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First published online 10 May 2006
doi: 10.1242/dev.02396
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Departments of Biology and Biochemistry/Microbiology, University of Victoria, POB 3020, STN CSC, Victoria, BC, V8W 3N5, Canada.
* Author for correspondence (e-mail: rburke{at}uvic.ca)
Accepted 6 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural development, Cell fate specification, Sea urchin, Animal plate, Ectoderm, Cellular signaling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Animal
plate ectoderm has limited potential, as the animal halves of eggs, or animal
quartets of blastomeres, differentiate into permanent blastulae containing
only ectoderm (Angerer and Angerer,
2003). Conversely, vegetal halves of eggs or embryos undergo
extensive regulation and nearly normal embryos can result
(Horstadius, 1973;
Maruyama et al., 1985).
However, the animal plate is a robust region that resists conversion to other
cell types. With only a few exceptions, expression of transcription factors or
signaling molecules alters cell fate in all cells except a small thickened
region at the animal pole (Wikramanayake
and Klein, 1995; Angerer et
al., 2000; Angerer et al.,
2001, Amore et al.,
2003; Duboc et al.,
2004; Bradham and McClay,
2006). In the absence of vegetal signaling, a permanent blastula
forms in which animal plate ectoderm expands, forming a hemisphere or more of
the embryo. Many authors consider the expanded ectoderm to be undifferentiated
or partially differentiated (Angerer and
Angerer, 2003). Some ectodermal markers are expressed, but the
thickened ectoderm does not express crucial indicators of either oral or
aboral ectoderm (Logan, 1999;
Wikramanayake and Klein, 1995;
Wikramanayake et al., 1998;
Takacs et al., 2004). The
region is thought to be determined by a set of maternally derived
transcription factors that endow the region with what is termed an animalizing
influence (Angerer et al.,
1998; Angerer and Angerer,
2000; Angerer and Angerer,
2003; Ettensohn and Sweet,
2000). The opposing, vegetalizing influence is known to be a
series of short-range signaling events initiated by activation of canonical
Wnt signaling in micromeres (Davidson et
al., 2002a; Davidson et al.,
2002b; Ettensohn and Sweet,
2000; Angerer and Angerer,
2003). The vegetal pole initiates progressive specification of
tiers of blastomeres to mesodermal, endodermal and ectodermal fates. However,
there is no satisfactory molecular explanation for the animalizing influence
centered at the opposite pole.
The larval nervous system is an array of neurons that control swimming and
feeding. There are several clusters of neurons with associated neuropil that
are thought to function as ganglia. The best know of these is the apical
organ. In the early larva it is an elliptical patch containing three types of
cells: four to six bilaterally positioned serotonergic neurons, a central
cluster of 10-12 non-serotonergic neurons and several non-neural support cells
(Nakajima, 1986;
Burke, 1983;
Nakajima et al., 1993;
Nakajima et al., 2004). The
neurons of the apical organ differentiate late in gastrulation and have been
revealed with antibodies to serotonin, probes for tryptophan hydroxylase and
antibodies to synaptotagmin (Bisgrove and
Burke, 1986; Bisgrove and
Burke, 1987; Nakajima et al.,
1993; Yaguchi and Katow,
2003; Nakajima et al.,
2004). Although we lack detailed lineage studies, topology of
sequential stages indicates that the apical organ is derived from the animal
plate (Burke, 1983;
Nakajima et al., 1993). In
gastrulae this region expresses the transcription factor SpNK2.1 and the
serotonergic cells form at the interface of the SpNK2.1 domain and the aboral
ectoderm (Tackacs et al., 2004; Nakajima
et al., 2004). Thus, the apical organ arises as a precisely
patterned neural structure from a defined ectodermal domain and provides an
opportunity to examine neural specification in sea urchin embryos.
