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First published online March 6, 2009
doi: 10.1242/10.1242/dev.032300
National Institute for Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
Author for correspondence (e-mail:
langerer{at}mail.nih.gov)
Accepted 29 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Microarray, Nodal, BMP2/4, Primary axis, Secondary axis, Canonical Wnt signaling, β-catenin, Gene expression profiling, Neural development, Transcription factor
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Levine and
Brivanlou, 2007; Wilson and
Houart, 2004). This early ectoderm is inherently biased toward
neural fate, as suggested by the observations that single cells derived from
Xenopus animal caps (Grunz and
Tacke, 1989; Sato and Sargent,
1989) or mouse embryonic stem (ES) cells, cultured in serum-free
media (Smith et al., 2008;
Smukler et al., 2006;
Watanabe et al., 2005) express
neural rather than epidermal markers and will maintain this state in the
absence of signals. This early neural-biased region is subsequently shaped
into a definitive neurogenic domain by several processes. One involves the
localized activities of the TGF-β cytokines, Nodal
(Camus et al., 2006) and BMP,
which promote epidermal fates, and of their antagonists, which inhibit these
fates. Additional signaling pathways, such as FGF and Wnt, regulate BMP
signaling activities by regulating the stability of the downstream effector,
Smad1/5/8 [(Fuentealba et al.,
2007) and references cited therein], but may have additional roles
in regulating neural versus non-neural ectodermal decisions. Another process
is the activation of the cell-autonomous neural gene regulatory program that
converts cells from an early neural bias to a pre-neural gene regulatory
state. These steps include not only the production of new neural regulatory
proteins but also the activation of mechanisms that exclude signaling and
allow the autonomous gene regulatory program to proceed.
The transitional state of pre-neural ectoderm is not well understood, but,
based on molecular marker expression, it appears to be forebrain-like
(Ang et al., 1994;
Ang and Rossant, 1993;
Yang and Klingensmith, 2006)
(reviewed by Foley et al.,
2000). In embryos lacking BMP and Nodal receptors, nearly all of
the ectoderm becomes anterior neural tissue (reviewed by
Levine and Brivanlou, 2007),
which expresses both general neural markers and those normally restricted to
forebrain, such as Six3, Dlx5 and Hesx1
(Camus et al., 2006).
Similarly, mouse ES cells cultured in serum-free conditions in the presence of
BMP and Nodal antagonists preferentially express several forebrain markers
(Ikeda et al., 2005). However,
there are relatively few known regulatory proteins, and their regulatory
connections are not yet understood.
The sea urchin embryo, which shares a common ancestor with the vertebrates,
is an excellent system to identify regulatory activities constituting the gene
regulatory state of early pre-neural ectoderm. This region, localized at the
animal pole, is specified by the early mesenchyme blastula stage
(Burke et al., 2006) and gives
rise to the animal plate, a disk of 40-60 cells in the ciliated band of the
3-day pluteus larva that contains serotonergic neurons on its aboral side and
non-serotonergic neurons on all sides
(Nakajima et al., 2004a), as
well as cells bearing long, immotile cilia. Gene expression patterns suggest
that the animal pole domain (APD) also includes immediately flanking
epithelial ectoderm cells (Burke et al.,
2006; Howard-Ashby et al.,
2006a; Howard-Ashby et al.,
2006b) (see also Results).
Ectoderm patterning in the sea urchin embryo is regulated by a series of
signaling events, beginning with a wave of canonical Wnt signaling that
originates in the most vegetal blastomeres of the 16-32-cell embryo, passes
upwards through vegetal tiers of blastomeres and specifies endomesodermal
tissues (Davidson et al.,
2002; Logan et al.,
1999) (reviewed by Angerer and
Angerer, 2003). Canonical Wnt signaling is also required for
Nodal-dependent patterning along the secondary axis by removing a repressor of
nodal expression, FoxQ2, from the lateral ectoderm
(Yaguchi et al., 2008). Nodal
is required for production of BMP2/4
(Duboc et al., 2004), which
activates the gene regulatory network for aboral ectoderm differentiation
(Angerer et al., 2000), and
Chordin (Bradham et al., 2009),
which inhibits BMP2/4 function. These events occur during blastula stages and
direct the lateral ectoderm to oral and aboral epidermal fates, while the APD
persists, marked first by the expression of foxq2
(Tu et al., 2006;
Yaguchi et al., 2008) followed
by homeobrain (hbn), achaete-scute (ac-sc)
and retinal anterior homeobox (rx)
(Burke et al., 2006;
Howard-Ashby et al., 2006a;
Howard-Ashby et al., 2006b).
