1 Department of Anatomy and Cell Biology, Institute for Biomedical Sciences, The
George Washington University, Washington, DC 20037, USA
2 Department of Ophthalmology, MEEI, Harvard Medical School, Boston, MA 02138,
USA
Author for correspondence (e-mail:
anasam{at}gwumc.edu)
Accepted 28 September 2004
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SUMMARY |
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Key words: Pre-placodal ectoderm, Neural crest, foxD3, zic2, sox2, sox3, keratin, dlx5, dlx6, Cell fate determination, Patterning, Xenopus
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Introduction |
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Several studies indicate that during gastrulation, embryonic ectoderm is
separated into fields with distinct fates (neural plate, neural crest, PPE,
epidermis; Fig. 1A) in response
to different concentrations of BMP. Genes expressed by the presumptive
epidermis are positively regulated by BMPs
(Suzuki et al., 1997;
Feledy et al., 1999
;
Beanan and Sargent, 2000
;
Luo et al., 2001a
;
Tribulo et al., 2003
), whereas
anti-BMP factors secreted from the organizer and the dorsal midline mesoderm
promote neural plate formation (Weinstein
and Hemmati-Brivanlou, 1999
;
Wilson and Edlund, 2001
). It
has been suggested that signals responsible for establishing the neural plate
also establish a lateral neurogenic ectoderm (LNE) that surrounds the neural
plate and gives rise to the intervening neural crest and PPE
(Baker and Bronner-Fraser,
2001
). The best-studied derivative of the LNE, the neural crest,
appears to be induced by intermediate concentrations of anti-BMP factors
(Morgan and Sargent, 1997
;
Marchant et al., 1998
;
Mayor et al., 1999
;
Mayor and Aybar, 2001
;
Aybar et al., 2002
). In
addition, neural crest induction requires signaling pathways that establish
the posterior axis of the neural plate (Wnt, FGF, retinoic acid)
(LaBonne and Bronner-Fraser,
1998
; Chang and
Hemmati-Brivanlou, 1998
; Mayor
and Aybar, 2001
; Villanueva et
al., 2002
; Monsoro-Burq et
al., 2003
; Glavic et al.,
2004a
). Whether similar signaling events are involved in
establishing the PPE has not been addressed because of a paucity of molecular
markers specific for this ectodermal domain.
|
Although placodes have long been recognized as important embryonic
structures, their transient nature and the lack of specific molecular markers
have made it difficult to study the mechanisms by which they form. Recently,
however, markers of the PPE during the initial induction of the placodes have
been identified in Xenopus. six1 is homologous to Drosophila sine
oculis; it is characterized by a homeobox DNA-binding domain and a
protein-protein interaction domain called the Six domain. It is initially
expressed in a band surrounding the anterior neural plate and later in all
neurogenic placodes (Pandur and Moody,
2000). eya1 is homologous to Drosophila eyes
absent (eya); it functions as a co-factor for Six genes of the
Six1/2 and Six4/5 subfamilies (Pignoni et
al., 1997
; Ohto et al.,
1999
; Ikeda et al.,
2002
) and is expressed in a pattern very similar to that of
six1 (David et al.,
2001
). We have used these markers to demonstrate that gradients of
both neural inducer and anteroposterior signals are required for proper PPE
formation. Moreover, we show that six1 expression is required for the
establishment of the PPE, and it promotes the PPE at the expense of the neural
crest and epidermis by both activating and repressing target gene expression.
Finally, we demonstrate that several genes expressed in the embryonic ectoderm
mutually influence each other to define its distinct subdomains.
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Materials and methods |
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RNA microinjection
Transcripts of six1 (400-600 pg), six1VP16 (100 pg),
six1EnR (100 pg), Dgroucho (400 pg), noggin (5-40
pg) (Smith and Harland, 1992),
chordin (5-40 pg) (Sasai et al.,
1994
), dnWnt8 (500 pg)
(Hoppler et al., 1996
),
frzb-1 (750 pg) (Wang et al.,
1997
), bmp4 (10-50 pg)
(Dale et al., 1992
),
eya1 (400 pg) (David et al.,
2001
), zic2 (100 pg)
(Brewster et al., 1998
),
foxD3 (25 pg) (Sasai et al.,
2001
) (from D. Kessler), dlx5 (200 pg)
(Luo et al., 2001a
),
dlx6 (200 pg) (Luo et al.,
2001a
) and sox2 (200 pg)
(Mizuseki et al., 1998
) (from
T. Grammar) were mixed with ß-galactosidase
(ß-gal) mRNA (100-200 pg) and microinjected into identified
blastomeres with known ectodermal fates
(Fig. 1C) as described
(Moody, 2000
).