We report here that the animal plate ectoderm that forms in permanent blastulae when vegetal signaling is blocked differentiates as neural tissue forming an expanded and unpatterned apical organ. Using expression of components of the pathways that specify oral, aboral and ciliary band ectoderm in embryos and chimeras in which vegetal signaling is blocked, we have investigated the signaling pathways that restrict the expansion of the animal plate and pattern neurogenesis. We propose that maternal determinants specify the animal plate as neurogenic and that it is restricted in size and patterned by specification of ectoderm.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microinjection of RNAs
De-jellied eggs were arrayed in rows on a plastic culture dish coated with
1% protamine sulfate. RNA was synthesized with mMessage mMachine kit (Ambion),
quantified by gel-electrophoresis, diluted in 22.5% glycerol, filtered and
loaded into injection needles. We used the following plasmids:
-cadherin (Logan et al.,
1999); stabilized ß-catenin (XBC69)
(Yost et al., 1996); normal
and kinase dead-Gsk3ß SU-G31, SU-G32
(Emily-Fenouil et al., 1998);
Antivin, Nodal (Duboc et al.,
2004); BMP (Angerer et al.,
2000); Noggin (Lamb et al.,
1993; Angerer et al.,
2000); and Gsc (Angerer et al.,
2001). Concentrations of RNA use in the injection needles were:
-cadherin (0.5 µg/µl), XBC69 stabilized ß-catenin (100
ng/µl), Gsk3ß (2.0 µg/µl), dominant-negative Gsk3ß (1.0
µg/µl), Antivin (40 ng/µl), BMP (2.0 µg/µl), Nodal (200
ng/µl), Noggin (1.0 µg/µl), Gsc (100 ng/µl) and about 1% of the
egg volume was injected. In all instances, previously reported phenotypes were
obtained. In some experiments, eggs were injected with -cadherin, and
after cleavage, one blastomere was re-injected with a second RNA mixed with
RNA encoding six iterations of the 9E10 myc epitope
(Evan et al., 1985). To inject
one blastomere of an eight-cell embryo, eggs were inseminated in filtered
seawater (FSW) containing 1 mM 3-amino1,2,4-triazol (ATA) and rinsed with FSW
after 30 minutes. Eight-cell embryos were put in
Ca2+Mg2+-free artificial seawater (CMFASW, 10 minutes),
injected and cultured in FSW.
Immunohistochemistry
Embryos were fixed with 4% paraformaldehyde in FSW for 10 minutes and
rinsed with phosphate-buffered saline with Tween [PBST; 0.8 mM
Na2HPO4-12H2O, 0.15 mM
KH2PO4, 14 mM NaCl, 0.27 mM KCl, 0.1% Tween-20 (pH 7.0)]
three times, blocked with 5.0% lamb serum in PBST (1 hour) and incubated with
rabbit anti-serotonin antibody (Sigma) or rabbit anti-SpNk2.1 antibody
(Takacs et al., 2004), and
mouse anti-synaptotagmin (1E11) (Nakajima
et al., 2004) or mouse anti-myc antibody (9E10)
(Evan et al., 1985) overnight
(4°C). After rinsing, specimens were incubated in secondary antibodies
conjugated with Alexa 488 or Alexa 568 (Molecular Probes) in PBST (2 hours).
Specimens were examined with a confocal laser-scanning microscope (Zeiss) or a
epifluorescence microscope (Zeiss or Leica). Serotonergic cells were counted
in through focus confocal stacks. A one-way analysis of variance (ANOVA) was
used with Tukey-Kramer or Dunnett multiple comparison post tests (Instat 3.0)
to analyze data.
Exogastrulated embryos
To make mechanically derived exogastrulae, hatched blastulae (33 hours)
were trapped under a coverslip with clay spacers and pressed until the
blastocoel was no longer visible. Embryos were left under the coverslip until
65 hours when exogastrulae had formed.
Whole-mount in situ hybridization
Embryos were prepared following the method of Yaguchi and Katow
(Yaguchi and Katow, 2003).
Digoxigenin (DIG: Roche)-labeled SpHnf6 antisense RNA probes were synthesized
from linearized plasmids. After proteinase K treatment (2 ng/µl) and
post-fix with 4% paraformaldehyde, overnight hybridization (47.5°C) in 50%
formamide, 5xDenhardt solution, 0.1 M MOPS (pH 7.0), 0.5 M NaCl, 1 mg/ml
bovine serum albumin (BSA), 0.1% Tween-20 with 0.1 ng/µl probes.