This region cannot be converted to squamous epidermal fates by misexpression
of Nodal or BMP2/4, unless FoxQ2 is removed
(Yaguchi et al., 2008). By
contrast, when Nodal-BMP2/4 and canonical Wnt signals are eliminated through
cadherin mRNA injection (Logan et
al., 1999; Wikramanayake et
al., 1998), then the animal plate expands, as shown by a greatly
increased number of serotoneric neurons distributed radially throughout a
large portion of the embryo (Yaguchi et
al., 2006).
The ability to greatly enlarge the early neurogenic ectoderm of the sea
urchin embryo experimentally offers a unique opportunity to identify
regulatory proteins that specify this region of the embryo. Many genes
expressed specifically in this territory are expected to be strongly
upregulated in cadherin-misexpressing embryos in comparison to normal
embryos, as has been shown for foxq2
(Yaguchi et al., 2008) and
nk2.1 (Takacs et al.,
2004). Here we use microarray analyses to identify such genes and
sort them according to their onset of expression
(Wei et al., 2006). In this
paper, we focus on six3 because it is one of the first of these genes
to be activated after fertilization and expressed in the APD at blastula
stage. We show that this factor is required for development of all neurons,
for antagonizing signals that inhibit neural differentiation and for the
expression of the large majority of regulatory genes expressed early in the
APD. Furthermore, misexpressed Six3 can convert nearly all cells of the embryo
to form an appropriately patterned APD. Consequently, six3 operates
at, or near the top of, the gene regulatory networks that control
specification of cell fates in the APD, and the Six3-dependent properties of
the APD suggest that this domain functions as a patterning center.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Microinjection of morpholino antisense oligonucleotides (MO) and mRNAs
Eggs were de-jellied by six passages through 74 µm Nitex and arrayed in
rows on a plastic culture dish coated with 1% protamine sulfate. After
insemination in ASW with 3-amino-1,2,4-triazole (Sigma, St Louis, MO),
approximately 2 pl of solution containing 22.5% glycerol and morpholinos
(Gene-Tools, Eugene, OR) or synthetic mRNAs at the following concentrations
were injected: cadherin mRNA (0.3 µg/µl), Six3 mRNA (0.2
µg/µl), Six3-MO1 (0.75 mM), Six3-MO2 (1.5 mM), FoxQ2-MO (0.8 mM),
Nodal-MO (0.6 mM). The morpholino sequences were: Six3-MO1,
5'-GGGCCGCTCTCATGGCGCCCCGGTC-3'; Six3-MO2,
5'-CCCCGGTCGCTGGGCGATGTTTCTG-3', 4 nt upstream of AUG; FoxQ2-MO,
5'-GGTTGTCAATGCTGAATAAAGTCAT-3'
(Yaguchi et al., 2008);
Nodal-MO: 5'-GATGTCTCAGCTCTCTGAAATGTAG-3', 56 nt upstream of AUG.
mRNAs were synthesized using mMESSAGE mMACHINE Kit (Ambion, Austin, TX),
according to the manufacturer's protocol, except that the RNA was precipitated
with three volumes of ethanol after addition of LiCl and glycogen.