Yeast two hybrid analysis
To determine if Xenopus six1 interacts with Dgroucho, the
Six domain (amino acids 9-123) was cloned into pGBKT7, and an N-terminal
region of Dgroucho (amino acids 1-247) was cloned into pGAD424
(Clontech). The yeast strain AH109 was transformed with both vectors and
assayed for reporter gene expression according to the MATCHMAKER kit
(Clontech).
Morpholinos
Morpholino antisense oligonucleotides (MO) were synthesized against two
different potential translational start sites in six1
(5'-GGAAGGCAGCATAGACATGGCTCAG-3' and
5'-CGCACACGCAAACACATACACGGG-3') (Gene-Tools). An equimolar mixture
of the two six1-MO, or a standard control MO
(5'-GGAAGGCAGCATAGACATGGCTCAG-3') was microinjected (10-16 ng). A
Myc-tagged construct (six1-myc) containing the six1
wild-type 5'UTR was generated to assess morpholino knock-down efficacy
by immunofluorescent detection of protein. A rescue construct
(six1-rescue) was generated by replacing the six1 wild-type
5'UTR with pCS2+ sequence and changing nine bases in the coding region
that would interfere with MO binding without altering amino acid sequence.
Animal cap explants
The animal pole was injected with mRNAs for noggin (50 pg),
cerberus (50 pg) (Bouwmeester et
al., 1996), bmp4 (50 pg), Wnt8 (50 pg)
(Hoppler and Moon, 1998
) or
constitutively activated fgfr1 (cfgfr1, 50 pg)
(Neilson and Friesel, 1996
).
Animal cap explants were dissected at stages 8.5-9 and cultured in NAM
(Messenger and Warner, 1979
),
in some cases supplemented with recombinant mouse Noggin protein (R & D
Systems). Explants were processed for either RT-PCR or in situ hybridization.
For each in situ hybridization experiment, control and an entire series of
Noggin-treated explants were processed in parallel so that staining
intensities could be compared. The intensity of reactivity in experimental
caps was compared with control caps, and then sorted into three groups of
staining intensity (none, moderate, high). Frequencies of caps in each group
were compared between treatments by Chi-square analyses.
RT-PCR
Total RNA from animal cap explants was isolated then subjected to first
strand cDNA synthesis using oligo(dT) primers. PCR was performed in the linear
range using six1 primers as described
(Pandur and Moody, 2000).
In situ hybridization
Full-length antisense RNA probes for six1
(Pandur and Moody, 2000),
eya1 (David et al.,
2001
), sox2 (Penzel
et al., 1997
) (from R. Grainger), sox3
(Zygar et al., 1998
),
sox11 (from T. Grammer and R. Harland), foxD3
(Sasai et al., 2001
) (from D.
Kessler), epidermal specific keratin
(Jonas et al., 1989
),
dlx5 and dlx6 (Luo et
al., 2001a
), and zic2
(Brewster et al., 1998
) were
transcribed in vitro, and embryos were processed by standard protocols
(Sive et al., 2000
). The
widths of the expression domains of marker genes were measured in whole-mount
preparations of ß-gal mRNA-injected control embryos and of
experimental transcript-injected embryos at 40 x with an eyepiece
micrometer, as described (Kenyon et al.,
2001
). Measurements were expressed as differences between injected
and uninjected sides of the same embryo, and the mean differences between
groups were analyzed by t-tests.
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Results |
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|
Neural inducers alone are not sufficient to induce six1 in the intact embryo
Consistent with the explant data, increased BMP signaling in the lateral
ectoderm of the intact embryo, achieved by injecting doses of bmp4
mRNA that do not disrupt axial patterning into ventrolateral blastomeres that
contribute significantly to the LNE (V1.2.1, V1.2.2;
Fig. 1C), reduced endogenous
six1 expression (Fig.
3A; 56% of cases at 20 pg, 67% at 40 pg). To test whether neural
inducer alone is sufficient to induce the PPE in the intact embryo, different
concentrations of noggin or chordin mRNA (10 pg, 20 pg and
40 pg) were microinjected into a ventral blastomere (V1.1.2;
Fig. 1C) that contributes
significantly to the ventral epidermis. These concentrations were used because
they induce neural genes in explants but infrequently re-pattern the embryo to
produce secondary axes. Two morphologies were observed: a dispersed clone with
no obvious patterning defects (Fig.