For combined immunolocalization and in situ hybridization, specimens prepared for in situ localization were rinsed in PBST, blocked with 5.0% lamb serum in PBST (1 hour), and incubated in anti-SpNk2.1 overnight at 4°C.
Chimeric embryos
Eggs co-injected with -cadherin and myc RNA and normal eggs were
inseminated in ATA-FSW, transferred to 1% agarose-coated dishes, and incubated
to 16 cells. Embryos were transferred to CMFASW (10 minutes). With fine glass
needles, the mesomere tier was removed from an RNA-injected embryo and grafted
to macromere and micromere halves of a normal embryo. Chimeras were cultured
in small indentations in an agarose-coated dish.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cadherin RNA prevents nuclear localization
of ß-catenin and interferes with activation of the endomesodermal gene
regulatory network (Logan et al.,
1999; Davidson et al.,
2002b). Embryos injected with -cadherin RNA form permanent
blastulae with a thickened epithelium in the animal hemisphere and a thin
epithelium at the vegetal pole (Fig.
1A). Preparations of 72-hour embryos with neuron-specific
antibodies reveal permanent blastulae contain 50 or more serotonergic neurons
scattered throughout the thickened epithelium of the animal pole
(Fig. 1B-D). The cells are
flask-shaped and have a short, apical dendritic pole and neurites projecting
basally. The neurites form a basiepithelial plexus surrounding the blastocoel.
By contrast, embryos injected with glycerol have an apical organ with four to
six serotonergic neurons (Fig.
1E). The serotonergic cells of the apical organ normally appear
after 55 hours; however, in -cadherin-injected embryos, they appear as
early as 48 hours (47% of 111 embryos). In 120-hour permanent blastulae
labeled with anti-synaptotagmin and anti-serotonin, all the serotonergic cells
co-localize with anti-synaptotagmin. However, there are cells that are only
synaptotagmin immunoreactive, which are similar in form to the serotonergic
cells but outnumber them by 2:1 (Fig.
1F). This is similar to 4- to 5-day-old plutei, where there are
non-serotonergic neurons in the apical organ.
Injection of RNA encoding Gsk3ß (Gsk) is thought to interfere with
vegetal signaling by enhancing the degradation of endogenous ß-catenin
(Emily-Fenouil et al., 1998).
Gsk RNA injection produces permanent blastulae similar to -cadherin RNA
injected embryos (Fig. 1G). In
preparations with neuron-specific antibodies of 72-hour permanent blastulae
injected with Gsk RNA there are numerous serotonergic neurons throughout the
expanded thickened ectoderm (Fig.
1G; 68% of 321 embryos). The thickened ectoderm of Gsk
RNA-injected embryos is smaller than that of -cadherin-injected embryos
and has fewer serotonergic neurons. An alternative means to produce permanent
blastulae is to isolate the animal half of eight-cell or 16-cell embryos
(Wikramanayake and Klein,
1995). As with permanent blastulae produced by RNA injection,
these embryos contain large numbers of serotonergic cells scattered throughout
the thickened epithelium (Fig.
1H, 100% of 21 embryos).
Takacs et al. (Takacs et al.,
2004) showed that the apical organ serotonergic neurons
differentiate at the edge of a region of ectoderm that expresses SpNk2.1 where
it contacts aboral ectoderm. Otim et al.
(Otim et al., 2004) showed
that SpHnf6 transcripts accumulate in cells of the ciliary band; a strip of
cells at the interface of oral and aboral ectoderm. The animal plate ectoderm
is the only region that co-expresses these two transcription factors
(Fig. 1I,J). In
-cadherin-injected embryos prepared for in situ hybridization and
immunolocalization, SpHnf6 mRNA and SpNK2.1 protein are present throughout the
thickened epithelium (Fig.
