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First published online 16 May 2007
doi: 10.1242/dev.004598
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1 JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125,
Japan.
2 Genome Resource and Analysis Subunit, Center for Developmental Biology, RIKEN,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
3 Department of Biophysics, Graduate School of Science, Kyoto University,
Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
* Author for correspondence (e-mail: hoda{at}brh.co.jp)
Accepted 27 March 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Chelicerate, Arthropod, Delta, Notch, Growth zone, Caudal lobe, Mesoderm, Caudal ectoderm, Segmentation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Similar modes of segmentation are seen outside the phylum Arthropoda (e.g. in
Chordata and Annelida). Whether segmentation in different bilaterian phyla
occurs by common or independent origins is the subject of controversy
(Davis and Patel, 1999;
Peel and Akam, 2003). Recent
work in spider has suggested that spider segmentation and vertebrate
somitogenesis share a mechanism involving Delta and Notch, which favors a
common origin model (Stollewerk et al.,
2003; Schoppmeier and Damen,
2005). However, it is still not clear whether the mesodermal
somites of vertebrates can be compared to arthropod segments, which consist of
ectoderm and mesoderm (Patel,
2003). Understanding of developmental processes occurring before
segmentation may be essential to resolve these issues.
Increasing numbers of studies in arthropods have focused on the growth
zone. In the well-studied insect Drosophila melanogaster, all
segments form almost simultaneously during early stages of embryogenesis.
Thus, the Drosophila embryo shows no area corresponding to the growth
zone at any developmental stage. Nonetheless, mechanisms regulating posterior
and terminal patterning in Drosophila
(St Johnston and Nüsslein-Volhard,
1992) have facilitated analysis of the development of the growth
zone. In diverse arthropod species, the growth zone is characterized by
specific expression of homologs of Drosophila caudal (cad)
(Mlodzik et al., 1985;
Macdonald and Struhl, 1986;
Moreno and Morata, 1999;
Schulz et al., 1998;
Akiyama-Oda and Oda, 2003;
Copf et al., 2004;
Shinmyo et al., 2005).
cad genes were shown to be essential for sequential production of the
posterior segments in some species (Copf et
al., 2004; Shinmyo et al.,
2005). In addition to the cad genes, homologs of
Drosophila maternal terminal genes, namely torso and
torso-like, are required to set up or maintain a functional growth
zone in the beetle Tribolium
(Schoppmeier and Schröder,
2005). Despite these significant studies, the cellular and
molecular events leading to establishment of the growth zone and the diversity
of mechanisms underlying its development among arthropods are poorly
understood.
Spiders, which are chelicerate arthropods, phylogenetically distant from
the insects, are classical organisms that have been used for developmental
study (Montgomery, 1909;
Holm, 1952;
Seitz, 1966;
Yoshikura, 1975). Modern
molecular techniques have been applied to two spider species, Cupiennius
salei and Achaearanea tepidariorum. In Achaearanea
development, the growth zone becomes morphologically apparent as a caudal lobe
during stages when the germ disc is converted to a germ band
(Montgomery, 1909;
Yamazaki et al., 2005). These
stages overlap with appearance of mesoderm cells expressing a homolog of
twist (twi), At-twi
(Yamazaki et al., 2005) and
caudal ectoderm cells expressing a homolog of cad, At-cad
(Akiyama-Oda and Oda, 2003).
Once formed, the caudal lobe is composed of an ectodermal and a mesodermal
cell layer (Montgomery, 1909).
Work with Cupiennius has revealed dynamic expression of
Delta among other genes at the caudal region
(Damen et al., 2000;
Stollewerk et al., 2003),
reminiscent of the molecular oscillation proposed for vertebrate somitogenesis
(Pourquié, 2003). RNA
interference (RNAi)-mediated knockdown of several components of the
Delta-Notch signaling pathway, including Delta, was shown to cause defects in
the pattern of the opisthosomal segments
(Stollewerk et al., 2003;
Schoppmeier and Damen, 2005).
