JT Biohistory Research Hall, 1-1, Murasaki-cho, Takatsuki, Osaka
569-1125, Japan
* Tsukita Cell Axis Project, ERATO, JST
PRESTO, JST
Author for correspondence (e-mail:
yasuko{at}brh.co.jp)
Accepted 16 January 2003
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SUMMARY |
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Key words: Spider, Chelicerate, Embryogenesis, Cumulus, dpp/BMP2/BMP4, fork head/HNF3, otd/otx, cad/cdx, Mad/Smad, Body axis formation
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INTRODUCTION |
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Cellular and molecular mechanisms that control early patterning have been
most extensively studied for the insect, Drosophila melanogaster. In
the early Drosophila embryo, AP and DV asymmetries pre-exist as
localized maternal components, which give rise to a region-specific zygotic
expression of genes (St Johnston and
Nüsslein-Volhard, 1992;
Driever, 1993
;
Chasan and Anderson, 1993
). The
DV polarity of the Drosophila embryo comes from a regulated nuclear
localization of the maternal transcription factor Dorsal. A gradient of
nuclear Dorsal concentration along the DV axis subdivides the embryo into a
series of domains with different fates: amnioserosa, dorsal ectoderm, ventral
(neurogenic) ectoderm and mesoderm (Rusch
and Levine, 1996
; Stathopoulos
and Levine, 2002
). The boundaries of these domains are established
by the activities of zygotic genes transcribed at different thresholds of the
Dorsal gradient. One such zygotic gene is decapentaplegic
(dpp), which is expressed in dorsal 40% of the cellular blastoderm
(St Johnston and Gelbart,
1987
). It encodes a secreted protein belonging to the TGFß
superfamily (Padgett et al.,
1987
). The dpp mutant embryo is strongly ventralized
(Irish and Gelbart, 1987
).
Conversely, dpp overexpression expands the dorsal area
(Ferguson and Anderson, 1992a
;
Ferguson and Anderson, 1992b
).
Thus, the Dpp protein acts as a dorsal morphogen in the Drosophila
embryo. In cells that receive the Dpp signal, the cytoplasmic Mothers against
dpp (Mad) protein is phosphorylated by activated Dpp receptors, and
translocated to the nucleus, where phosphorylated Mad (pMad) regulates
transcription of downstream genes (Raftery
and Sutherland, 1999
; Rushlow
et al., 2001
). Another zygotic gene, short gastrulation
(sog), is expressed at lateral regions next to the
dpp-expressing domain
(François et al.,
1994
). This gene encodes an extracellular protein
(François et al., 1994
)
that antagonizes the Dpp activity
(Marqués et al., 1997
;
Ashe and Levine, 1999
). The
dorsal region is established by the relative activities of Dpp, Sog and other
factors (Ashe et al.,
2000
).
Striking similarities in the mechanisms of DV patterning are known between
Drosophila and vertebrates. Vertebrate homologs of dpp and
sog, BMP2/4 and chordin, respectively, function to organize the DV
pattern in a similar fashion although the ventral side of vertebrates
corresponds to the dorsal side of Drosophila
(Holley et al., 1995;
De Robertis and Sasai, 1996
;
Ferguson, 1996
;
Holley and Ferguson, 1997
).
Combined with the distant phylogenetic relationship between
Drosophila and vertebrates, it is generally thought that the origin
of the DV axis is shared by most bilaterians. However, there are very few
studies investigating how the embryonic DV axis is specified in bilaterian
animals other than insects and vertebrates. Although homologs of
dpp/BMP2/4 have been isolated in a wide range of metazoans, including
the amphioxus, ascidian, sea urchin, gastropod, planarian and coral
(Miya et al., 1997
;
Panopoulou et al., 1998
;
Orii et al., 1998
;
Angerer et al., 2000
;
Darras and Nishida, 2001
;
Hayward et al., 2002
;
Nederbragt et al., 2002
),
developmental analyses on these genes have not revealed plausible scenarios
for the evolution of DV axis formation in the metazoans. It may be important
to try to figure out the ancestral mode of DV patterning for the respective
phyla or classes.