Microarray methods
Microarrays and data processing were described previously
(Wei et al., 2006). RNA from
800-1000 embryos was purified using Nucleospin columns (Macherey-Nagel,
Bethelem, PA) and amplified with the MessageAmp aRNA Kit (Ambion, Austin, TX)
according to the manufacturer's instructions. The amplified antisense RNA
(aRNA) was further converted to double-stranded cDNA as follows: for
first-strand cDNA synthesis, 5 µg of aRNA was mixed with random primers
(1.25 µg), incubated at 70°C for 5 minutes, transferred to ice and then
reverse transcribed with Superscript III Reverse Transcriptase (Invitrogen,
Carlsbad, CA) in the presence of 1 mM dNTP and 1.3 U/µl RNase Out at
50°C for 2 hours. Template aRNA was removed with 5 units of RNaseH at
37°C for 30 minutes and the cDNA was purified with a QIAquick PCR
purification column and eluted with 30 µl water. The cDNA was mixed with
oligo dT (2 µM final concentration), incubated for 5 minutes at 70°C
and second-strand cDNA was synthesized with DNA polymerase I (E.
coli) (0.4 U/µl) in the presence of 1 mM dNTP at 16°C for 2 hours.
The double-stranded cDNAs were purified by QIAquick chromatography and
labeled, hybridized and scanned by Nimblegen microarray services.
Quantitative PCR
Quantitative PCR (QPCR) was performed as described previously
(Ransick, 2004) with some
modifications. Total RNA from 300 to 1000 injected embryos was purified using
NucleoSpin columns (Macherey-Nagel, Bethelem, PA) and reverse transcription
was performed using Surperscript III (Invitrogen, Carlsbad, CA). iQ SYBR Green
Supermix (Bio-Rad,
http://www.biorad.com)
was used for PCR reactions carried out with an iQ thermal cycler (Bio-Rad).
Details of the primer sets used for QPCR are available upon request. Relative
concentrations of mRNA were normalized to mitochondrial 12sRNA Ct
values.
Whole-mount in situ hybridization
Embryos were fixed in 4% paraformaldehyde in ASW for 1 hour at RT and
washed four times in MOPS buffer (0.1 M MOPS, pH 7.0, 0.5 M NaCl, 0.1%
Tween-20). Pre-hybridization, hybridization and staining were as described
previously (Minokawa et al.,
2004). Two-color fluorescent in situ hybridization was carried out
as described previously (Yaguchi et al.,
2008). The foxq2 probe was labeled with FITC and detected
with Cy3-TSA and the Hbn probe was labeled with digoxygenin and
detected with FITC-TSA.
Immunohistochemistry
Primary antibodies were incubated overnight at 4°C using the following
dilutions: serotonin (1:1000, Sigma, St Louis, MO), Nk2.1 (1:800), Gsc [1:600
(Kenny et al., 2003)], Spec1
[1:80 (Wikramanayake and Klein,
1997)], synaptotagmin [1e11, 1:800
(Nakajima et al., 2004b)].
Bound primary antibodies were detected by incubation with Alexa-coupled
secondary antibodies for 1 hour. The embryos were observed using a Zeiss
microscope (Axiovert 200M). Optical sections were obtained with an ApoTome
unit (Zeiss, Thornwood, NY) and stacked images were prepared using Adobe
Photoshop.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and from
others (Howard-Ashby et al.,
2006a; Howard-Ashby et al.,
2006b; Materna et al.,
2006; Tu et al.,
2006), which documented APD expression patterns of many genes
encoding transcription factors and/or that were orthologs of factors known to
function in neural tissue in embryos of other species. The experimental
approach compared representation of RNAs in cadherin mRNA-injected
versus normal embryos at the hatching blastula stage. Embryos lacking nuclear
β-catenin consist almost exclusively of animal pole ectoderm, as shown by
the expression patterns of foxq2 and hbn. In normal embryos,
these mRNAs are restricted to the animal pole beginning at blastula stages
(Burke et al., 2006;
Tu et al., 2006;
Yaguchi et al., 2008) and the
domains of their expression at mesenchyme blastula stage overlap at the animal
pole (Fig. 1B-D). As
development proceeds through gastrulation, the domain of
hbn-expressing cells forms a ring that surrounds cells expressing
foxq2 (Fig. 1F). When
canonical Wnt, Nodal and BMP signaling are all eliminated by cadherin
mRNA injection, the animal half of the embryo expresses foxq2 while
most of the remainder of the embryo expresses hbn
(Fig. 1H). These patterns
indicate that embryos lacking canonical Wnt and Nodal-BMP2/4 signaling consist
almost entirely of a dramatically expanded APD, the outer borders of which are
defined by hbn expression. Foxq2 and hbn
transcripts appear in the APD during late cleavage/early blastula stages,
indicating that specification of the APD occurs at least by this time.
