EDN3 (Sigma) at 10
nM. When mentioned, KITL (R&D Systems) was added at 5 nM,
12-O-Tetradecanoylphorbol 13-acetate (TPA) at 40 nM and endothelin 1 (EDN1,
Sigma) at 40 nM. For in vitro reconstitution experiments, embryonic NT
explants were isolated at E9.5 from the following matings: +/+ x +/+;
EdnrblacZ/+ x
EdnrblacZ/+; Mitf
miew/Mitf miew x
Mitf miew/Mitf miew; or
KitlSl/+ x KitlSl/+. Neural
crest cell cultures were established separately for each embryo and the
embryos were genotyped. Twenty-four hours later, NTs from
EdnrblacZ/EdnrblacZ cultures were
removed and replaced with NTs from either wild type,
EdnrblacZ/EdnrblacZ, Mitf
miew/Mitf miew or
KitlSl/KitlSl cultures. For each reconstitution
experiment, the medium contained 10 nM EDN3.
Antibodies and immunostaining
At various days in culture, neural crest cells were fixed in 4%
formaldehyde in PBS (pH 7.5) for 25 minutes at room temperature, and then
permeabilized with 0.1% Triton-X-100 for 5 minutes. For double indirect
immunolabeling, the cells were incubated with rabbit anti-Mitf
(Opdecamp et al., 1997) or
anti-tyrosinase antisera (Vincent Hearing) for 60 minutes, respectively, and
then with anti-ß-galactosidase (ß-gal) monoclonal antibody (Promega)
for 30 minutes. These antibodies were revealed with RITC-coupled goat
anti-rabbit (Fab)2 and FITC-coupled goat anti-mouse
(Fab)2.
Identification of ß-gal-positive cells and pigmented melanocytes
The cultured neural crest cells were fixed at 14 days in 4% formaldehyde in
PBS. X-gal staining was performed in X-gal buffer (2 mM MgCl2,
0.02% NP-40, 2 mM K3Fe(CN)6, 2 mM
K4Fe(CN)6, 0.05% X-gal in PBS, pH 7.5), and postfixed in
4% formaldehyde. The appearance of melanocytes was examined at various time
points in live cultures. Their differentiation from neural crest cells was
judged by the presence of melanin granules (one of the markers of mature
melanocytes) and the characteristic dendritic morphology under phase-contrast
and bright-field microscopy.
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Results |
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Despite the fact that lineage development is initiated in the absence of
functional Ednrb, the cultures do not generate pigmented cells. As
the rate-limiting step in pigmentation is tyrosinase activity, we reasoned
that cells devoid of functional Ednrb might not reach the
tyrosinase-positive stage. Indeed, double-labeling for ß-gal and
tyrosinase showed that even after 14 days in culture, i.e. 8 days after the
usual onset of tyrosinase expression (Hou
et al., 2000),
EdnrblacZ/EdnrblacZ NTs did not
generate tyrosinase-positive cells, while EdnrblacZ/+ NTs
did (Fig. 2E,F). This result
suggested that in vitro, Ednrb signaling is required for the
development of cells to the differentiated, tyrosinase-positive stage and
hence explains why EdnrblacZ/EdnrblacZ
cells never become pigmented.
Ednrb +/+ NTs induce melanoblast differentiation in EdnrblacZ/EdnrblacZ neural crest cells
To test whether Ednrb-deficient melanoblasts may be rescued in the
presence of wild-type NT or neural crest cells, we performed in vitro
recombination experiments in which the
EdnrblacZ/EdnrblacZ NTs, after a 24
hour culture period during which the neural crest cells emigrated, were
removed and replaced with Ednrb+/+ NTs. For these
experiments, we used Ednrb+/+ NTs from embryos homozygous
for the Mitf allele, Mitf miew, which
cannot generate melanoblasts that survive for more than 24-48 hours and hence
neither contain tyrosinase-positive nor pigmented cells
(Opdecamp et al., 1997). In
this way, we could avoid the potential complication that a transfer of
melanosomes from pigmented Ednrb+/+ cells to the
non-pigmented EdnrblacZ/EdnrblacZ
cells might have created. As a control,
EdnrblacZ/EdnrblacZ NTs were exchanged
for EdnrblacZ/EdnrblacZ NTs obtained
from parallel cultures, and, as expected under these conditions, no
tyrosinase-positive cells were seen (Fig.