In non-insect arthropods, there is only limited knowledge concerning
cellular and molecular mechanisms of early patterning. Molecular phylogenetics
suggests that within the Arthropoda the Chelicerata are the living group
phylogenetically most distant from the Insecta
(Friedrich and Tautz, 1995;
Hwang et al., 2001
;
Giribet et al., 2001
;
Cook et al., 2001
). Comparative
analysis is expected to provide data for understanding the ancestral
developmental mechanisms of the arthropods. An increasing number of
developmental genes have been isolated and examined in the spider and other
chelicerates, illuminating similarities and differences between
Drosophila and chelicerates
(Telford and Thomas, 1998a
;
Telford and Thomas, 1998b
;
Damen and Tautz, 1998
;
Damen et al., 1998
;
Damen et al., 2000
; Abzhanov
et al., 1999; Stollewerk et al.,
2001
; Damen, 2002
;
Dearden et al., 2002
).
In this study, in order to compare the mechanisms patterning the AP and DV
axes of Drosophila and spider embryos, we used Achaearanea
tepidariorum, which is easily accessible to analyzing the early
embryogenesis. First, we describe a cluster of mesenchymal cells at the
cumulus, which migrate from the center to the rim of the germ disc resulting
in the transition from radial to axial symmetry. Next, we isolated spider
genes homologous to Drosophila early patterning genes, dpp, fork
head (fkh), orthodenticle (otd; oc
FlyBase) and caudal (cad), and examined their expressions.
In the Drosophila cellular blastoderm, dpp is expressed to
specify the dorsal part (Irish and
Gelbart, 1987; St Johnston and
Gelbart, 1987
; Ferguson and
Anderson, 1992a
; Ferguson and
Anderson, 1992b
), and fkh, otd and cad are
expressed to specify the anterior and posterior terminal domains
(Weigel et al., 1989
;
Cohen and Jürgens, 1990
;
Finkelstein and Perrimon,
1990
; Macdonald and Struhl,
1986
; Wu and Lengyel,
1998
). The expression patterns of the spider genes helped
understand the early development of the embryo. We found that the mesenchymal
cells at the cumulus expressed the dpp homolog. Furthermore, using
the crossreacting antibody against phosphorylated Mad (pMad), we showed that
the Dpp signal was received by germ disc epithelial cells. We suggest that at
the cumulus, progressive mesenchymal-epithelial cell interactions involving
the Dpp-Mad signaling cascade generate DV polarity in accordance with the AP
axis formation. Our findings offer molecular support for the functional
importance of the cumulus in the axial pattern formation of the spider
embryo.
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MATERIALS AND METHODS |
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Scanning electron microscopy
Spider eggs were dechorionated with commercial bleach, followed by careful
washing with distilled water. For fixation, the eggs were incubated for
several hours or over night in a two-phase solution of heptane, 4%
paraformaldehyde and 0.7% glutaraldehyde in C & G's balanced saline (55 mM
NaCl, 40 mM KCl, 15 mM MgSO4, 5 mM CaCl2 and 10 mM
Tricine, pH 6.9) at 4°C. The fixative was replaced by phosphate-buffered
saline (PBS) with 0.1% Tween20 (PBS-Tween), and the vitelline membranes were
removed with forceps and glass needles. Some of the samples, used to observe
the inside of the egg, were cut by a razor blade, followed by a removal of the
yolk. Then, the samples were post-fixed with 2.5% glutaraldehyde in PBS for 1
hour or longer. After several washes with PBS, they were dehydrated through an
ethanol series, followed by a replacement with t-butylalcohol. They
were then critical-point dried using a freeze-drying device, JFD-300 (JEOL),
sputtered with an ion sputtering device, JFC-1500 (JEOL), and examined at 10
kV in a scanning electron microscope (SEM), JSM-5300LLV (JEOL). Photographs
were taken with Polapan 572 (Polaroid).
Phalloidin staining
Eggs were fixed in the same fixative as used for the SEM preparation, or in
a two-phase fixative of heptane and 4% paraformaldehyde in PBS for several
hours or overnight at 4°C. After the removal of the vitelline envelopes,
the eggs were stained with 1 U/ml phalloidin-fluorescein (Molecular Probes).
The eggs fixed in the gulutaryaldehyde-containing fixative were structurally
well preserved, but stained weakly as compared with those fixed in the other.