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cadherin mRNA- versus
glycerol-injected embryos at the hatching blastula stage using a microarray
representing all gene predictions found in the sea urchin genome sequence
(Wei et al., 2006). This stage
marks the transition between cleavage and the onset of morphogenesis and is
shortly after the time when the earliest markers for the APD have been
detected (Burke et al., 2006;
Howard-Ashby et al., 2006a;
Howard-Ashby et al., 2006b;
Tu et al., 2006;
Yaguchi et al., 2008). A set
of genes encoding transcription factors and components of signaling pathways
with an average of at least 3-fold higher expression in two batches of
cadherin mRNA-injected embryos was identified. These partially
overlapped with the set of candidate genes identified by the bioinformatics
approach. The combined sets contain 27 genes and constitute a provisional
early APD regulatory gene set (E-APD)
(Table 1).
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).
Patterns were verified by comparison with published data
(Burke et al., 2006;
Croce et al., 2006;
Howard-Ashby et al., 2006a;
Howard-Ashby et al., 2006b;
Materna et al., 2006;
Sweet et al., 2002;
Takacs et al., 2004;
Tu et al., 2006;
Yaguchi et al., 2008) and by
our whole-mount in situ hybridizations (our unpublished observations). The
genes were sorted into three groups: those represented maximally in the
maternal RNA population (Fig.
2A), those strongly upregulated during cleavage stages
(Fig. 2B,C) and those
upregulated between late cleavage and early gastrula stages
(Fig. 2D). We first focused on
members of the early embryonic expression group. As shown below, using both
loss- and gain-of-function assays, we identified one, Sp-Six3
(hereafter Six3), which operates at or near the top of the APD gene
regulatory hierarchy.
Six3 is expressed early in the APD
The identification of sea urchin six3 is unambiguous because its
sequence is very highly conserved in the homeodomain (98% identical to
HsSix3), the six domain (91%) and the groucho interaction domain (71%) (see
Fig. S1 in the supplementary material). We confirmed previous studies showing
that Six3 is expressed in a dynamic pattern during development of
Paracentrotus lividus (Poustka et
al., 2007), a species closely related to Strongylocentrotus
purpuratus used in this study. The most important features are that
Six3 transcripts are expressed in the animal hemisphere during late
cleavage (Fig. 3A), in the APD
by the hatched blastula stage (Fig.
3B) and then in two rings (Fig.
3G,H), one near the periphery of the APD
(Fig. 3C-F,H) and the other in
the endomesoderm (Fig. 3C-G),
during mesenchyme blastula stages. During gastrulation, Six3 is
expressed in some secondary mesenchyme cells scattered throughout the
blastocoel and at the tip of the archenteron
(Fig. 3I, arrow), as reported
by Howard-Ashby et al. (Howard-Ashby et
al., 2006b), in cells in the animal pole
(Fig. 3I,J) and oral ectoderm
(Fig. 3J,K). At pluteus stage,
Six3 RNA is detected in two clusters of cells flanking the mouth
(Fig. 3K, arrows) and in the
ciliated band (Fig. 3K).
Six mRNA overall levels at early mesenchyme blastula stage do not
significantly change upon cadherin mRNA injection
(Table 1). In situ
hybridizations are consistent with this result and show that the distribution
differs in cadherin mRNA-injected embryos, being absent from vegetal
cells and retaining the broad animal hemisphere expression that is established
before restriction by processes dependent on canonical Wnt (see Fig. S2 in the
supplementary material).