However, as double-stranded RNA (dsRNA) was applied to Cupiennius
embryos around the blastula stage by injection in to the perivitelline space
(Schoppmeier and Damen, 2001),
it is unclear whether target genes were silenced during stages before
initiation of segmentation.
In this study, we examined the cellular and molecular events giving rise to
a caudal lobe in Achaearanea, by taking advantage of parental RNAi,
which can suppress target gene function during early stages of embryogenesis
(Akiyama-Oda and Oda, 2006). We
found that transcripts of a Delta homolog are expressed at high
levels in what are likely to be prospective mesoderm cells arising from around
the blastopore. Based on gene expression data and phenotypes produced by
RNAi-mediated knockdown of Delta and other components of the Notch signaling
pathway, we propose that Delta-Notch signaling is essential for caudal lobe
formation in Achaearanea, preceding initiation of segmentation. Our
findings reveal that formation of mesoderm and caudal ectoderm is a single
event that sets up a functional caudal lobe in 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).
cDNA cloning
An Achaearanea germ-band stage embryo cDNA library
(Akiyama-Oda and Oda, 2003) was
used to generate more than 9000 expressed sequence tags (ESTs), which were
sequenced from the 5' end. These include two sequences derived from a
single gene closely related to Drosophila Delta [clone identification
(ID), At_eW_009_P16 and At_eW_018_P16]. A full-length cDNA clone for this gene
was isolated by re-screening the library, its nucleotide sequence was
determined, and the deduced amino acid sequence of the Delta-related
cDNA analyzed by BLASTP (hit against Drosophila Delta with an E-value
of 1e-156), BLAST 2 sequences, ClustalW version 1.81 and PHYLIP version 3.5.
Analysis confirmed that the gene is the closest relative of known
Delta genes, and it was designated At-Delta. The EST
collection also included Notch (clone ID, At_eW_027_D04) and
Suppressor of Hairless [Su(H)] (clone ID,
At_eW_016_A20) homologs, designated At-Notch and
At-Su(H), respectively. The nucleotide sequences of a
partial At-Notch and a partial At-Su(H) cDNA were
determined. The deduced amino acid sequence of the At-Notch cDNA was
hit against Drosophila Notch with an E-value of 4e-147 and that of
the At-Su(H) cDNA against Drosophila Su(H) with an
E-value of 9e-35. A short fragment of a homolog of Drosophila
hedgehog (hh), designated At-hh, was isolated by PCR
with the following primers: forward, 5'-GARGARGGNACNGGNGCNGA-3',
and reverse, 5'-ACCCARTCRAANCCNGCYTC-3'. A full-length
At-hh cDNA clone was isolated by cDNA library screening. Phylogeny of
Hh proteins including At-Hh was analyzed in a previous study
(Simonnet et al., 2004).
Sequences are available from GenBank under the accession numbers:
At-Delta, AB287420; At-Notch, AB287421;
At-Su(H), AB287422; and At-hh, AB125742.
Antibodies
A DNA fragment encoding the carboxyl-terminal 182 amino acids of
Achaearanea Forkhead (At-Fkh)
(Akiyama-Oda and Oda, 2003) was
amplified by PCR and inserted between the EcoRI and SalI
sites of pET-28a(+) vector (Novagen, Madison, WI). Expression of the fusion
protein was induced in BL21 (DE3) cells, and cell lysates were fractionated by
SDS-PAGE and electroeluted from the gel. Purified protein was employed as an
antigen to immunize two guinea pigs. As antiserum from one of the two animals
specifically reacted to bacterially expressed At-Fkh fused to maltose-binding
protein (see Fig. S1 in the supplementary material) and showed nuclear
staining consistent with the expression patterns of At-fkh
transcripts previously reported
(Akiyama-Oda and Oda, 2003;
Akiyama-Oda and Oda, 2006), it
was used for experiments. A commercially available rabbit anti-ß-catenin
antiserum (C2206, Sigma-Aldrich, St Louis, MO) was used.
Staining of embryos
For immunostaining, embryos were fixed as described
(Oda et al., 2005).