3A). The reconstitution with Mitf miew/Mitf
miew NTs, however, led to the generation of
ß-gal/tyrosinase double-positive cells in
EdnrblacZ/EdnrblacZ cultures
(Fig. 3B). This result
suggested that the Ednrb-positive neural crest cells provided an
exogenous signal capable of rescuing tyrosinase expression in the co-cultured
Ednrb-negative cells, and that melanocytes were not required for this
rescue. Nevertheless, the possibility, however remote, that at least some of
the double-positive cells might have been the product of cell fusions cannot
formally be excluded with this experiment.
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KITL is both required and sufficient to rescue EdnrblacZ/EdnrblacZ melanocyte precursors to the tyrosinase-positive stage
As the above reconstitution experiments suggested that KITL is required to
allow tyrosinase expression in Ednrb-negative melanocyte precursors,
we then asked whether it was sufficient. In fact, we found that the treatment
of EdnrblacZ/EdnrblacZ cultures with
soluble KITL from the day of NT explantation led to the generation of
tyrosinase-expressing cells (Fig.
4). Thus, KITL was both required and sufficient to promote the
differentiation of Ednrb-deficient melanocyte precursors.
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Discussion |
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At first sight, a role for Ednrb in melanocyte differentiation
would seem to contradict previous in vivo findings that showed, based on
conditional downregulation, that Ednrb is no longer needed for
melanocyte development after E12.5 (Shin
et al., 1999); at this developmental time point, tyrosinase is not
yet expressed, at least not in the trunk area, and pigmentation also is not
seen until several days later. We have to consider, though, that our in vitro
results are based on analyzing isolated NT and neural crest cells, not intact
embryos. Conceivably, the role of Ednrb in melanocyte differentiation
may not be revealed in vivo because of compensatory mechanisms that operate
efficiently only in the embryo. Thus, parallel signaling pathways, whether
acting cell-autonomously or not, might substitute for the lack of
Ednrb beyond E12.5 in vivo. In fact, Ednrb-deficient mice
can have pigmented spots on the head and at the base of the tail, indicating
that in vivo, melanocytes can be generated, albeit rarely, in the complete
absence of Ednrb. The fact that, in vitro, conditions can be found
that rescue Ednrb-negative cells to the fully mature, pigmented stage
is entirely consistent with the appearance of these pigmented spots in vivo,
and suggests that they are derived from a few somatically variant precursor
cells that have crossed a threshold of sensitivity to rescuing pathways that
are similar, or identical, to those we have identified in vitro. Although it
was believed that signaling through EDNRA might be responsible for these
pigment spots (Reid et al.,
1996
), this is unlikely, because our in vitro studies with EDN1
provide no evidence for a role for EDNRA in pigmentation.
There are several alternative explanations for the seeming discrepancy between a role for EDNRB beyond the equivalent of E12.5 in vitro and not in vivo. It is possible, for example, that the conditional downregulation of the Ednrb mRNA in vivo was not followed by an equally rapid downregulation of EDNRB protein or of its activated downstream targets. In addition, the conditional downregulation of Ednrb, even if complete in melanoblasts, may not have been complete in other cell types, and incomplete downregulation in other cell types may then have added indirectly to the rescue of melanocytes as suggested by our in vitro analysis.
The cell non-autonomous action of Ednrb during melanocyte
development lends support to previous observations in chimeric mice in which a
wild-type environment helped Ednrb mutant enteric neuroblasts to
colonize the large intestine (Kapur et
al., 1995). The details of the underlying mechanisms may differ,
though, as it has been suggested that EDNRB signaling may normally delay
neuroblast differentiation (Wu et al.,
1999
), allowing for prolonged cell proliferation and migration,
and not promote cell differentiation, as described here for melanoblasts. In
any event, cell non-autonomous actions of growth factor receptors are not
without precedents. A telling example are embryos deficient in a tyrosine
kinase receptor, fibroblast growth factor receptor 1 (Fgfr1), which
is expressed in several cell types, including presomitic mesoderm precursors.
In such embryos, somites fail to form, suggesting Fgfr1 plays a
cell-autonomous role in somite specification, but a chimeric analysis showed
that upon development in a wild-type environment, the mutant cells can
contribute to somites (Rossant and Spence,
1998
).