1K-M). Thus, treatments that block or interfere with vegetal
signaling result in permanent blastulae in which the thickened ectoderm in the
animal hemisphere differentiates as neurons. The markers expressed indicate
that this is an expansion of the animal plate ectoderm that normally gives
rise to the apical organ.
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; Wikramanayake et al.,
1998). Embryos injected with stabilized ß-catenin form
exogastrulae with most cells converted to endomesodermal lineages. We did not
detect neurons in the small remaining region of ectoderm
(Fig. 2A-C; 78% of 45 embryos).
Control embryos injected with 22.5% glycerol form normal prisms with four to
six serotonergic neurons (Fig.
2J-L). Similarly, a kinase-dead form of Gsk3ß (dnGsk) acts as
a dominant negative and produces exogastrulae with excess vegetal lineage
cells and reduced ectoderm (Emily-Fenouil
et al., 1998). Neither serotonin immunoreactive cells nor
anti-synaptotagmin immunoreactive cells are detected in these embryos
(Fig. 2D-F; 98% of 104
embryos). To ensure that neurogenesis is not in some way dependant on
gastrulation, we made exogastrulae with physical pressure. These exogastrulae
have a normal complement of four to six serotonergic neurons, indicating it is
not internalizing the archenteron that is necessary for neurogenesis
(Fig. 2G-I). In stabilized
ß-catenin injected embryos, SpNk2.1 protein is not detected in the
ectoderm (Fig. 2M-O). We
conclude that enhanced vegetal signaling suppresses formation of the animal
plate.
Vegetal signaling acts indirectly to restrict the size of the animal plate
Vegetal signaling is initiated by micromeres at the vegetal pole and the
embryo is patterned by a series of short-range signaling events that
sequentially specify adjacent tiers of blastomeres
(Davidson et al., 1998).
Although ß-catenin has not been detected in the nuclei of animal
blastomeres, a direct role for this factor in suppressing neurogenesis in
animal blastomeres can not be excluded. To distinguish between direct and
indirect signaling, we prepared chimeric embryos. Mesomeres of a 16-cell
embryo injected with -cadherin and myc RNA as a lineage marker were
grafted onto the vegetal half of an uninjected embryo
(Fig. 3A). Chimeras developed
into embryos in which the archenteron and mesenchyme cells have no myc signal
and ectoderm that is anti-myc immunoreactive
(Fig. 3C-E). In all the
chimeras, serotonergic neurons are detected that are identical in number and
position to control embryos, or embryos injected with myc RNA alone (4
embryos). We further tested this by injection of -cadherin and myc RNA
into one cell of an eight-cell embryo (Fig.
3B). Embryos in which -cadherin and the myc lineage marker
were introduced into a vegetal octomere had deficiencies in their morphology
(data not shown; 33% of 46 embryos had defects). In some octomere-injected
embryos 1/4 of the ectoderm contained -cadherin and the myc lineage
marker (Fig. 3F-H). These
embryos appear normal, express SpNK2.1 normally, and have four to six neurons
in the apical organ (Fig. 3F-K,
67% of 46 embryos). We conclude that vegetal signaling has an indirect effect
on differentiation of the animal pole, most probably through its role in
specification of ectoderm.
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).
Expression of SpNK2.1 is restricted to the central region of the thickened
ectoderm and SpHnf6 is expressed throughout the entire thickened ectoderm
(Fig. 4A,B; 91% of 299
embryos). Serotonergic neurons form a ring of cells at the periphery of the
SpNK2.1 domain (Fig. 4C). The
number of serotonergic neurons formed in 72-hour-old embryos is similar to
that of control embryos, but by 96 hours, there are significantly more
serotonergic neurons than in uninjected controls
(Fig. 6). Ciliary band
associated neurons revealed by anti-synaptotagmin (SpSynB) are spread
throughout the thickened ectoderm (Fig.
4D).
SB203580 is a p38 kinase inhibitor that has been shown to interfere with
Nodal expression and specification of ectoderm
(Bradham and McClay, 2006).
Embryos treated with SB203580 are radially symmetric and have significantly
more serotonergic neurons than control plutei
(Fig. 4E,
Fig. 6). The serotonergic
neurons lack bilateral symmetry.