Anti-At-Fkh and anti-ß-catenin antisera were diluted 1:1000. Donkey
anti-guinea pig IgG labeled with Cy3 (Chemicon, Temecula, CA) and donkey
anti-rabbit IgG labeled with Cy5 (Chemicon) were used as secondary antibodies
diluted 1:200. Samples were counterstained with 1 U/ml phalloidin-fluorescein
(Molecular Probes, Eugene, OR) and 0.5 mg/ml DAPI (Sigma). Stained samples
were examined with an Olympus IX71 microscope equipped with a cooled CCD
camera (CoolSNAP HQ, Roper Scientific, Tucson, AZ) controlled by MetaMorph
version 6.1 (Universal Imaging, Downington, PA).
Single- and dual-color whole-mount in situ hybridizations were performed as
described (Lehmann and Tautz,
1994; Akiyama-Oda and Oda,
2006), followed by DAPI staining. All stained samples except for
those shown in Fig. 1L,L'
were flat-mounted after removal of excess yolk. Embryos were photographed
using a stereomicroscope (SZX12, Olympus) equipped with a color CCD camera
(C7780-10, Hamamatsu Photonics, Hamamatsu, Japan), and an Axiophot2 (Carl
Zeiss). Cell death was visualized using a TACS 2 TdT-DAB kit (Trevigen,
Gaithersburg, MD).
Parental RNAi
The 1054 bp (nucleotide [nt] 136-1189) and 965 bp (nt 1406-2370) regions of
At-Delta cDNA were used to synthesize At-DeltaEB
and At-DeltaHH dsRNAs. Control dsRNA was synthesized using
the entire coding region of jellyfish green fluorescent protein
(gfp) gene (Quantum), At-Notch dsRNA with a 1190 bp region
(nt 30-1219) of a partial At-Notch cDNA, and
At-Su(H) dsRNA with a 580 bp region (nt 1-580) of a partial
At-Su(H) cDNA. Synthesis and injection of dsRNA were
performed as described (Akiyama-Oda and
Oda, 2006). dsRNAs were used at concentrations of 1.5-2.0
µg/µl for injection. dsRNA solution (1-2 µl) was repeatedly
introduced into the opisthosoma of each female at 2-3 day intervals. The
injection was repeated five times for At-DeltaEB, gfp and
At-Su(H)dsRNA, three (the individuals #7 and #8) or four
(the individual #9) times for At-DeltaHH dsRNA, and six
times for At-Notch dsRNA.
Time-lapse recording of live embryos
After dechorionation with 100% bleach, embryos were attached to glass
coverslips with double-sided sticky tape and covered with halocarbon oil 700
(Sigma). Images were taken every 5 minutes with the above color CCD camera and
processed in MetaMorph version 6.1, ImageJ version 1.32 and Adobe Premiere
version 6.0 to produce the movie.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
This site of cell internalization is referred to as the blastopore
(Fig. 1B,D)
(Montgomery, 1909). We
designated At-fkh-positive cells situated below the central area of
the germ disc epithelium as central endoderm (cEND) cells. Cumulus mesenchymal
(CM) cells, characterized by decapentaplegic (dpp)
expression (Akiyama-Oda and Oda,
2003), appeared to originate as a subpopulation of cEND cells. In
accordance with this, strong ß-catenin concentration at specific sites of
cell-cell contact was observed in the cEND cell cluster at late stage 4
(Fig. 1F, arrow) and in the
migrating CM cell cluster during stage 5 (data not shown). After CM cells left
the center of the germ disc, remaining cEND cells were scattered
(Fig.
