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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02400
1 JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125,
Japan.
2 PRESTO, Japan Science and Technology Agency, Saitama, Japan.
* Authors for correspondence (e-mail: yasuko{at}brh.co.jp and hoda{at}brh.co.jp)
Accepted 10 April 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Spider, Embryogenesis, dpp, sog, Antagonist, Body axis formation, RNAi
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Kimelman and Bjornson,
2004). The dorsal lip of the blastopore in the amphibian embryo,
known as Spemann's organizer, can organize surrounding cells to form a
complete body axis (Spemann and Mangold,
1924). This organizer itself contributes to the axial
(dorsal-most) mesoderm becoming the notochord, concomitant with the patterning
of the more ventral mesoderm and neural tissues. Molecular studies have
suggested that the organizing activity is exerted by antagonizing bone
morphogenetic protein (BMP) and Wnt signals
(Piccolo et al., 1996;
Harland and Gerhart, 1997;
De Robertis et al., 2000).
Chordin, noggin and follistatin are BMP antagonists expressed at the organizer
(Lamb et al., 1993;
Hemmati-Brivanlou et al.,
1994; Sasai et al.,
1994; Fainsod et al.,
1997). Simultaneous depletion of these three BMP antagonists leads
to a gross failure in the development of dorsal structures, including the
notochord and neural tissues (Khokha et
al., 2005).
In the fruit fly Drosophila melanogaster, a homologue of
vertebrate BMP2/4, Decapentaplegic (Dpp), and a homologue of chordin, Short
gastrulation (Sog), have also been shown to function antagonistically in
dorsoventral (DV) pattern formation of the embryo
(Irish and Gelbart, 1987;
Padgett et al., 1987;
Ferguson and Anderson, 1992a;
Ferguson and Anderson, 1992b;
François et al., 1994).
Sog is the only Dpp antagonist that is known to function in the early
Drosophila embryo, and two different roles for the molecule have been
identified. The first role is to prevent the Dpp signal from invading the
neuroectoderm that forms at the ventrolateral region
(François et al., 1994;
Biehs et al., 1996). The second
role is to contribute to the formation of a sharp stripe of Dpp signaling
activation at the dorsal-most region that will become the extra-embryonic
amnioserosa (Ashe and Levine,
1999; Decotto and Ferguson,
2001). Although the latter role is considered to be unique to the
fly, the former role is markedly similar to that of BMP antagonists in the
development of vertebrate dorsal structures. This similarity has raised the
hypothesis that the orientation of the Drosophila DV axis is opposite
to that of the vertebrate DV axis, assuming that these axes had a common
origin (Holley et al., 1995;
De Robertis and Sasai, 1996;
Ferguson, 1996;
Holley and Ferguson, 1997;
Bier, 1997). Despite the
fascinating implications this would have for DV axis evolution, however, the
null mutation for Drosophila sog only slightly reduces the
neuroectoderm and barely affects the differentiation of the ventral midline
(Fig. S1 in supplementary material)
(François et al., 1994)
not validating the potential importance of Dpp/BMP antagonism in DV axis
specification in the common ancestor of Drosophila and vertebrates.
The only organism other than Drosophila and vertebrates in which a
Dpp/BMP antagonist has been studied is the ascidian, in which function of the
antagonist has not been related to DV axis development or neural induction
(Darras and Nishida, 2001).
In the phylum Arthropoda, twinned embryos can be produced spontaneously
(see Fig. S2 in the supplementary material) or experimentally in spiders,
horseshoe crabs and short-germ insects
(Holm, 1952;
Sekiguchi, 1957;
Seitz, 1970;
Sander, 1976;
Itow et al., 1991). These may
indicate the existence of different modes of axis specification from that of
Drosophila. A study of the Dpp-Sog system within Arthropoda would
contribute to a better understanding of DV axis evolution. Spiders and
horseshoe crabs are chelicerate arthropods that are phylogenetically distant
from the insect Drosophila
(Friedrich and Tautz, 1995;
Hwang et al., 2001;
Giribet et al., 2001). In
early development of spiders, such as Achaearanea tepidariorum, the
future DV axis becomes predictable by the onset of directional movement of a
cellular thickening called the cumulus, which is formed at the center of the
radially symmetrical germ disc and then shifts centrifugally to the rim
(Akiyama-Oda and Oda, 2003).