The existence of cell non-autonomous mechanisms would predict that genetic
modifiers causing phenotypic variabilities in receptor mutants, including
Ednrb mutants of mice (Pavan et
al., 1995) and humans, need not necessarily influence the
expression or activity of the corresponding signaling pathways in the very
cell types that cause disease. It is often unknown, however, what cell types
might be responsible for such indirect rescue. In the case of Ednrb,
we have only demonstrated that the melanocyte lineage, at least its later
tyrosinase-positive cell population, is not involved, and we can but speculate
as to the cell type that is involved (see below). In addition, we do not know
whether in the Ednrb-deficient NTs and their derivatives, the
respective `helper' cell type(s) are missing altogether, or are present but
simply unable to respond to EDN3. Regardless of the nature of the responsible
cell type, our results clearly indicate that the helper cells need to provide
at least one factor KITL.
The rational for testing KITL as the prime rescue factor for
Ednrb-deficient cells was based on a number of previous studies that
showed that Ednrb and Kit are both required for melanocyte
development (Baynash et al.,
1994; Geissler et al.,
1988
) and that EDN3 and KITL cooperate to effect melanogenesis
(Hou et al., 2000
;
Reid et al., 1996
). Moreover,
we have previously found that although Kit-deficient neural crest
cells respond to EDN3 with enhanced survival, they do not develop to the
tyrosinase-positive state (Hou et al.,
2000
), and we report here that Ednrb deficiency does not
interfere with Kit expression. Last, variant alleles of the
Kitl locus on mouse chromosome 10 serve to phenotypically modify the
hypomorphic, non-lethal Ednrbs (piebald) allele
(Pavan et al., 1995
;
Rhim et al., 2000
). Taken
together, these findings provided strong indications that it was the lack of
sufficient amounts of KITL that was responsible for the absence of tyrosinase
expression in Ednrb-deficient melanocyte precursors. Indeed, KITL was
able to induce tyrosinase expression in Ednrb-deficient cells, as was
the addition of Ednrb+/+ NTs, even
Ednrb+/+ NTs that were incapable of generating melanocytes
of their own as long as they could synthesize KITL. It appears, then, that
EDNRB, by still unknown mechanisms, stimulates KITL synthesis and/or
secretion. A likely source for this KITL is the neural tube or its derivatives
where both Ednrb (Lee et al.,
2003
) and Kitl (Guo et
al., 1997
; Wehrle-Haller and
Weston, 1999
) are expressed.
Although the above results showed a role for KITL in rescuing the
Ednrb-deficient cells to the tyrosinase-positive stage, KITL was not
sufficient to render them pigmented. This was unlikely to be because of
insufficient expression of tyrosinase; the intensity of its immunofluorescent
signal in KITL-treated EdnrblacZ/EdnrblacZ
cells was similar to that observed in equally treated
EdnrblacZ/+ cells. Rather, it appears that tyrosinase is
not properly activated in the rescued cells. In fact, even in
Ednrb-wild type cultures, KITL is insufficient to stimulate
melanogenesis (Morrison-Graham and Weston,
1993; Murphy et al.,
1992
; Reid et al.,
1996
), but the addition of TPA
(Murphy et al., 1992
) or EDN3
(Reid et al., 1996
) induces
pigmentation. Similarly, the addition of TPA induces pigmentation in our
rescued cells. A major site of action of both EDNRB signaling and TPA is the
activation of protein kinase C (PKC), which has been shown to activate
tyrosinase by serine phosphorylation (Park
et al., 1999
). If indeed melanogenesis in Ednrb-deficient
cells requires the stimulation of PKC, then our results strongly suggest that
for melanin synthesis to occur, the cells also depend on cell-autonomous EDNRB
signaling pathways. In fact, during terminal differentiation, the
KITL-mediated cell non-autonomous action of Ednrb may be replaced by
its cell-autonomous signaling. This view is consistent with the observation,
based on the in vivo use of neutralizing KIT antibodies, that KIT signaling is
no longer needed for melanogenesis after
E14.0 when melanocytes begin
their terminal differentiation in the epidermis
(Nishikawa et al., 1991
).
Although our observations do not assess the relative importance of cell non-autonomous Ednrb signaling during melanocyte development in wild-type embryos in vivo, they clearly show that such mechanisms exist. This fact suggests, therefore, that the well-known dual dependency of the melanocyte lineage on two major signaling pathways, G-protein coupled signaling through EDNRB and tyrosine kinase receptor signaling through KIT, may actually result from complex interplays between cell-autonomous and cell non-autonomous actions. As schematically shown in Fig. 6, these different modes of action probably operate at distinct stages in lineage development, allowing for an intricate mechanism to fine-tune the proper deposition of melanocytes in the different bodily compartments.
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ACKNOWLEDGMENTS |
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