SB431542 treatment is thought to block the Nodal receptor
(Duboc et al., 2005) and in
sea urchins produces embryos that are similar to Antivin RNA-injected embryos.
In embryos treated with SB431542 from hatching, more serotonergic neurons form
than in untreated controls and the serotonergic neurons form around the edge
of the SpNk2.1-expressing cells of the animal plate
(Fig. 4F,G;
Fig. 6).
BMP RNA injection produces radially symmetric embryos that have a small
animal plate that bulges outward (Angerer
et al., 2000). Expression of SpNK2.1 is most intense in the small
region of thickened ectoderm, but there are cells adjacent that have nuclear
SpNK2.1 protein (Fig. 4H).
SpHnf6 expression is restricted to the thickened ectoderm
(Fig. 4I). Serotonergic cells
are scattered throughout the thickened ectoderm
(Fig. 4J; 86% of 79 embryos).
The number of serotonergic neurons in BMP-injected embryos is similar to
control embryos at 72 hours, but by 96 hours, the number of neurons is almost
twice that of controls (Fig.
6).
Enhancing oral ectoderm specification reduces serotonergic neurons
Several reagents have been identified that enhance oral ectoderm
specification. Injection of eggs with RNA encoding Nodal produces radialized
embryos with the distinctive shape of a top
(Duboc et al., 2004). SpNK2.1
is expressed in the narrowed pole region of these embryos and SpHnf6
expression is restricted to a subset of these cells at the pole
(Fig. 4K-M). The region
expressing both markers corresponds to the animal plate cells that are
surrounded by a region corresponding to the region of oral ectoderm that
expresses SpNK2.1 (Takacs et al.,
2004). In Nodal-injected embryos, serotonergic cells rarely
differentiate (Fig. 4M; 94% of
123 embryos lack serotonergic neurons).
Noggin, which is thought to antagonize BMP specification of aboral ectoderm
produces radially symmetric embryos with a small animal plate and lack aboral
ectoderm markers (Angerer et al.,
2000). In these embryos, SpNK2.1 protein is in the nuclei of cells
in the small region of thickened animal plate ectoderm
(Fig. 4N,O; 100% of 146
embryos). In 96-hour embryos injected with Noggin RNA, significantly fewer
serotonergic cells form at either 72 or 96 hours
(Fig. 4O,
Fig. 6).
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;
Duboc et al., 2004). Embryos
expressing SpGsc form blastulae that lack a thickened animal plate ectoderm
and express the oral ectoderm marker EctoV
(Angerer et al., 2001). In
these permanent blastulae, SpNk2.1 is expressed throughout embryos injected
with SpGsc and there are no serotonergic cells formed
(Fig. 5J-L; 100% of 150
embryos). We are unable to detect SpHnf6 mRNA in SpGsc-injected embryos
(Fig. 5K). Thus, enhancing
SpGsc expression produces embryos that have features of the region of oral
ectoderm adjacent to the animal plate that expresses SpNk2.1. With the exception of SpGsc, all treatments that respecify ectoderm produce embryos in which an animal plate, defined by co-expression of SpHnf6 and SpNK2.1, is formed. When the animal plate is surrounded by ciliary band or aboral ectoderm, more serotonergic neurons are formed, but bilateral patterning is lost. This response is not clear until 96 hours and may be an indirect effect. When the thickened animal plate cells are surrounded by oral ectoderm, fewer serotonergic neurons form.