Graft experiments using Agelena labyrinthica showed that the cumulus
has the ability to induce a secondary body axis
(Holm, 1952). The shift of the
cumulus is followed by formation of the extra-embryonic area and
rearrangements of the germ disc cells. These morphogenetic events transform
the germ disc into the bilaterally symmetrical germ band. Our previous study
showed that, in the spider Achaearanea tepidariorum, a cluster of the
mesenchymal cells at the cumulus (CM cells) is the source of Dpp signals
(Akiyama-Oda and Oda, 2003). In
this study, we investigated the roles of dpp and sog in DV
axis development of the Achaearanea embryo.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Yamazaki et al., 2005).
At-dpp depleted embryos, which exhibited gross malformations, could
not be staged on the basis of morphological characteristics; instead, they
were staged based on the time past stage 5.
cDNA cloning
We cloned Achaearanea sog (At-sog), Pholcus sog
(Pp-sog), Artemia sog (Af-sog), and the
Achaearanea genes single minded (At-sim),
prospero (At-pros), engrailed (At-en) and
optomotor blind (At-omb). Details of cDNA cloning are
presented in Table 1. The
sequences are available from the DNA Data Bank of Japan with the following
accession numbers: At-sog, AB236147; Pp-sog, AB236148;
Af-sog, AB236149; At-sim, AB236150; At-pros,
AB236151; At-en, BAD01489; At-omb, AB177876. To characterize
the deduced amino acid sequences of the cloned genes, BLASTP, BLAST 2
sequences
(http://www.ncbi.nlm.nih.gov/blast/),
PHYLIP version 3.5 and HarrPlot 2.0 (GENETYX Mac version 12) were used. BLASTP
search analyses revealed that the sequences of At-Pros and At-En are very
close to those of Cs-Pros (Weller and
Tautz, 2003) and Cs-En (Damen
et al., 1998), respectively, which were previously reported in
another spider species Cupiennius salei. Molecular phylogenetic trees
were constructed for At-Sim and At-Omb (see Fig. S3 in the supplementary
material).
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;
Oda et al., 2005). Whole-mount
in situ hybridization was performed as described previously
(Lehmann and Tautz, 1994;
Akiyama-Oda and Oda, 2003). For
single-staining, digoxigenin (DIG)-labeled probe was used. For
double-staining, embryos were incubated with a mixture of DIG- and
fluorescein-labeled probes. The DIG-labeled probe was visualized in the same
way as the single staining, followed by post-fixation and subsequent
inactivation of alkaline phosphatase with 0.1 M glycine-HCl (pH 2.2). Then,
the fluorescein-labeled probe was visualized using an alkaline
phosphatase-conjugated rabbit anti-FITC antibody (DAKO, 1:200 dilution) and
INT/BCIP solution (Roche). For nuclear staining, DAPI (Sigma) was used. To
detect phosphorylated Mothers against dpp (pMad) protein for visualizing
nuclei of cells responding Dpp signals, the PS1 antibody
(Persson et al., 1998) was
used as described (Akiyama-Oda and Oda,
2003). For sectioning, stained Achaearanea embryos were
dehydrated in a graded ethanol-xylene series, embedded in TissuePrep (Fisher
Scientific), and then serially sectioned at 5 µm.
Double-stranded RNA preparation
Double-stranded RNAs (dsRNAs) for the 706-bp [nucleotide (nt) 334-1039] and
1041-bp (nt 1947-2987) regions of the At-sog cDNA, the 736-bp (nt
1005-1740) region of At-dpp
(Akiyama-Oda and Oda, 2003),
and the coding region of the gene for jellyfish green fluorescent protein
(gfp) (Quantum) were prepared according to Niimi et al.