Some of the samples were counterstained with 1 µM TOTO-3 (Molecular
probes). The stained samples were observed under a Zeiss Axiophoto 2
microscope equipped with a BioRad laser confocal system (MRC1024).
cDNA cloning
To isolate spider homologs of the Drosophila genes, dpp, fkh,
otd and cad, we initially performed degenerate PCR. Primers used
for amplification of the genes were as follows:
cDNA prepared from segmentation stage embryos was used as a template for the PCR amplifications. The cycles of PCR for dpp were: one cycle of 95°C for 5 minutes, 55°C for 2 minutes 30 seconds and 72°C for 40 seconds; and 35 cycles of 95°C for 40 seconds, 55°C for 40 seconds and 72°C for 40 seconds. For amplification of fkh, otd and cad, the annealing temperatures were changed to 45°C, 50°C and 50°C, respectively. Amplified fragments were subcloned using a TA-cloning® kit dual promoter (Invitrogen) and sequenced. Among them, candidates for the spider homologs of the Drosophila genes were found.
For cDNA library construction, two pools of polyA+ RNA were prepared from three egg sacs containing embryos at different stages and just hatched prelarvae of Achaearanea tepidariorum using a QuickPrepTM Micro mRNA Purification Kit (Amersham Pharmacia Biotech). From each RNA pool, oligo-dT primed cDNA libraries were constructed using SuperScriptTM Lamda System for cDNA Synthesis and Cloning (Gibco BRL) and Gigapack®III Gold Packaging Extract (Stratagene).
For library screening, digoxigenin (DIG)-labeled DNA probes for the PCR-amplified fragments were made using a PCR DIG Probe Synthesis Kit (Roche). The embryo and prelarvae cDNA libraries were screened with the probes in a high stringent condition to obtain full-length cDNAs for the candidate genes. Both strands of representative cDNA clones were sequenced and their open reading frames were determined. As shown in the text, the isolated genes were concluded to be A. tepidariorum orthologs of Drosophila dpp, fkh, otd and cad, which were designated At.dpp, At.fkh, At.otd and At.cad, respectively. The sequences are available from the DNA data bank of Japan (DDBJ) with the following Accession Numbers: At.dpp, AB096072; At.fkh, AB096073; At.otd, AB096074; At.cad, AB096075.
Phylogenetic analysis
The deduced amino acid sequences of the C-terminal region of the Dpp
protein, the winged-helix region of Fkh and the homeodomains of Otd and Cad
were manually aligned with the corresponding amino acid sequences of proteins
from other species that were found using the BLAST search. Aligned sequences
were used to construct phylogenetic trees by the neighbor-joining method
(Saitou and Nei, 1987) using
PHYLIP.
In situ hybridization and antibody staining
RNA probes for in situ hybridization were prepared using T7 RNA polymerase
(Gibco BRL, or Stratagene) and DIG RNA Labeling Mix (Roche) according to the
standard method. After dechorionation with bleach, embryos were fixed in a
two-phase solution of heptane and 5.5% formaldehyde in PEMS (100 mM PIPES, 1
mM EDTA, 2 mM MgSO4, pH 6.9). After fixation, the embryos were
washed with PBS-Tween, followed by gradual replacement with methanol, and then
the vitelline membranes were removed with forceps and glass needles.
Alternatively, the vitelline membranes were removed in PBS-Tween before
replacement with methanol. The former procedure protected the yolk mass from
destruction, but the cumulus was hardly visible in the resultant samples. To
observe the cumulus carefully, we followed the latter procedure, which allowed
us to remove the yolk mass with the germ disc preserved. In the resultant
samples, the cumulus was easily visible even after staining. Hybridization,
washes and detection were performed in the same way as those for the
Drosophila embryos (Lehmann and
Tautz, 1994). For antibody staining, embryos were prepared in the
same way as for the in situ hybridization staining. PS1 antibody raised
against the phosphorylated human Smad1 C-terminal peptide
(Persson et al., 1998
) was
used at a dilution of 1:500 to detect pMad protein. For the secondary
antibody, biotin-conjugated antirabbit IgG (Amersham) was used at a dilution
of 1:200. For detection, the elite ABC peroxidase kit (Vectastain) was used.
For simultaneous detection for At.dpp RNA and the pMad protein, the
in situ hybridization staining was followed by the antibody staining.
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RESULTS |
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Marked differences in morphologies were also observed among the epithelial
cells. The epithelial cells located in the trail of the CM cells extended
long, thin cytoplasmic projections from their basal side onto the surface of
the CM cells (Fig. 2B, thin
arrows). The epithelial cells positioned very close to the CM cells had short,
thick projections. These cytoplasmic projections were faintly detectable by
phalloidin-fluorescein (Fig.