Six3 function is required for APD formation and differentiation of neurons
To test the function of Six3, we injected into fertilized eggs two
different Six3-translation-blocking morpholinos, each of which elicited the
same developmental defects. Embryos at 3 days assumed a rounded morphology
(Fig. 4A,B) and spicules were
either reduced or absent. The animal pole ectoderm lacked the thickened
epithelial morphology characteristic of the animal plate
(Fig. 4, compare A,B with C,
brackets). In some embryo batches, gastrulation occurred normally, although it
was delayed, and the position of the archenteron showed that oral/aboral
polarity was established (Fig.
4A). In other batches, most of the embryos exogastrulated. In
embryos lacking Six3, neural differentiation was strongly inhibited, as
assayed by immunostaining for neural markers normally expressed during late
gastrula and pluteus stages (Fig.
4, compare A,B with C). The majority (2/3) of embryos contained no
serotonergic neurons and the remainder had a reduced number
(Fig. 4D) compared with the
normal number at this stage (3-5). The same was true for all other neurons,
assayed by the pan-neural marker synaptotagmin (1e11)
(Fig. 4A,B), which are found
within the APD and ciliated band of normal 3-day embryos
(Fig. 4C). Significantly, the
expression of hbn was reduced to a level undetectable by in situ
hybridization (Fig. 4F versus
Fig. 4E). QPCR measurements
confirmed this observation and showed that the levels of both hbn and
foxq2 mRNAs, which mark the outer boundaries and inner portions of
the APD, respectively, were reduced 8- to 10-fold in Six3 morphants at the
mesenchyme blastula stage (Fig.
5).
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cadherin-injected embryos. As shown in
Fig. 5A, loss of Six3 in these
embryos completely eliminated development of all of the excess neurons found
in cadherin-injected embryos and no thickened epithelium formed, again
demonstrating an important role for Six3 in APD development. The microarray
data identified a surprisingly large number of gene predictions (682) that
were strongly downregulated (at least 4-fold) in embryos doubly injected with
cadherin mRNA and Six3-MO, compared with embryos injected with
cadherin mRNA alone. Furthermore, more than 60% of all genes in the
previous microarray screen that were upregulated at least 3-fold in
cadherin mRNA-injected embryos, and therefore likely to be expressed in
the APD, were significantly downregulated by loss of Six3. Importantly,
microarray data indicated that the majority of the E-APD genes were sensitive
to Six3 (Fig. 5B, yellow). In
good agreement with this, QPCR measurements in two other batches of embryos
showed that only 5 E-APD genes did not depend significantly on Six3
(Fig. 5B, red, blue).
Six3 overexpression is sufficient to expand the APD
These results strongly support the idea that Six3 functions early in APD
gene regulatory networks, and raise the possibility that it might be
sufficient to induce other cells in the embryo to adopt APD fates.
Misexpression of Six3 resulted in embryos displaying an extraordinary change
in morphology. A horseshoe-shaped band of densely packed cells extended in the
vegetal direction from the animal pole
(Fig. 6, compare B,C with A).
Serotonergic neurons, normally restricted to the animal plate
(Fig. 6D), increased 4-fold in
number and were distributed along the dense band
(Fig. 6G). Furthermore, the
columnar shapes and arrangement of neural projections in
synaptotagmin-containing cells (1e11) are similar to those in the animal plate
of control embryos (Fig. 6E,
white dashed box versus Fig.
6H). All of these cells contain in their nuclei NK2.1
(Fig. 6J-M, green), a
transcription factor normally expressed in the animal plate and adjacent
supra-oral ectoderm (Fig.
6I,I'; green circles in
Fig. 6U), as well as a few
cells in the foregut (Takacs et al.,
2004). A chain of 1e11-positive neural cells bisects the band
(Fig. 6K, red) and cells on the
inner side of the NK2.1-positive band also express Gsc
(Fig. 6L,N, red). The
combination of NK2.1 and Gsc uniquely marks the supra-oral facial epithelium
at the oral edge of the animal plate (Fig.