(Niimi et al., 2005), except
that the dsRNAs were denatured at 95°C for 5 minutes, and annealed. The
dsRNAs were used at concentrations of 1.5 to 2.5 µg/µl.
dsRNA injection to the spider Achaearanea tepidariorum
Mature spider females, either virgins or postmated, were used for dsRNA
injection. Under a stereomicroscope, 1-2 µl of dsRNA solution was
introduced into the opisthosoma of each female from the dorsal side using a
pulled glass capillary whose tip was broken with forceps. This injection
process was repeated two to six times at intervals of 2-3 days. The virgin
females were mated after the second cycle of injection. However, the timing of
mating essentially did not affect the final results. Egg sacs made by the
injected females were each analyzed separately. Some eggs from every egg sac
were submerged in halocarbon oil 700 (Sigma) to examine the development of the
embryo. To evaluate the effects of At-sog dsRNA injection, the
numbers of segments bearing unseparated limb buds at stage 9 (a normal spider
embryo has six pairs of limb buds) were counted after dechorionation with
commercial bleach. Embryos with four to six unseparated limb buds were
classified as having the severe phenotype, and those with one to three
unseparated limb bud(s) as having the mild phenotype. Achaearanea
embryos show spontaneous developmental errors at frequencies depending on
individuals or egg sacs. To minimize non-specific effects, egg sacs in which
more than 40% of eggs failed to form a germ band were discarded. Females that
repeated such abnormal egg production three or more consecutive times were
also discarded.
RT-PCR
Semi-quantitative RT-PCR was performed to compare the levels of gene
expression at stage 9 (for At-sog dsRNA injected and non injected),
or stage 5 (for At-dpp dsRNA injected and non injected) embryos.
Total RNA was extracted from 30 eggs of each type using a MagExtractor RNA kit
(Toyobo). The total RNA was treated with DNaseI (Stratagene), and a
first-strand cDNA was prepared using an oligo(dT) primer and SuperScript II
reverse transcriptase (Invitrogen). The cDNA was used as a template for PCR
reactions. The PCR conditions were as follows: 20 or more cycles of 95°C
for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. The primer
sets used are as follows: for At-sog in At-sog RNAi
experiments, tacgaactggaggagaga and tgttccgtatgcctctgt; for At-sog in
At-dpp RNAi experiments, aagtgcgatcgaatcacg and gttgccgtacctttctgt;
for At-sim, taggaagccagaacctct and aggtcctaaggcacttgt; for
At-dpp, ttgatcctacaaggaaaggc and gacttgattccacctatgagg; for
histone H3, taccaagcaaacagctcg and gcttttcggctgcttgtaa; for
EF1
, tggacacaagtgaaccac and tctgaccaggatggttca. The sequences
for histone H3 and EF1 were designed according to
data from our Achaearanea EST project (unpublished results).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The signals appeared in the germ disc as a ring
(Fig. 2A). This ring of
At-sog expression was broken by the insertion of an
At-sog-negative area (Fig.
2B), which corresponded to the presumptive extra-embryonic area as
revealed by double staining for At-sog transcripts and pMad
(Fig. 2J,J'). During
stages 6 and 7, when the extra-embryonic area is expanded and the germ disc
cells are dynamically rearranged, a large part of the embryonic area,
excluding the forming caudal lobe and the periphery of the embryonic area,
expressed At-sog (Fig.
2B-E). As the germ band formed and extended, the At-sog
expression domain progressively narrowed
(Fig. 2F). This narrowing
At-sog expression domain was complementary to the pMad expression
domain expanding from the dorsal side (Fig.
2K,L,L'). Eventually, the At-sog expression was
confined to the ventral midline area (Fig.