1H,J,J',K,K'). The rim of the germ disc (Fig. 1G) was another site of cell internalization. At mid stage 5, but not earlier, some cells expressing At-fkh transcripts were found below the germ disc epithelium close to the rim (Fig. 1I,I'). These internalized cells, which did not express At-twi transcripts (Fig. 1L,L'), were designated peripheral endoderm (pEND) cells. At mid- to late stage 5, some At-fkh-expressing cells located at the rim of the germ disc began to express At-twi transcripts (Fig. 1L,L'). These cells did not enter the inside until CM cells almost reached the rim of the germ disc (late stage 5). At approximately the beginning of stage 6, when the extraembryonic area begins to differentiate (Fig. 1M), an increasing number of At-twi-expressing cells was seen at and near the rim of the germ disc (Fig. 1N). All of these peripheral At-twi-positive cells except cells at the rim were located below the surface epithelium (data not shown). At-twi-positive cells situated below the peripheral epithelium were designated peripheral mesoderm (pMES) cells. After internalization, pMES cells appeared to migrate centripetally (Fig. 1N,T-W).
As previously described (Yamazaki et
al., 2005), some cells around the center of the germ disc, which
were apparently distinct from cEND cells, began to express At-twi
transcripts starting at the beginning of stage 6
(Fig. 1N-Q,O'-Q').
These cells showed an evenly spaced distribution, although many were seen in
pairs. Visualization of nuclei showed that At-twi-positive cells were
at the surface or internalizing (Fig.
1O,O',P,P'). At-twi-positive cells that
situated below the central epithelium were designated central mesoderm (cMES)
cells. New cMES cells continued to arise from an area adjacent to the growing
At-cad expression domain (Fig.
1R-W). Our previous studies showed that in the formed caudal lobe,
in addition to ectoderm cells, some mesoderm cells express At-cad
transcripts and that expression of At-twi transcripts disappears
(Akiyama-Oda and Oda, 2003;
Yamazaki et al., 2005).
Molecular characterization of a Delta homolog in Achaearanea
EST analysis of Achaearanea germ-band-stage embryos identified a
homolog of Drosophila Delta, designated At-Delta. Subsequent
in situ hybridization analysis revealed that the At-Delta expression
pattern was, in part, similar to that of At-twi at around stages 5
and 6 (see below).
The deduced amino acid sequence of At-Delta showed a Delta/Serrate/LAG-2
(DSL) domain and nine EGF-like repeats
(Fig. 2A,B). The overall
protein structure more closely resembled Drosophila Delta than
Drosophila Serrate, and phylogenetic analysis using unambiguously
alignable amino acids in the DSL domain and in EGF-like repeats 1-3 confirmed
this observation (Fig. 2C). It
also showed that At-Delta most closely resembled Cs-Delta1, one of two Delta
homologs reported in another spider, Cupiennius salei
(Fig. 2B,C)
(Stollewerk, 2002).
Expression patterns of At-Delta transcripts
Expression patterns of At-Delta transcripts were examined by
whole-mount in situ hybridization. From before mid-stage 4, At-Delta
transcripts were detected in cells located near, but not at, the rim of the
forming germ disc (Fig. 3A-C).
No expression was detectable around the center of the germ disc at mid-stage 4
(Fig. 3A). In a late-stage 4
embryo, however, several cells located at the rim of the forming germ disc and
all non-germ-disc surface cells expressed At-Delta
(Fig. 3D). In the same embryo,
some surface cells around the blastopore distinct from the cEND cells
expressed At-Delta (Fig.
3D-F). During stage 5, the area displaying interspersed
At-Delta expression expanded (Fig.
3G). At-Delta-expressing cells, many of which were seen
in pairs, were located at the same level as the remaining cells in the
epithelium (Fig. 3H). About a
quarter of the surface cells at the central area displayed high-levels of
At-Delta expression. Both paired and unpaired
At-Delta-positive cells were evenly spaced.
By early stage 6, At-Delta expression began to fade from around the center of the germ disc (Fig. 3I). This process was followed by the appearance of At-twi transcripts, probably in prospective cMES cells (Fig. 3L,M). In support of this idea, cells positive for low levels of both At-Delta and At-twi were found in that area as At-Delta expression faded (Fig. 3M, arrows). Expression of At-cad was observed in the growing At-Delta-negative domain at mid-stage 6 but not early stage 6 (Fig. 3I,J). Changing expression patterns of At-Delta, At-fkh, At-twi and At-cad preceding caudal lobe formation are summarized schematically in Fig. 3N.