Nodal inhibits formation of serotonergic neurons
Treatments that enhance oral or aboral specification are carried out in
embryos in which normal vegetal specification occurs and there is a background
of endogenous signaling. To isolate the effects of treatments, we have
produced embryos in which vegetal signaling is blocked by co-injection of
-cadherin RNA. Embryos in which Antivin or BMP is co-expressed with
-cadherin form permanent blastulae with a thickened animal plate. These
embryos are like the -cadherin-only controls; SpNK2.1 is expressed in
the thickened ectoderm and up to 50 serotonergic neurons differentiate
(Fig. 5A-C; Antivin, 82% of 106
embryos; BMP, 90% of 104 embryos with phenotype). Co-expression of Nodal with
-cadherin produces permanent blastulae that have a broad, thickened
animal plate. The animal plate ectoderm expresses SpNK2.1 and SpHnf6, as in
embryos injected with -cadherin alone
(Fig. 5D,E). However, there are
only a few serotonergic cells that differentiate
(Fig. 5F; 99% of 156 embryos
with this phenotype). In chimeras in which one half of the embryo expresses
-cadherin, and other half expresses -cadherin and Nodal,
serotonergic neurons form in animal plate of the half expressing
-cadherin only. However, the serotonergic neurons that form are mostly
two to three cell diameters from the interface with the half expressing Nodal,
and they are reduced in density (Fig.
5I; 100% of 45 embryos). Chimeras with Antivin or BMP RNA
co-expressed in one half of the embryo have serotonergic neurons in both
halves, as in controls expressing -cadherin only
(Fig. 5G,H; Antivin, 98% of 59
embryos; BMP, 85% of 41 embryos). Thus, when the animal plate is adjacent to
cells expressing Nodal, there is a suppression of serotonergic neuron
formation in cells of the plate that are close to the interface.
The effect of suppressing neuron formation in adjacent cells could result
directly from secretion of Nodal protein or from an indirect effect of half of
the embryo expressing Nodal and becoming oral ectoderm. SpGsc lies downstream
of Nodal and expression appears to induce an oral ectoderm phenotype
(Angerer et al., 2001;
Duboc et al., 2004). In
embryos that are expressing -cadherin to block vegetal signaling and
SpGsc to specify oral ectoderm, permanent blastulae form that are similar to
those formed when SpGsc is expressed alone. The embryos lack a thickened
animal plate, express SpNK2.1 uniformly, yet they do not express neural
markers, and SpHnf6 can not be detected with in situ hybridization probes
(Fig. 5M-O; 100% of 150 embryos
have this phenotype). Thus, without vegetal signaling, SpGsc induces the
SpNk2.1 expressing region of oral ectoderm. We prepared chimeras, in which
vegetal signaling is blocked throughout the entire embryo, and half of the
embryo is converted to SpNk2.1-expressing oral ectoderm by co-expression of
SpGsc. In the half of the embryo co-expressing SpGsc and -cadherin, no
serotonergic neurons form. In the half expressing -cadherin alone,
there are abundant serotonergic neurons
(Fig. 5P; 77% of 22 embryos
have this phenotype). The serotonergic neurons form in cells directly at the
interface with cells co-expressing SpGsc and -cadherin, indicating that
there is no suppression of serotonergic neuron in adjacent cells as a
consequence of SpGsc specification of SpNK2.1-expressing oral ectoderm. This
supports a model in which Nodal, or a signal produced as a consequence of
Nodal, but not part of the Gsc pathway, acts to suppress neuron formation.
To further assess this, we used SB431542 to block Nodal receptors. Embryos
co-injected with -cadherin and Nodal have an average of two
serotonergic neurons (Fig. 5F,
Fig. 6). When we treat these
Nodal/-cadherin co-injected embryos with SB431542, there is an average
of 17 serotonergic neurons scattered throughout the thickened animal plate
ectoderm (Fig. 5R,
Fig. 6). Thus, blocking the
Nodal receptor is able to partially rescue the suppression of serotonergic
neuron formation by Nodal. To control for SB431542 having an independent
enhancing effect on serotonergic neuron formation in the absence of Nodal, we
treated embryos that have vegetal signaling blocked by injection of
-cadherin. An average of 40 serotonergic neurons form in an unpatterned
apical organ that is similar to that of embryos expressing -cadherin
alone (Fig. 5Q,
Fig. 6).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Emily-Fenouil et al.,
1998; Logan et al.,
1999). The thickness of the ectoderm and the long cilia suggest
that, in these embryos, the animal plate is expanded. In normal embryos, the
animal plate ectoderm is unique in being the only region where there is an
overlap in the expression of SpNK2.1 and SpHnf6. These transcription factors
are expressed throughout the thickened animal plate ectoderm in permanent
blastulae, providing specific molecular markers that indicate this is an
expansion of the animal plate. The neural markers indicate that the thickened
cap of ectoderm differentiates as a field of neurons. The region lacks the
characteristic bilateral symmetry and a well-organized neuropil, but the form
and types of neurons suggest this is an expanded, but poorly patterned, apical
organ. Expression of stabilized ß-catenin and dnGsk enhances
nuclearization of ß-catenin and converts animal blastomeres to
endomesodermal fates (Wikramanayake et
al., 1998; Emily-Fenouil et
al., 1998). We are not able to identify neurons in these
vegetalized embryos. Thus, treatments that block vegetal signaling result in
an expansion of the animal plate and treatments that enhance vegetal signaling
seem to repress it.