2G). Sectioning revealed that At-sog staining was
localized at the surface ectoderm, with no signal detectable in the mesodermal
layer (Fig. 2H,I). Although the
cells at the ventral midline area were morphologically indistinguishable from
the neighboring ectoderm cells at this stage, this ventral midline area was
characterized by specific expression of At-fork head
(At-fkh) (Akiyama-Oda and Oda,
2003) and a sim homologue (At-sim)
(Fig. 2M,N). The onset of
At-fkh expression at the ventral midline area was at late stage 7,
whereas the onset of At-sim expression was at stage 9. The
expressions of At-sog, At-fkh, and At-sim were initially
observed in nearly the same cell population
(Fig. 2M,N). Later, however,
different patterns of expression became evident
(Fig. 3). This appeared to
reflect the differentiation of several cell types within the ventral midline
area.
The At-sog expression domain expanded anterior to the
At-orthodenticle (At-otd)
(Akiyama-Oda and Oda, 2003)
expression domain (Fig. 2O,P).
The At-sog expression domain also expanded posteriorly in accordance
with the production of segments in the growing opisthosomal region
(Fig. 2G,L), although the
caudal lobe did not express At-sog. The axial pattern of
At-sog expression in the germ-band stage embryo appeared to result
from expansion of the expression along the emerging anterior-posterior (AP)
axis and reduction of the expression along the emerging DV axis in accordance
with dynamic cell rearrangements converting the germ disc to the germ
band.
Similar to At-sog, Drosophila sog shows restricted expression at
the ventral midline in the germ-band stage embryo
(François et al.,
1994). Similar ventral midline expression was also observed for
Pp-sog (Fig. 2Q,R) and
Af-sog (Fig.
2S,S'). The ventral midline expression of sog genes
may be a conserved feature of the phylum Arthropoda.
Depletion of At-sog expression by RNA interference
To investigate the function of At-sog, we depleted At-sog
expression from early spider embryos by parental RNA interference (pRNAi).
Adult females were repeatedly injected with dsRNA corresponding to a 706-bp
region (nt 334-1039) of At-sog cDNA, and their eggs were examined. In
control experiments, dsRNA for gfp was used. Embryos derived from
females injected with At-sog dsRNA, but not those from females
injected with gfp dsRNA, showed abnormal development of limbs in the
prosomal region (Fig. 4A-C).
Time-lapse microscopy of live embryos and DNA staining of fixed embryos
revealed that the abnormalities were due to lack of separation of extending
limb buds in the individual segments (see Movie 1 in the supplementary
material; Fig. 4A,B). The
phenotypes of the abnormal embryos were classified as `severe' and `mild'
based on the number of segments bearing unseparated limb buds
(Fig. 4A,B,F-P; see Materials
and methods). Such abnormal embryos were first found in the second or third
round of egg laying after the first dsRNA injection
(Fig. 4F-M). Whole-mount in
situ hybridization revealed that, in the embryos exhibiting severe phenotypes,
the level of At-sog mRNA was drastically reduced compared to the
control embryos (Fig. 4D,E). A
specific reduction in At-sog expression caused by pRNAi treatment was
also confirmed by RT-PCR (Fig.
4Q). In addition, injection of dsRNA synthesized using another
section of the At-sog cDNA (nt 1947-2987) also resulted in
unseparated limbs (data not shown). The phenotypes obtained with injection of
At-sog dsRNA were different from those obtained with injection of
At-dpp dsRNA (see below), suggesting gene-specific effects. Taken
together, these data suggest that pRNAi works in Achaearanea to
deplete the expression of specific genes, as has been reported in other animal
species (Fire et al., 1998;
Bucher et al., 2002;
Liu and Kaufman, 2003;
Mito et al., 2005). Embryos in
which At-sog expression was depleted by pRNAi, designated
At-sog RNAi embryos hereafter, were further analyzed to understand
the role of At-sog in the early development of the spider.
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; Weller and Tautz,
2003). The dorsal area of each limb-bearing hemisegment is
subdivided into a limb and a non-limb section. One At-pros-positive
cell cluster, tentatively called the dorsal-most cluster, was present at a
regular position in each non-limb section
(Fig. 5G arrows). In
At-sog RNAi embryos exhibiting very severe phenotypes, the
At-pros-positive neural cell clusters were mostly missing except for
a few regularly aligned clusters located at similar AP positions to those of
the dorsal-most clusters (Fig.