6I,I', yellow cells and
Fig. 6U, yellow circles). The
densely packed columnar cells of the expanded band surround more flattened
epithelial cells that express NK2.1, but not Gsc
(Fig. 6M,N), as do cells of the
upper regions of the mouth of normal embryos
(Fig. 6I, mouth, m). Further
evidence that these cells are similar to those near the mouth of normal
embryos is that they express hbn mRNA, which, in normal 3-day
embryos, accumulates around the margin of the animal plate and extends into
the supra-oral ectoderm (Fig.
1, Fig. 6O). In
Six3-misexpressing embryos, hbn-positive cells are concentrated in
the thin epithelium at the vegetal pole and thus located in the same position
relative to the expanded animal plate and the Nk2.1-positive cells in the
upper foregut as in normal embryos (Fig.
6P,Q). The side opposite the oral region differentiates as a thin
squamous epithelium, expresses the aboral ectoderm marker, Spec1
(Fig. 6R-T) and may correspond
to the hbn-positive strip of aboral ectoderm adjacent to the animal
plate. Collectively, these gene expression patterns lead to the remarkable
conclusion that Six3 is sufficient to re-specify the fates of cells in most of
the rest of the embryo, generating a 3-day embryo consisting of a greatly
expanded, but correctly patterned, animal pole domain with oral/aboral
polarity. The APD in normal and Six3-misexpressing embryos is marked by the
blue circles in Fig. 6U.
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).
Surprisingly, Six3 does not suppress bmp2/4 mRNA accumulation as much
as it reduces nodal, although bmp2/4 expression requires
nodal function in normal embryos
(Duboc et al., 2004). This
apparent paradox may result from a combination of reduced Chordin-mediated
inhibition of BMP2/4 signaling coupled with diffusion of BMP2/4 and subsequent
autoactivation, as has been demonstrated in other systems
(Biehs et al., 1996;
Jones et al., 1992).
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)
(Fig. 1). Because Nodal and BMP
depend on canonical Wnt signals, we eliminated both TGF-β ligands with a
Nodal-MO (Yaguchi et al.,
2008) to test whether they were responsible for the Wnt-dependent
restriction of the APD. Fig.
7B,F shows that this is not the case because serotonergic neurons
remain restricted to the APD in these embryos. However, when Nodal morphants
are provided with exogenous Six3, then serotonergic neurons increase greatly
in number and appear throughout the animal half of the embryo
(Fig. 7D), as is observed in
cadherin-misexpressing embryos
(Yaguchi et al., 2006), which
lack canonical Wnt signaling. Therefore, ectopic expression of Six3 can
override the other signals, presumably Wnt, that restrict these neurons to the
APD of normal embryos. Although serotonergic neurons do not form in the lateral ectoderm in Nodal morphants, some non-serotonergic neurons do (Fig. 7B). This results primarily, if not exclusively, from loss of BMP2/4 signaling, as the same result is obtained in BMP2/4-MO-injected embryos (S.Y., J.Y., L.M.A., R.C.A and R. D. Burke, unpublished). Because development of all neurons depends on Six3 in the normal embryo, we asked whether the ectopic neurons in Nodal morphants also depend on Six3 by co-injecting Nodal-MO and Six3-MO. As expected, the serotonergic neurons present at the animal pole in Nodal morphants were lost in the double morphants (Fig. 7G,H). These embryos do not contain differentiated non-serotonergic neurons with axonal processes, although some 1e11-immunoreactive spots were observed. These might indicate the presence of incompletely differentiated neurons or reflect an initial neural bias of ectoderm cells that is normally overridden by TGF-β signaling.
Six3 can antagonize Wnt signaling
The facts that the APD does not expand in Nodal morphants but does in
cadherin-injected embryos and that Six3 can overcome canonical
Wnt-dependent effects in the lateral ectoderm raise the possibility that Six3
represses Wnt signaling. In support of this hypothesis, we find that Six3
misexpression downregulates most of the genes encoding Wnt ligands that are
expressed during early development (Table
3, left) (Fig. 8).