Our results with chimeras expressing -cadherin in animal blastomeres
and octomere injections of -cadherin indicate that repression of
neurogenesis is not directly dependent on Wnt/ß-catenin signaling. It
appears that the embryo is patterned by signals that specify successive tiers
of cells, beginning at the vegetal pole and ultimately specifying oral and
aboral ectoderm (Davidson et al.,
1998). The endomesoderm specification pathway is unlikely to be
involved in restricting the size of the animal plate, as suppression of
Notch/Delta signaling does not affect development of the apical organ
(Sherwood and McClay, 1999;
Sweet et al., 2002) (S.Y.,
J.Y. and R.D.B., unpublished). We do not know how vegetal signaling initiates
expression of p 38 or Nodal, but it has been demonstrated that oral ectoderm
specification depends on vegetal signaling
(Duboc et al., 2004;
Bradham and McClay, 2006).
The animal plate is restricted in size by signals that specify ectoderm
Blocking vegetal signaling produces embryos that have an expanded animal
plate, suggesting that there is a process in the embryo that normally
restricts neurogenesis to a small region. Restriction of the size of the
animal plate ectoderm does not appear to be a consequence of Nodal or BMP
signaling, because with vegetal signaling blocked, expression of these
signaling molecules results in embryos with expanded animal plates. The thin
ectoderm that is formed expresses markers that indicates it is either oral,
when Nodal is expressed, or aboral, when BMP is expressed
(Duboc et al., 2004;
Angerer et al., 2000). However,
in embryos in which the vegetal pathway is not altered, but Nodal expression
is blocked with a morpholino, or counteracted by expression of Antivin or
treatment with SB431542, the resulting embryos have a small animal plate. In
these embryos, the small animal plate is surrounded by ectoderm that expresses
markers for ciliary band; Pl
2 tubulin
(Duboc et al., 2004), SpHnf6
and ciliary band neurons. Similarly, blocking BMP signaling with a morpholino,
or counteracting it with Noggin, in embryos in which vegetal signaling is
intact also produces embryos with a restricted animal plate surrounded by
ectoderm expressing oral ectoderm markers
(Angerer et al., 2000;
Duboc et al., 2004). Thus,
expression of BMP and Nodal when vegetal signaling is blocked cannot rescue
restriction in the size of the animal plate. However, blocking specification
of oral and aboral ectoderm by inhibiting Nodal and, consequently, BMP results
in embryos in which there is a restricted animal plate surrounded by ectoderm
with a ciliary band phenotype. These observations suggest that there is a
signal, or signals, that are ß-catenin dependent that restrict the size
of the neurogenic animal plate and produce ectoderm with a ciliary band
phenotype. Duboc et al. (Duboc et al.,
2004) propose that the initial specification of ectoderm is to a
ciliary band phenotype and that oral and aboral domains arise later. Bradham
and McClay (Bradham and McClay,
2006) propose that p38MAPK acts upstream of Nodal and is necessary
for oral specification. Embryos in which p38MAPK is inhibited or a kinase
inactive form is overexpressed have a small animal plate, indicating p38MAPK
is not necessary for restricting the size of the animal plate. Although we do
not know the molecular basis of this signal, it appears specification of
ectoderm that precedes Nodal and BMP specification of oral and aboral ectoderm
restricts neurogenesis to a small domain near the animal pole
(Fig. 7).