3B) or an elongated clone associated with a ventrally located
putative secondary axis (Fig.
3C,D). Staining with sox2 confirmed that every embryo
with an elongated clone contained ectopic neural tissue
(Fig. 3C), whereas none with a
dispersed clone did. Regardless of concentration of injected mRNA, in no case
was there ectopic six1 expression associated with a dispersed clone
(Fig. 3B), but in every
elongated clone, ectopic six1 expression occurred at its anterior tip
(Fig. 3D). Thus, in contrast to
explants, in which Noggin alone induces six1 expression, in whole
embryos the additional presence of an axis is necessary for six1
expression.
|
six1 expression expands placode gene expression at the expense of epidermis and neural crest, and is necessary for PPE formation
At neural plate stages, sox2/sox3 neural plate expression domains
are separated from the six1/eya1 placodal domains by the intervening
foxD3/slug neural crest domain
(Schlosser and Ahrens, 2004;
Glavic et al., 2004b
).
Epidermis-specific keratin is expressed lateral to the six1
domain (Fig. 4A) and another
PPE marker, sox11, is expressed in the neural plate and in lateral
crescents that overlap with those of six1/eya1
(Fig. 4D). To test whether
six1 regulates the expression of any of these genes, wild-type
six1 (six1-WT) was overexpressed in the LNE region
by mRNA injection into ventrolateral blastomeres. keratin expression
was significantly repressed at the sites of increased six1 expression
(Fig. 4B; 86.8% of embryos;
Table 1). By contrast, genes
expressed in the PPE domain were expanded; sox11 (75%) and
eya1 (81%) domains were significantly larger than in controls
(Fig. 4E;
Table 1). The foxD3
domain was significantly reduced (Fig.
4H; 73.7%; Table
1). These changes in gene expression in the lateral ectoderm did
not significantly alter the extent of the neural plate domain, as defined by
the size of the expression domains of sox2 or sox3
(Fig. 4K;
Table 1). These results
demonstrate that elevated six1 expression in the lateral ectoderm
promotes PPE genes at the expense of epidermal and neural crest genes, but has
minimal direct effect on the neural plate domain.
|
|
six1 affects ectodermal genes via both transcriptional activation and repression
Six1/2 type factors likely function as both transcriptional activators and
repressors depending upon the presence of co-factors
(Silver et al., 2003). To
determine whether the above effects are via transcriptional activation or
repression, both activating (six1VP16) and repressive
(six1EnR) constructs were made. The keratin expression
domain was dramatically repressed by six1VP16
(Fig. 5A; 96.3%;
Table 1). The lateral
expression of six1EnR also caused a significant reduction in
keratin expression (Fig.
5C; 100%, Table 1).
PPE genes (sox11, 95.5%; eya1, 75%) were significantly
expanded by six1VP16, and significantly reduced (68.8% and 90%,
respectively) by six1EnR (Fig.
5E,G; Table 1). The
foxD3 expression domain was significantly expanded by
six1VP16 (90.9%), and significantly reduced by six1EnR
(Fig. 5I,K; 81.8%;
Table 1). A neural plate gene
(sox2) was unaffected by either construct expressed in the lateral
ectoderm (Table 1). These
results predict that: (1) keratin expression is repressed by Six1
both directly and indirectly because the six1-WT and both activating
and repressing constructs reduce its domain; (2) PPE genes are
transcriptionally activated by Six1 because the effect of six1VP16
mimics six1-WT and the effect of six1EnR is the reverse; (3)
foxD3 is transcriptionally repressed by Six1 because the effect of
six1EnR mimics six1-WT and the effect of six1VP16
is the reverse.