One of these is Wnt8, a crucial vegetal signal required for normal
endomesoderm development (Wikramanayake et
al., 2004), a result that is consistent with the lack of vegetal
development in Six3-misexpressing embryos. Collectively, these results suggest
that the borders of the APD are determined by Six3/Wnt antagonism.
Six3 is not sufficient to repress expression of wnt and nodal in the APD
The ability of Six3 to strongly downregulate (directly or indirectly) genes
encoding Wnt ligands, as well as nodal, lefty and chordin,
raises the possibility that it normally prevents expression of these genes in
the APD. To evaluate this, we first examined the effects of Six3 on gene
expression in cadherin-injected embryos in order to eliminate possible
counteracting effects of Six3 function in the endomesoderm. Both microarray
and QPCR data show that, in embryos consisting mostly of the APD, Six3
suppresses expression of the Wnt genes, and nodal, lefty and
chordin (Table 3;
Fig. 8). However, in normal
embryos (glycerol-injected), Six3 repression of these genes cannot be detected
(Table 3;
Fig. 8), indicating that
additional mechanisms protect the APD from Wnt and TGF-β expression in
the normal embryo.
Six3 regulation of other signaling pathways
Six3 also positively regulates (either directly or indirectly) genes
encoding proteins that function in other signaling pathways
(Table 3). These include
delta, the Notch ligand that mediates lateral inhibition, a crucial
process in neural development, as well as other potential regulators of
neurogensis. The latter include fgf9/16/20, fgfr-like, a
membrane-bound receptor lacking the tyrosine kinase domain, and frizzled
5/8, a Wnt receptor that may transduce non-canonical signals, but whose
function in the APD is unknown (Croce et
al., 2006). Future studies will examine how the activities of
these signaling pathways and early Six3-dependent transcription factors
interact in the APD gene regulatory network.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The known functions of sea urchin Six3-dependent genes or their orthologs
in other embryos are consistent with its requirement for specification of the
multiple cell types that differentiate in animal pole ectoderm. For example,
NK2.1 and its regulator, FoxQ2 (Yaguchi et
al., 2008), are necessary in cells that produce the long, immotile
apical tuft cilia (Dunn et al.,
2007). FoxQ2 also supports neural differentiation
(Yaguchi et al., 2008) and
several other Six3-dependent genes are subject to lateral inhibition through
Notch signaling (our unpublished data), which is a cardinal feature of
neurogenic genes (reviewed by Lewis,
1996). Sp-Rx is likely to function specifically in
serotonergic neurons because it is exclusively co-expressed with serotonin
during the early stages of differentiation (see Fig. S3 in the supplementary
material). Orthologs of Sp-Zic2 and Sp-Ac-Sc function in
neural development in other embryos
(Andreazzoli et al., 2003;
Grinberg and Millen, 2005;
Kageyama et al., 1995) and
hbn is expressed in the anterior ectoderm of Drosophila
(Walldorf et al., 2000) and
polychaete annelids (Frobius and Seaver,
2006). Thus, Six3 is necessary for the differentiation of the
non-neural cells producing long specialized cilia, serotonergic and
non-serotonergic neurons and cells in the flanking ectoderm that express
hbn. The large number of Six3-dependent regulatory genes reinforces
the conclusion that Six3 functions at or near the top of the APD gene
regulatory networks that specify animal ectoderm cell types.
The establishment of the APD depends on Six3, whereas specification and
patterning of all other regions of the embryo ultimately depends on canonical
Wnt signaling, initially at the opposite pole in the most vegetal blastomeres.
Our results show that canonical Wnt-dependent suppression of APD fates in the
lateral ectoderm does not work through TGF-β signaling because the animal
plate does not expand when Nodal and BMP2/4 are eliminated but Wnt signaling
is maintained. However, the fact that it does expand in a TGF-β-deficient
environment when Six3 is misexpressed (Fig.