5G,I). In At-sog RNAi embryos exhibiting milder
phenotypes, however, At-pros-positive cell clusters formed around the
sites where At-sog was persistently expressed
(Fig. 5H). These results
suggest that the severe phenotypes caused by At-sog pRNAi were due to
an almost complete loss of the ventral structures, including the ventral
midline area and the CNS. The milder phenotypes generated by At-sog
RNAi treatment imply that the ventral midline and the CNS form concomitantly
by a mechanism requiring At-sog activity.
A homologue of omb, designated At-omb, was specifically
expressed at the dorsal side of each limb bud that had started to extend
(Fig. 5J), as described for
Cupiennius omb (Prpic et al.,
2003). In At-sog RNAi embryos exhibiting severe
phenotypes, the domains of At-omb expression were fused to cover the
entire width of the germ band (Fig.
5L). Similar alterations in the expression pattern were observed
in the opisthosomal region (Fig.
5K,M). As shown previously, At-twist (At-twi) is
expressed in mesodermal cell populations
(Yamazaki et al., 2005).
During stage 7, At-twi-expressing mesodermal cells become segmentally
arranged in the prosoma. During later stages, additional stripes of
At-twi-expressing cells appear in the opisthosoma. Each stripe of
At-twi-expressing cells initially displays little unevenness along
the predictable DV axis and then dorsally splits into two separate clusters at
the sites of appendage formation (Fig.
5N,O), with At-twi-expressing cells becoming absent in
the ventral areas. However, in At-sog RNAi embryos, in which the
At-twi-expressing mesoderm developed normally until at least stage 7
(not shown), no dorsal separation of the mesodermal cells was observed in the
prosoma or opisthosoma (Fig.
5P,Q). Taken together, these data indicated that the loss of the
ventral structures caused by At-sog pRNAi appeared to result from
gross dorsalization of the germ band.
Persistent radial symmetry in At-dpp RNAi embryos
Next, we conducted pRNAi experiments for At-dpp, which is
initially expressed in the CM cells and later expressed in the dorsal region
of the germ band where limb buds are formed
(Akiyama-Oda and Oda, 2003). A
specific reduction in At-dpp expression caused by the pRNAi treatment
was confirmed by RT-PCR, although a very small amount of At-dpp
transcripts was still detectable (Fig.
6I). In At-dpp RNAi embryos, the cumulus appeared and
shifted normally (Fig. 6).
However, the At-dpp RNAi embryos began to exhibit defects from the
beginning of stage 6, when the extra-embryonic area starts to differentiate.
DNA staining revealed that the extra-embryonic area, which can be easily
recognized by the sparse distribution of nuclei
(Fig. 7A), was reduced in size
(Fig. 7B) or lost
(Fig. 7C). The defective germ
disc was extended longitudinally to envelop the entire yolk mass.
Consequently, in severe cases, morphologically monotonous embryos were formed
with little asymmetry (Fig. 6,
Fig. 7C). Staining of such
At-dpp RNAi embryos for a homologue of en, designated
At-en, revealed rings of At-en expression instead of the
two-ended bands of At-en expression observed in normal embryos
(Fig. 7D-F). Similarly, a ring
of expression was observed for At-otd in severely defective
At-dpp RNAi embryos (Fig.
7G-I). These persistent circular patterns of gene expression
probably reflected the loss of the extra-embryonic area. Striped expression of
At-en (Fig. 7E) and
anterior and posterior expression of At-otd and At-caudal
(Akiyama-Oda and Oda, 2003),
respectively, in At-dpp RNAi embryos
(Fig. 7G-L) imply that
At-dpp RNAi had little effect on AP patterning, although there were
fewer At-en stripes than normal.