|
Subdomains within the LNE depend upon mutual gene interactions
It has been proposed that interactions between the border of the neural
plate and the non-neural ectoderm are crucial for establishing fates within
the LNE (Luo et al., 2001a;
McLarren et al., 2003
;
Woda et al., 2003
). We
investigated whether expanded six1 expression affects genes whose
domains lie at the border between the PPE and neural plate (zic2) or
the border between the PPE and epidermis (dlx5, dlx6). Increased
six1-WT expression significantly expanded the patch of zic2
that is expressed lateral to the neural plate
(Fig. 6A,B; 73.1%;
Table 1), without affecting the
neural plate domain (data not shown). This effect was mimicked by both the
six1VP16 construct (100%) and co-injection of
six1-WT+eya1-WT mRNAs (85.7%), but not by the
six1EnR construct or co-injection of
six1-WT+Dgroucho mRNAs
(Table 1). These results
indicate that Six1 positively regulates the lateral zic2 domain via
transcriptional activation. However, reduction of Six1 protein by
six1-MO injections also expanded zic2 expression
(Fig. 6C; 90%;
Table 1), indicating that other
factors that are antagonized by Six1 probably also positively regulate
zic2 (see below). Increased six1-WT expression had two
effects on Dlx expression. First, it caused the lateral stripes of
dlx5 and dlx6 expression to be located further laterally
(Fig. 6E; 71.4% and 70.4%,
respectively; Table 1). Second,
the dlx5 (78.6%) and dlx6 (51.9%) stripes either had gaps of
full repression or were diffuse and less intense compared with the uninjected,
control side (Fig. 6E). The
six1VP16 construct (93.3%), co-injection of
six1-WT+eya1-WT mRNAs (90.0%), the six1EnR
construct (80.0%) and co-expression of six1-WT+Dgroucho
mRNAs (65.0%) all caused a phenotype similar to six1-WT for
dlx6 (Table 1). Both
dlx5 (84.6%; Table 1)
and dlx6 (Fig. 6F;
100%; Table 1) were moved more
laterally or repressed by reduction of Six1 by MO injection, suggesting that
other factors antagonized by Six1 also negatively regulate the dlx5/6
genes (see below).
|
|
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Discussion |
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We investigated the potential roles of these mechanisms in PPE specification. Placodes have long been recognized as important embryonic anlage for cranial sensory structures, but the mechanisms by which the early, transient PPE that gives rise to all individual placodes is induced and segregated from neighboring ectodermal domains have not been elucidated. Our findings support the involvement of all three mechanisms in PPE specification (Fig. 7): (1) gradients of both neural inductive and anteroposterior signaling are necessary to induce and appropriately position the PPE; (2) six1 acts as a placodal fate specifying gene that positively regulates the expression of other PPE markers, and negatively regulates neural crest and epidermal identities: and (3) interactions between several transcription factors expressed in the LNE border zone modulate the relative sizes of the different ectodermal subdomains.
Induction of PPE and neural crest by differential responses to neural inducing and anterior-posteriorizing factors
The lateral neurogenic ectoderm (LNE), which comprises neural crest and
PPE, is located between the neural plate and the epidermis in an intermediate
region of a proposed gradient of BMP activity
(Weinstein and Hemmati-Brivanlou,
1999; Wilson and Edlund,
2001
). It has been shown that neural crest markers are induced by
concentrations of neural inducers lower than those required for neural plate
markers (Morgan and Sargent,
1997
; Marchant et al.,
1998
), and we demonstrate that even lower concentrations elicit
the highest levels of expression of two placodal markers (six1,
eya1). This differential responsiveness to Noggin concentrations in
explants supports the idea that an endogenous gradient of neural inducing
signals provides a first step in separating the embryonic ectoderm into its
different domains (Fig. 7).
However, in the intact embryo, in which several signaling pathways intersect
to influence the fate of the cell, intermediate BMP levels are not sufficient
to induce either neural crest or placode fates. The neural crest, which is
absent from the most anterior pole of the neural plate but extends to its
posterior tip, requires Wnt and FGF signaling
(LaBonne and Bronner-Fraser,
1998
; Chang and
Hemmati-Brivanlou, 1998
; Mayor
and Aybar, 2001
; Villanueva et
al., 2002
; Monsoro-Burq et
al., 2003
; Glavic et al.,
2004a
). These pathways are thought to act both to posteriorize the
axis and to directly promote neural crest fate
(Monsoro-Burq et al., 2003
;
Lewis et al., 2004
). We
demonstrate that these same signals antagonize six1 expression, and
propose that their posterior expression restricts placode formation to the
head (Fig. 7). In the intact
embryo, the expression of endogenous Wnt and FGF antagonists in anterior
regions may additionally contribute to restricting placode formation
(Leyns et al., 1997
;
Glinka et al., 1998
;
Piccolo et al., 1999
;
Bradley et al., 2000
;
Nutt et al., 2001
;
Zhang et al., 2001
;
Yamaguchi, 2001
;
Tsang and Dawid, 2004
).
These data indicate that PPE and neural crest formation require the
combined activities of neural inducing and anterior-posteriorizing factors.