7D) suggests that Six3 can antagonize Wnt-dependent processes. It
can strongly repress genes encoding Wnt ligands, as is observed in both
Six3-misexpressing and cadherin-injected embryos, and it is required
for APD-specific expression of sFRP1/5
(Fig. 5; our unpublished
observations), which may restrict Wnt signaling from the APD if it behaves as
in the mouse embryonic forebrain (Houart
et al., 2002; Lagutin et al.,
2003). These observations suggest that the border between the APD
and lateral ectoderm depends, at least in part, on interactions between Six3
and canonical Wnt-dependent processes.
Our data indicate that a primary role of TGF-β signals is to promote the differentiation of epidermal oral and aboral epithelia in the lateral ectoderm by suppressing the innate neural bias of early ectoderm. If Nodal/BMP signaling is knocked down, then neural cells can begin to differentiate in the lateral ectoderm. When these signals are present, then lateral ectoderm cells become epidermal except at the oral/aboral border, where the ciliated band forms and where non-serotonergic neurons develop. Where and when these ciliated band neurons are specified is not clear, but they do require the function of Six3. An interesting question for future experiments will be to determine whether they are originally specified throughout the lateral ectoderm but continue to differentiate only in the ciliated band, a site of late six3 expression, or whether they are originally specified in the APD and then migrate to this site.
The Six3 misexpression phenotype is striking. Expression of marker genes indicates that Six3 is sufficient to convert nearly the entire embryo into animal pole ectoderm that is specified by the APD gene regulatory state during blastula stages and is molecularly and morphologically patterned into the cell types found in the 3-day pluteus larva. Furthermore, expression of most, if not all, of these genes requires Six3 in the normal embryo. These observations support the view that Six3 is not only upstream in neural GRNs, but also in those that specify other tissues in animal pole ectoderm. Collectively our findings strongly support a model in which the APD is a third patterning center, along with those operating in vegetal and oral blastomeres. Although the APD regulome controls the development of a discrete region of the embryo, its activity combines with that of the Nodal-dependent oral patterning center to specify the supra-oral ectoderm defined by the combination of gsc (Nodal-dependent) and nk2.1 (Six3-dependent) expression. Similarly, we anticipate the existence of Six3/BMP combinatorial control in the aboral portion of the APD.
|
|
; Yaguchi et al.,
2008). We propose that as Nodal/BMP signaling increases during
blastula stages, Six3 and FoxQ2 concentrations continue to rise in APD cells,
creating a TGF-β- and canonical Wnt-free zone necessary for APD
patterning and neurogenesis. Here we focused on the role of Six3 in the APD, but it is also expressed in endomesoderm cells starting at mesenchyme blastula stage. Its role in this context is not known, but undoubtedly is controlled by the vegetal regulatory state.
The anterior neuroectoderm of vertebrate embryos and the APD of sea urchin
embryos contain the initial neurogenic domains and the development of each
requires Six3. Consequently, these embryos may share significant portions of
the same gene regulatory network. In support of this, a significant number of
the sea urchin Six3-dependent early-APD regulatory genes have orthologs
expressed in either the forebrain or eye field, or both. These include
zic2, rx, achaete-scute, nkx2.1, fez, dkk3 and sFRP1/5
(Andreazzoli et al., 2003;
Diep et al., 2004;
Elms et al., 2004;
Ferreiro et al., 1993;
Guillemot and Joyner, 1993;
Houart et al., 2002;
Jeong et al., 2007;
van den Akker et al., 2008).
It is likely that Six3-dependent regulatory proteins function in the core of
an ancient GRN specifying the forebrain neural identity, which suggests that
other newly identified sea urchin APD regulatory genes may also function in
vertebrate early forebrain development. Consequently, the relatively advanced
state of GRN analysis in the sea urchin embryo
(Oliveri et al., 2008) and the
discovery of a large set of Six3-dependent APD genes offers a potential
blueprint for elucidating the control circuits that guide specification of
neurogenic domains in other embryos and will provide a foundation for
understanding how they evolved.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1179/DC1
|
Footnotes |
|---|
* Present address: Shimoda Marine Research Center, University of Tsukuba,
Shimoda, Shizuoka 415-0025, Japan 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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