|
|
To determine how oral suppresses serotonergic neuron formation, we prepared
embryos in which vegetal signaling is blocked by injection of
-cadherin. We believe this to be a model in which we can examine the
effects of ectoderm specification on the expanded animal plate without the
influence of endogenous vegetal signaling. When Nodal is co-expressed in these
embryos, there is a marked reduction in serotonergic neurons, suggesting Nodal
acts to suppress this phenotype. The experiments with chimeras indicate that
Nodal affect cells adjacent to the cells expressing it. By contrast, cells
expressing SpGsc, although apparently oral, have no effect on the animal plate
cells adjacent to them. In vertebrates, the inhibitor SB431542 blocks the
activity of Alk4, Alk7 (nodal/activin type I receptor) and Alk5 (TGFß
type I receptor) (Inman et al.,
2002), and has been demonstrated to block Nodal function in sea
urchin embryos (Duboc et al.,
2005). When embryos are treated at 19 hours with SB431542, the
result is a phenotype similar to the Nodal morpholino or expression of antivin
(Duboc et al., 2004). These
embryos have extra serotonergic neurons in the apical organ and bilateral
symmetry is lost. When we use SB431542 to treat embryos with vegetal signaling
blocked and Nodal expression suppressing serotonergic neuron formation, there
is a significant rescue of serotonergic neurons. This suggests that blocking
perception of Nodal in animal plate cells is sufficient for specification of
serotonergic phenotype to proceed. Nodal clearly has roles in specification of
oral ectoderm and establishing the position of the adult rudiment
(Duboc et al., 2004;
Duboc et al., 2005). However,
in ascidians and vertebrates, Nodal has also been demonstrated to function in
patterning neural tissues (Thisse et al.,
2000; Hudson and Yasuo,
2005). Our experiments indicate that, in urchins, Nodal secreted
by oral ectoderm acts as a paracrine factor that represses the differentiation
of serotonergic neurons at the interface with the animal plate.
|
The animal plate is a neurogenic ectoderm
The animal plate is resistant to re-specification, and expands in the
absence of vegetal signaling (Angerer and
Angerer, 2003). The observation that the expanded animal plate
differentiates as neural tissue indicates that the animal plate is a region of
neurogenic ectoderm. The robust phenotype of the cells that resist
respecification to other cell types is neural, and the animalizing influence,
which researchers have attributed to this region of the embryos for over 100
years, is a neuralizing influence. The vegetal influences, which we now
interpret as the effects of nuclearization of ß-catenin, appear to be
specification of cell types that restrict the expansion of this neurogenic
region of ectoderm.
In vertebrates, ectoderm has a tendency to differentiate into neural
tissue, and epidermis results only under the influence of BMP. As BMP4
signaling is widespread, the neural plate forms only where this signaling is
directly inhibited, by molecules such as noggin, follistatin and chordin
(Hammati-Brivanlou et al., 1994; Hammati-Brivanlou and Melton, 1997). This
hypothesis, termed the default model, is currently being revised to
accommodate observations that indicate there may be an early requirement for
fibroblast growth factor signaling
(Delaune et al., 2005;
Stern, 2005). In
Drosophila embryos, dorsal is established initially during oogenesis
but is maintained by dorsal cells secreting a BMP4 ortholog, decapentaplegia;
ventral neural ectoderm is protected from its dorsalizing effect by secretion
of Short gastrulation, a molecule similar to Chordin
(Biehs et al., 1996). The
suggestion has been made that anti-neural inhibition is a mechanism of neural
specification that will be found throughout the metazoans
(Bier, 1997). In echinoids,
unspecified ectoderm appears to become neurogenic animal plate ectoderm. Thus,
a default neuralizing influence appears to be a shared metazoan mechanism of
ectoderm specification that is retained in a deuterostome with a
planktotrophic larval form.
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ACKNOWLEDGMENTS |
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