A second wave of At-Delta expression was observed in the nascent
caudal lobe at late stage 6/early stage 7
(Fig. 3K). Similar to previous
observations for Cupiennius Delta genes
(Stollewerk et al., 2003), an
increase in the number of stripes of At-Delta expression was observed
in the developing opisthosomal region (Fig.
3O,P). In addition, one relatively broad band of At-Delta
expression became prominent in the prosomal region during early germ band
stages (Fig. 3O,P). In the
limb-extending germ band (stage 9), At-Delta expression was observed
in neural precursors (Fig. 3Q),
as reported for Cs-Delta1
(Stollewerk, 2002).
Parental RNAi against At-Delta results in defects in caudal lobe formation
To examine the function of At-Delta in early spider embryos, we
used parental RNAi. Two types of dsRNA were prepared from non-overlapping
regions of At-Delta cDNA, designated At-DeltaEB
and At-DeltaHH dsRNAs
(Fig. 2A). Females injected
with At-DeltaEB or At-DeltaHH dsRNA,
but not with control gfp dsRNA, produced embryos displaying
morphological defects starting at early stage 6
(Fig. 4A-E; see Movie 1 in the
supplementary material). In affected embryos, an unusual large invagination
was formed around the center of the germ disc, resulting in abnormal
thickening of cells at the emerging caudal region
(Fig. 4B). For most females
injected with At-DeltaEB or At-DeltaHH
dsRNA, the penetrance of this `invagination' phenotype was 100% (excluding
embryos showing non-specific developmental defects) 2 to 3 weeks after the
first injection (Fig. 4D,E).
This condition lasted for another few weeks. Neither the frequency of
egg-laying nor the quality of eggs was significantly affected.
Embryos displaying invagination phenotypes at stage 6 developed into various forms. Many formed either the correct or a reduced number of limbs, but formation of the germ band and extension of limbs were markedly delayed (see Movie 1 in the supplementary material). Phenotypes of late embryos were categorized as normal or consisting of three defective classes (classes 1 to 3; Fig. 5). Despite early defects, embryos of the normal class recovered a normal form (Fig. 5E). The class 1 phenotype showed a reduced opisthosoma with the prosoma developing rather normally (Fig. 5B). In many class 1 embryos, extension of limbs corresponding to the third and fourth walking legs was delayed. The class 2 phenotype displayed a loss of the entire opisthosoma, with the prosomal limbs frequently reduced in number (Fig. 5C). The class 3 phenotype showed a significant reduction of the prosoma with a disorganized germ band configuration, in addition to loss of the entire opisthosoma (Fig. 5D). Based on late phenotypes, the effect of At-DeltaHH dsRNA injection was more significant than that of At-DeltaEB dsRNA injection (Fig. 5E).
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Mesoderm and caudal ectoderm phenotypes of At-Delta RNAi embryos
We further investigated the cause of caudal lobe defects in
At-Delta RNAi embryos using molecular markers of early cell types. In
At-Delta RNAi embryos, the At-fkh-expressing cEND cells,
including CM cells, formed and behaved normally
(Fig. 7A,B). However, the
number of At-fkh-expressing cells at the periphery of the stage 5
germ disc was markedly reduced in At-DeltaHH RNAi embryos
(Fig. 7A,B), but less so in
At-DeltaEB RNAi embryos (data not shown). At stage 6,
there was no significant difference in the expression of At-fkh
between control and At-DeltaEB RNAi embryos
(Fig. 7C,D). By contrast,
expression patterns of At-twi and At-cad were grossly
defective in At-DeltaEB RNAi embryos at late stage 6
(Fig. 7E-H). All cells
participating in abnormal invagination seen in At-Delta RNAi embryos
expressed high levels of At-twi
(Fig. 7F). Conversely, no cells
in the emerging caudal region of At-Delta RNAi embryos expressed
significant levels of At-cad transcripts
(Fig. 7G,H). Finally,
At-Delta RNAi embryos at stage 6 did not show At-twi
transcripts in the peripheral region, as did control embryos
(Fig. 7E,F).