However, different concentrations of BMP antagonists favor one fate over the
other, and posteriorizing factors promote neural crest over placodal fate.
Thus, it is likely that the LNE is initially competent to give rise to both
placodal and crest derivatives, and the acquisition of the specific fate of a
LNE cell results from local concentrations of the pertinent signaling
pathways. This hypothesis is consistent with: (1) observations that although
six1/eya1 are most highly expressed at Noggin concentrations
lower than those for foxD3, there is significant overlap in the two
dose-response curves; (2) fate maps showing that otic placode precursors are
intermingled with future neural crest precursors
(Streit, 2002); and (3)
observations that the expression domains of some neural crest and placodal
marker genes partially overlap (McLarren
et al., 2003
; Schlosser and
Ahrens, 2004
; Glavic et al.,
2004b
).
six1 acts as a placodal fate specifying gene
Studies in vitro have previously shown that Six proteins can function as
transcriptional activators and repressors
(Kobayashi et al., 2001;
Ohto et al., 1999
;
Silver et al., 2003
;
Li et al., 2003
). However,
only evidence for repression has been documented in vivo, specifically for the
related factors Optx2/Six6 and Six3 (Zuber
et al., 1999
; Kobayashi et
al., 2001
). We find that placodal fate specification by Six1
requires both activating and repressing activities. Analyses of
six1-WT and six1-MO injected embryos show that Six1
positively regulates the expression of PPE factors and negatively regulates
other ectodermal genes (Fig.
4). By using Six1VP16 and Six1EnR constructs, we further
demonstrate that these effects result from both activating and repressing
activities of Six1 in vivo (Fig.
5). These activities may be mediated, at least in part, by the
association of Six1 with the transcriptional activator Eya1 or transcriptional
repressors of the groucho family (Fig.
5). Whether endogenous Six1 functions as an activator or repressor
is probably determined by the relative levels and activities of the different
co-factors within six1-expressing cells.
Definition of sub-domains within the embryonic ectoderm
Our studies also predict complex in vivo interactions between six1
and other ectodermal genes. sox2 and sox3 are both induced
in presumptive neural ectoderm by neural inductive signaling, and promote
stabilization of a neural fate (Mizuseki
et al., 1998; Penzel et al., 1998). Later, they are both expressed
in neural stem cells (Graham et al.,
2003
). We show that endogenous six1 expression in the LNE
precedes sox2/3 placodal expression, indicating that sox2/3
are not upstream of six1. six1 overexpression in the LNE has no
significant effect on sox2/3 neural plate expression, indicating that
its effects in this border domain are cell autonomous and not due to
intermediate signaling. However, when six1 is reduced in the lateral
ectoderm, sox2/3 expression expands laterally. This could be a
secondary result of the expansion of foxD3, which in turn expands
sox2/3 (Sasai et al.,
2001
) (Fig. 5)
and/or a mutual antagonism between six1 and sox2/3. The
latter possibility is supported by the observations that six1
expression in the neural plate dramatically represses sox2/3 (data
not shown) and that expression of sox2 in the LNE represses
six1. At later stages sox2/3 are expressed in placodal
domains that presumably overlap with six1 expression. How these genes
interact at this later phase of placode development remains to be
determined.
foxD3 is required for neural crest formation, and both explant and
in vivo studies show that it induces neural crest and neural plate marker
genes (Sasai et al., 2001). We
show that foxD3 and six1 have a mutually antagonistic
relationship; the over-expression of one gene causes the repression of the
other. Conversely, the reduction of Six1 causes the foxD3 domain to
expand, and the reduction of foxD3 by six1 overexpression
causes expansion of other placodal markers. It is not yet known whether the
interactions between foxD3 and six1 are direct or indirect;
because six1 can repress both foxD3 and sox2
expression domains and foxD3 can expand sox2/3 domains
(Sasai et al., 2001
) (data
herein), the interaction could be via sox2/3 regulation.