Furthermore, we examined At-sog expression in At-dpp RNAi embryos at different stages. RT-PCR showed that there was no detectable difference in the level of At-sog transcripts between untreated and At-dpp RNAi embryos at late stage 5 (Fig. 6I). Expression patterns of At-sog transcripts were observed in more than 29 late stage 5 embryos derived from the egg sac that was used for the RT-PCR experiment. It was found that all of the embryos showed more or less reduced asymmetry of the At-sog expression pattern (not shown), but none displayed complete symmetry, indicating that At-sog transcription was still affected by the shifting cumulus even in the At-dpp RNAi embryos. However, in severely defective At-dpp RNAi embryos at the later stages (stages 8 and 9) the entire surface ectoderm except for the posterior terminal area expressed At-sog transcripts at evenly high levels (Fig. 7M-O). Little asymmetry was recognized in the At-sog expression pattern. At-dpp RNAi embryos at the later stages were further examined for genes whose expression patterns reflect differences along the DV axis in the normal germ band. Staining for At-twi showed rings of At-twi-expressing mesodermal cells (Fig. 7P-R). No significant staining for At-pros, At-sim, or At-omb was obtained (Fig. 7S; not shown for At-sim and At-omb). In addition, the patterns of At-fkh expression, which varied among embryos, were radially symmetrical (not shown). Taken together, these data suggested that the At-dpp-depleted embryos were prevented from breaking the radial symmetry.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Depletion of At-dpp prevented the embryo from breaking the radial
symmetry, therefore, the At-Dpp-mediated specification of the extra-embryonic
area is crucial for radial-to-axial symmetry transformation of the spider
embryo (Fig. 8, yellow).
Despite the asymmetric patterns of At-sog expression from late stage
5 onward (Fig. 2),
At-sog function appears not to be involved in the formation of the
extra-embryonic area or the germ band (Fig.
5A). However, our data cannot exclude the possibility that the
suppressed At-sog is sufficient to play a role in the early events.
Since we failed to observe At-dpp RNAi embryos showing radially
symmetrical At-sog expression at late stage 5, it remains unclear
whether At-dpp is needed to make the initial At-sog
expression asymmetric. The ubiquitous expression of At-sog
transcripts at high levels in the later At-dpp RNAi embryo
(Fig. 7N) suggested that
At-sog transcription is negatively regulated by Dpp signaling. The
gradual expansion of the pMad-positive area from the dorsal side
(Fig. 2K,L) might be achieved
by the combination of positive feedback loops of Dpp signaling, as proposed in
Drosophila (Biehs et al.,
1996), antagonism of Dpp signaling by Sog and progressive
repression of At-sog transcription by the Dpp signals.
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). The germ-disc stage embryo has an axis that is probably
equivalent to the animal-vegetal axis in other phyla, but does not yet have
the AP axis specified. AP axis specification is considered to involve two
processes; one is development (or refinement) of AP positional information,
and the other is specification of the anterior pole. The former, which was
little affected in At-dpp RNAi embryos
(Fig. 7), probably does not
depend on Dpp signals, but the latter does depend on Dpp signals. In the
normal germ disc, an anterior pole is predicted to be the most distant point
from the extra-embryonic area induced by Dpp signals
(Akiyama-Oda and Oda, 2003).
This rule may be extended to account for duplicated AP axes caused by grafting
of the cumulus in Holm's experiments
(Holm, 1952). In this
experimental situation, two anterior poles are predictable at the halfway
points along the germ disc rim between the native and ectopic extra-embryonic
areas. The later At-dpp RNAi embryo had a recognizable anterior pole
despite lacking the extra-embryonic area
(Fig. 7C). However, this
anterior pole simply resulted from symmetric longitudinal extension of the
germ disc, the periphery of which converged at the pole of the yolk side. In
addition, the longitudinal extension of the At-dpp RNAi germ disc
implies that Dpp signaling may not be necessary to induce the dynamic cell
rearrangements during germ band formation.
Comparison of axis specification between fly and spider
There are two major differences in the roles of Drosophila and
Achaearanea Sog. First, fly Sog is necessary for specifying the
extra-embryonic area (Ashe and Levine,
1999; Decotto and Ferguson,
2001) but spider Sog probably is not
(Fig. 5A). Second, spider Sog
(Fig. 5D-I) but not fly Sog
(François et al., 1994)
(see Fig. S1 in the supplementary material) is essential for specifying the
ventral tissues that run axially through the germ band. These differences may
reflect evolutionary changes in the gene regulatory networks.