Most At-DeltaEB RNAi embryos formed a bilaterally
symmetrical germ band of reduced length. Such germ bands displayed a rather
normal arrangement of At-twi-expressing cells in most of the prosomal
region, but persistently exhibited a large mass of At-twi-expressing
cells in the caudal region (Fig.
7J). This was in contrast to the normal germ band, in which
At-twi expression is absent from the caudal lobe
(Fig. 7I)
(Yamazaki et al., 2005). In
addition, staining for At-hh showed that
At-DeltaEB RNAi embryos did not generate opisthosomal
segments despite the rather normal segmentation seen in the prosomal region
(Fig. 7K,L). These observations
accounted for the class 2 phenotype. Moreover, TUNEL staining showed that
there were many dying cells in the caudal region of At-Delta RNAi
embryos, but only a few in the caudal lobe of control embryos
(Fig. 7M,N).
Parental RNAi against Notch and Su(H) homologs results in similar phenotypes
To examine whether other components of the Delta-Notch signaling pathway
function in Achaearanea caudal lobe formation, we performed parental
RNAi against At-Notch and At-Su(H). As seen with
At-Delta dsRNAs, injection of At-Notch or At-Su(H)
dsRNA resulted in embryos displaying unusual invagination around the center of
the germ disc and subsequent thickening of cells at the emerging caudal region
(Fig. 8A,B). However, the
penetrance of the invagination phenotype in individual egg sacs after the
injection of At-Notch and At-Su(H) dsRNA was not 100% (see
Fig. S2 in the supplementary material). Nonetheless, expression patterns of
At-Delta, At-twi and At-cad transcripts were affected in
both At-Notch and At-Su(H) RNAi embryos derived from egg
sacs with the high penetrance of the invagination phenotype
(Fig. 8C-H). In these embryos,
the interspersed pattern of At-Delta expression around the center of
the germ disc was partially or completely disrupted, although levels of
At-Delta transcripts were higher than in At-Delta RNAi
embryos (Fig. 8C,D). At later
stages, there was an increased number of At-twi-expressing cells and
a reduced number of At-cad-expressing cells at the caudal region.
This phenotype was similar to that seen in At-Delta RNAi embryos. In
addition, in At-Notch but not At-Su(H) RNAi embryos, many
At-twi-expressing cells probably corresponding to pMES cells were
observed outside the caudal region (Fig.
8E,F), indicative of a difference between At-Delta and
At-Notch RNAi embryos.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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High levels of Delta transcripts are expressed in prospective mesoderm cells arising from around the blastopore
We described five distinguishable cell types that internalize during stages
4-7, namely cEND, CM, pEND, pMES and cMES cells. The first three are
categorized as endoderm, and the last two as mesoderm. CM cells are regarded
as a derivative of the cEND cells. Formation of the Achaearanea
blastopore is associated with cEND cell internalization. Both mesodermal cell
types express At-twi, a homolog of the Drosophila
mesoderm-determining gene, suggesting that Achaearanea mesoderm is
evolutionarily related to Drosophila mesoderm
(Yamazaki et al., 2005).
At-twi-expressing cMES cells appear to originate from multiple sites
around the blastopore independently of cEND cells. This idea is reinforced by
observation of evenly spaced cells expressing At-Delta in the central
area of the germ disc epithelium at earlier stages
(Fig. 3D-I) in combination with
simultaneous detection of At-Delta and At-twi transcripts
(Fig. 3L,M). It is likely that
in normal development, the cells expressing At-Delta begin to express
At-twi and enter the mesodermal pathway.
As is evident by time-lapse microscopy (see Movie 1 in the supplementary material), most surface cells of the germ disc do not significantly change positions during stages 4 and 5. Therefore, altering At-Delta expression patterns in the germ disc is not the result of cell movement but of progressive activation of At-Delta transcription. This process might be governed by a relay of short-range signals, by diffusion of long-range signals, or by graded activity of a maternal transcription factor.