The zic1, zic2 and zic3 genes, which are likely to be
functionally redundant, are first expressed throughout the entire presumptive
neural epithelium and then become restricted to the lateral border of the
neural plate and neural crest (Nakata et
al., 1997; Nakata et al.,
1998
; Brewster et al.,
1998
; Kuo et al.,
1998
; Mizuseki et al.,
1998
; LaBonne and
Bronner-Fraser, 1999
). The Zic genes appear to be important for
the initial phase of both neural plate and neural crest development, and all
three can induce ectopic expression of neural crest markers. In explants,
zic1 induces foxD3 and slug, and foxD3
induces zic1 and zic2, leading to the proposal that Zic
genes act upstream of foxD3 and slug to initiate neural
crest fate, and that Zic gene and foxD3 expression is maintained in
the neural crest by mutual interactions
(Sasai et al., 2001
). We
corroborate a mutual positive interaction between zic2 and
foxD3 by in vivo expression assays
(Fig. 6). However, in the LNE
foxD3 and the Zic genes do not have identical expression patterns
(Sasai et al., 2001
) (herein),
indicating that they also may be interacting through intermediary genes. We
demonstrate a similarly complex interaction between six1 and
zic2. Overexpression of six1 expands the zic2
lateral domain, but reduction of Six1 also expands it. We propose that the
former phenotype is caused by activation/maintenance of zic2 by
six1, whereas the latter phenotype is most probably caused by the
expansion of foxD3, which subsequently expands the zic2
domain.
Members of the Dlx gene family represent some of the earliest genes
expressed at the border between the neural plate and epidermis
(Feledy et al., 1999;
Luo et al., 2001a
;
Luo et al., 2001b
;
Beanan and Sargent, 2000
). In
chick, dlx5 is expressed at the neural/non-neural border, overlapping
with eya2 and six4 in the pre-placodal thickening where it
is proposed to create a border zone in which lateral neurogenic fates can be
expressed (McLarren et al.,
2003
). In Xenopus there is a low level of dlx5/6
expression along the border of the neural plate, but the most intense stripe
is adjacent to six1 expression along the anterior neural ridge, and
overlapping with the lateral edge of the crescent of six1 PPE
expression (Luo et al., 2001a
;
Luo et al., 2001b
) (herein).
In chick, dlx5 overexpression results in a weak upregulation of
six4 expression (McLarren et al.,
2003
), whereas in Xenopus wild-type
dlx5/dlx6 both strongly reduce six1 expression
(this study), and activator Dlx constructs cause a loss of six1
expression (Woda et al.,
2003
). It is not clear whether these differences are due to
species differences in the precise patterning of the embryonic ectoderm, as
has been proposed for neural induction
(Aybar and Mayor, 2002
), or due
to the fact that Six1 and Six4 belong to different subclasses of the Six gene
family (Kawakami et al.,
2000
).
Regardless, it is clear that in frog six1 has two effects on
dlx5/6 expression. Most prominently, overexpression of six1
in the LNE pushes the dlx5/6 stripe laterally away from the neural
plate midline. This phenotype is probably due to six1 causing an
expansion of the PPE (eya1, sox11) and reduction of epidermis
(keratin), resulting in the formation of a new border between the
expanded LNE and the epidermis. This interpretation is consistent with the
effects of six1 overexpression on keratin, and further
suggests that the effect is not due to movement of the neural plate border
because the sox2/3 domains do not change. Likewise, dlx5/6
negatively regulate six1 expression. A previous study also
demonstrated a mutual regulation between Dlx genes and six1
(Woda et al., 2003):
inhibition of endogenous Dlx activity relocated the six1 expression
domain more laterally, whereas activation relocated it more medially. The
second effect of six1 is complete repression of dlx5/6
expression in those cells expressing six1. This may result from Six1
either repressing dlx5/6 gene expression or causing changes in gene
expression in the affected cells that secondarily create an environment that
is not compatible with dlx5/6 expression. Interestingly,
foxD3 overexpression has similar effects on dlx5/6, which
could be direct, or, unlike six1, could be due to the expansion of
sox2/3. We further observed paradoxically similar dlx5/6
phenotypes by activator and repressor six1 construct expression and
six1-MO injections. We predict that these can be explained by
six1 effects on foxD3. The injection of six1VP16,
six1-WT+eya-WT and six1-MO may indirectly reduce
dlx5/6 by expansion of foxD3, whereas six1-WT alone
and six1EnR constructs may directly reduce dlx5/6. These
results support the proposal that dlx5/6 contribute to forming the
LNE border zone (McLarren et al.,
2003
; Woda et al.,
2003
), and additionally demonstrate that they do so by
participating in a complex interplay with several genes expressed in adjacent
domains. It will be important to determine the precise molecular interactions
between these various gene pathways to fully understand their roles in
specifying LNE fates.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Department of Biology, Hobart and William Smith Colleges,
Geneva, NY 14456, USA
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REFERENCES |
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