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; Ray et al.,
1991; Stathopoulos and Levine,
2002; Stathopoulos and Levine,
2004). This fly system could have reduced the contribution of
cell-cell interactions, explaining the relatively minor role of
Drosophila Sog in ventral specification. However, the Dorsal
gradient, which peaks at the ventral-most site, may have a lower resolution in
the dorsal half than in the ventral half
(Jiang and Levine, 1993). A
Sog-mediated mechanism that sharpens the Dpp-activating domain at the
dorsal-most region was recently proposed
(Ashe, 2005;
Shimmi et al., 2005;
Wang and Ferguson, 2005). This
mechanism might have been needed to compensate for the low resolution of the
Dorsal gradient in this dorsal region.
In contrast to the Dorsal-based mechanism that organizes the DV axis in
Drosophila, a mechanism controlling the direction of CM cell
migration is important for initial DV polarity in Achaearanea,
although its molecular basis remains unclear. As is evident from this study,
the spider system appears to require a series of cell-cell interactions
involving At-Dpp and At-Sog to initiate ventral-specific gene expression.
These cell-cell interactions may well account for the regulative nature of
body axis formation of spider embryos proposed by classical experiments
(Holm, 1952;
Sekiguchi, 1957;
Seitz, 1970), although the
mechanism of secondary axis induction in spiders remains to be studied. Based
on the results obtained in the analyses of spider dpp and
sog, we propose that one of the most fundamental differences in the
mechanisms that pattern the early fly and spider embryos is ventral
specification. This difference may reflect different degrees of contribution
made by maternal determinants and zygotic cell-cell interactions.
In addition, the mesoderm originates from between two separate lines of
presumptive ventral midline cells in the Drosophila embryo
(Leptin, 2004). This situation
is completely different from that of the Achaearanea mesoderm, which
was suggested by At-twi expression patterns at stages 5 and 6 to have
radially symmetrical origins at the peripheral and central areas of the germ
disc (Yamazaki et al., 2005).
In spider development, independent gene regulatory networks appear to specify
the DV pattern and the mesoderm.
Comparison of axis specification between vertebrates and spider
Our discovery of the differences between the fly and spider provide an
opportunity to rethink how arthropod embryos can be compared with vertebrate
embryos from the viewpoint of developmental evolution. Interestingly, the
spider situation in which sog activity is essential to specify the
ventral domains is similar to the vertebrate situation in which the antagonism
of Dpp/BMP signaling plays a central role in dorsal specification
(De Robertis et al., 2000;
Oelgeschläger et al.,
2003; Khokha et al.,
2005). The ventral midline area in the spider embryo is comparable
to the presumptive notochord area in the vertebrate embryo in that both areas
are the centers of the Dpp/BMP antagonism and adjoin the CNS. In addition, the
axial expression of At-fkh and At-sim
(Fig. 2M,N) is reminiscent of
the expression of their homologues in chordate notochords
(Ruiz i Altaba et al., 1993;
Sasaki and Hogan, 1993;
Strähle et al., 1993;
Shimauchi et al., 1997;
Shimeld, 1997;
Terazawa and Satoh, 1997;
Mazet and Shimeld, 2002).
Whether the spider ventral midline is homologous to the vertebrate notochord
is the emerging question. To satisfactorily answer this question, we will need
to consider the reason why the arthropod ventral midline is ectodermal and the
vertebrate notochord is mesodermal. The milder phenotypes of At-sog
RNAi embryos (Fig. 5E,H)
appeared to reflect concomitant development of the ventral midline and the
CNS. It is intriguing to investigate similarities and differences in the
mechanisms of neural induction in the spider and vertebrate embryos. Finally,
the Achaearanea experimental system could contribute to a better
understanding of the evolutionary relationships between the development of
arthropod and vertebrate embryos.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/12/2347/DC1
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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