Delta-Notch signaling is required to restrict the number of mesoderm cells
Defects seen after injection of At-DeltaEB,
At-DeltaHH, At-Notch and At-Su(H) dsRNA were
similar to each other, but completely different from those previously obtained
with dsRNAs of two Achaearanea genes, At-dpp and
At-short gastrulation (At-sog)
(Akiyama-Oda and Oda, 2006).
This situation suggests that the defects reveal specific developmental
processes requiring canonical Delta-Notch signaling. Like Cupiennius,
however, Achaearanea might have a second Delta gene and/or a
second Notch gene. The possibility that such genes might have been
additionally silenced in the RNAi experiments is not excluded.
Although it is a useful marker of future cMES cells, At-Delta does
not seem to be necessary for differentiation into mesoderm or internalization.
Defects in At-Delta RNAi embryos suggest that Delta-Notch signaling
restricts the number of cMES cells, a function reminiscent of mechanisms
controlling cell fate decisions in Drosophila early neurogenesis
(Campos-Ortega, 1993). The
evenly spaced but non-stereotyped arrangement of At-Delta-positive
cells in the germ disc epithelium as well as the ratio of
At-Delta-positive to -negative cells could be explained by lateral
inhibition (Meinhardt and Gierer,
1974; Honda et al.,
1990). As the lateral inhibition hypothesis would predict,
suppression of the inhibitory Delta signal as well as of At-Notch and
At-Su(H) function abolishes interspersed expression of
At-Delta and concomitantly causes overproduction of a single cell
type (cMES cells) at the expense of other (caudal ectoderm cells) cell types
(Fig. 7F,H;
Fig. 8E-H). The cells that
receive the At-Delta signal may be inhibited from transcribing
At-Delta and At-twi in response to a different signal and
entering the mesodermal pathway. The effect of blocking Delta-Notch signaling
is that all the germ disc cells in the central area are allowed to transcribe
At-Delta and At-twi and take the mesodermal fate.
|
;
Martín-Bermudo et al.,
1995; Morel and Schweisguth,
2000; Bardin and Schweisguth,
2006; De Renzis et al.,
2006). Thus, in both Drosophila and Achaearanea,
emerging mesoderm cells influence the fate of neighboring cells via
Delta-Notch signaling. It is tempting to speculate that Drosophila
mesectoderm specification and Achaearanea caudal ectoderm
specification share an evolutionary link. This hypothesis would require that
drastic changes in the mechanisms of ventral and caudal patterning occurred
after divergence of the lineages leading to Drosophila and
Achaearanea (Akiyama-Oda and Oda,
2006).
|
|
;
Henrique et al., 1995), in
which the proneural genes promote Delta expression to confer neural competence
(Campuzano and Modolell, 1992;
Kunisch et al., 1994), a
mesoderm-promoting gene(s) might exist upstream of At-Delta.
Interestingly, in the pre-involuted mesoderm of the Xenopus gastrula,
the muscle-determining factor MyoD has been identified as a direct positive
regulator of Delta-1 expression
(Wittenberger et al., 1999).
Another example comes from studies of sea urchin embryos, in which a double
repressor system involving pmar1 activates Delta transcription
(Oliveri et al., 2002;
Revilla-i-Domingo et al.,
2004), which is necessary for secondary mesoderm cell
specification (Sweet et al.,
2002). Moreover, in the stem cell zone of the chick embryo spinal
cord, FGF signaling functions to induce Delta1 expression
(Akai et al., 2005). Future
studies of spider embryos should identify a gene(s) activating
At-Delta transcription around the blastopore.
In the early Drosophila embryo, the highest nuclear concentrations
of the maternal transcription factor Dorsal directly activate zygotic
transcription of the mesoderm-determining gene twi
(Jiang et al., 1991).
Characterization of differences in Drosophila and
Achaearanea gene cascades leading to activation of twi
transcription should also be the focus of future studies.
Delta-Notch signaling is required to induce the caudal ectoderm
Our results show that Delta-Notch signaling is essential to induce and
specify caudal ectoderm in Achaearanea embryos. Caudal ectoderm is
characterized by At-cad expression. Loss of the entire opisthosoma in
embryos showing potent At-Delta RNAi phenotypes may be attributed to
almost complete failure to generate At-cad-expressing caudal ectoderm
cells. Our data indicate that zygotic At-cad transcription is
activated downstream of Delta-Notch signaling. However, as there is a
significant time lag (more than 10 hours) between initial elevation of
At-Delta transcription around the closing blastopore (late stage 4)
and the onset of At-cad transcription at the forming caudal lobe (mid
stage 6), it is unlikely that At-cad is a direct target of
Delta-Notch signaling.
|
|
),
At-cad transcripts were not expressed in overproduced mesoderm cells
in At-Delta RNAi embryos. Although At-twi transcripts
disappear in the normally formed caudal lobe
(Fig. 7I)
(Yamazaki et al., 2005),
overproduced mesoderm cells continued to express At-twi in the
At-Delta RNAi embryos (Fig.
7J). These data suggest that regulation of gene expression in
caudal lobe mesoderm may be influenced by ectoderm.
Opisthosomal defects that we observed in At-Delta RNAi embryos are
due to failure in caudal lobe formation rather than in segmentation.
Curiously, in previous Cupiennius studies, investigators described
morphological abnormalities of the caudal region in embryos injected with
dsRNAs targeted to components of the Delta-Notch signaling pathway
(Stollewerk et al., 2003;
Schoppmeier and Damen, 2005).
The cause of those segmentation defects is unclear, and, in addition, mesoderm
development has been poorly described in Cupiennius. Potential
discrepancies between the Cupiennius and Achaearanea studies
may arise from developmental variations between the species and/or from
different experimental approaches to performing RNAi
(Schoppmeier and Damen, 2001;
Akiyama-Oda and Oda, 2006).
Mesoderm and caudal ectoderm formation are a single event required to establish a normal caudal lobe
We propose that in the process of caudal lobe formation, At-Delta functions
both to prevent cells from adopting the mesoderm fate and to specify caudal
ectoderm fate. These two outcomes may represent interdependent outputs of
Notch-mediated genetic circuits that amplify small differences between
initially equivalent neighboring cells
(Artavanis-Tsakonas et al.,
1999). Therefore, mesoderm and caudal ectoderm formation in
Achaearanea are not independent events but a single one achieved by
Delta-Notch signaling progressively activated in the germ disc epithelium from
around the blastopore. Previous work on Delta-Notch signaling in spider
revived the hypothesis that arthropod segmentation and vertebrate
somitogenesis have a common origin
(Stollewerk et al., 2003;
Tautz, 2004;
Patel, 2003). However, an
unanswered question is how formation of mesodermal somites in vertebrates is
related to arthropod segments consisting of ectoderm and mesoderm. Our finding
that mesoderm and caudal ectoderm form as a single event in spider embryos may
be relevant to this issue.
|
Evolution of early patterning in arthropods
In Drosophila embryos, cascades of maternal and zygotic
transcription factors make a major contribution to anterior, posterior,
terminal and dorsoventral patterning, into which germ-layer specification is
integrated. These patterning systems are achieved in a syncytial environment.
Similar aspects of early patterning have been reported in another insect,
Tribolium (Sommer and Tautz,
1994; Chen et al.,
2000). By contrast, our previous and present studies with the
spider Achaearanea highlight the importance of cell-cell
communication mediated by Dpp and Sog in dorsoventral patterning
(Akiyama-Oda and Oda, 2006) and
by Delta-Notch signaling in mesoderm and caudal specification. The necessity
for cell-cell communication in early spider embryos may be due to early
completion of cellularization (Kondo,
1969; Suzuki and Kondo,
1995). Based on Achaearanea studies, the blastopore
region is particularly important, because cells producing Dpp signals
originate from that region and progressive activation of Delta-Notch signaling
starts there. Possibly, localized sources of intercellular signals, rather
than gradients of maternal transcription factors, play central roles in early
patterning in spider. Further studies with Achaearanea should
contribute to a better understanding of the diversity of arthropod development
and its ancestral mode.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2195/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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