2F). These observations remind one of the cytonemes described in
Drosophila larval imaginal discs
(Morata and Basler, 1999;
Ramírez-Weber and Kornberg,
1999
; Ramírez-Weber and
Kornberg, 2000
). In most other epithelial cells of the germ disc,
no cytoneme-like projections were found
(Fig. 2C).
Cloning of spider homologs of Drosophila dpp, fkh, otd and
cad
To search for developmental genes expressed in early spider embryos, we
isolated cDNA clones for A. tepidariorum homologs of Drosophila
dpp, fkh, otd and cad. Details for each cDNA clone are described
below and in the Materials and Methods. Phylogenetic trees were constructed
from the alignments shown in Fig.
3 (see Fig. S1 at
http://dev.biologists.org/supplemental/).
|
At.fkh
Drosophila fkh is a winged-helix domain (forkhead domain)
containing transcription factor expressed in several embryonic tissues such as
hindgut, stomodeum and yolk (Weigel et
al., 1989). The cDNA clone isolated from the spider predicts a
protein of 406 amino acids which contains a winged-helix domain 82% identical
to that of Drosophila Fkh and 83% to that of zebrafish Axial. The
protein is more closely related to Drosophila Fkh than to other
Drosophila winged-helix domain-containing proteins
(Fig. 3B). In addition to the
winged-helix domain, two other short domains at the C termini, which were
conserved in most of the known Fkh/HNF3 class proteins
(Lai et al., 1991
), were found
in the spider protein (not shown). Phylogenetic analysis based on the
winged-helix domains confirmed that the isolated gene was an ortholog of
Drosophila fkh. It was designate as At.fkh.
At.otd
Drosophila otd encodes a Paired-type homeodomain protein, which is
expressed in the head region (Finkelstein
et al., 1990; Cohen and
Jürgens, 1990
;
Finkelstein and Perrimon,
1990
). The cDNA clone isolated from the spider encodes a protein
of 303 amino acids that contains a homeodomain. This homeodomain closely
resembles those of Drosophila and other animal Otd/Otx proteins, but
less closely resembles those of Drosophila Orthopedia (Otp), Paired
(Prd) and Gooseberry (Gsb) (Fig.
3C). The C-terminal sequence of six amino acids, LKFESL, has an
affinity to the sequence, W(K/R)FQVL, which is found at the C termini of most
of the known Otd/Otx proteins except insect Otd proteins. Phylogenetic
analysis based on the homeodomains confirmed that the isolated gene was an
ortholog of Drosophila otd. It was designated as At.otd.
At.cad
Drosophila cad encodes a homeodomain protein that is expressed
from the earliest stage of embryogenesis and is required for normal posterior
development (Mlodzik et al.,
1985; Macdonald and Struhl,
1986
; Mlodzik and Gehring,
1987
; Wu and Lengyel,
1998
). The cDNA isolated from the spider encodes a protein of 300
amino acids that contains a homeodomain. This homeodomain is highly similar to
those of Drosophila and other animal Cad/Cdx proteins, but less
similar to those of Drosophila Deformed (Dfd), Sex comb reduced (Scr)
and Antennapedia (Antp) (Fig.
3D). Phylogenetic analysis based on the homeodomains confirmed
that the isolated gene was an ortholog of Drosophila cad. It was
designated as At.cad.
Expression of dpp in the CM cells
We examined the expression patterns of the isolated genes in stage 4-7
embryos by whole-mount in situ hybridization. Transcripts for At.dpp
were not detected in stage 4 embryos, but were detected in stage 5 embryos.
The signal was seen as a spot in the germ disc. The spot of At.dpp
expression, which was found at different positions from embryo to embryo
(Fig. 4A-D), corresponded to
the position of the cumulus. The variation is probably due to variation in the
age of the embryos. Even in embryos with the cumulus positioned almost at the
center of the germ disc, At.dpp transcripts were already detectable
(Fig. 4A,B), although we are
not sure whether the At.dpp signal was asymmetric or not with respect
to the position of the blastopore. Magnified images showed that
At.dpp transcripts were expressed in the CM cells, but not in the
surface epithelial cells (Fig.
4E,F). The number of At.dpp-positive cells was
9.3±2.0 (n=16), which was comparable with the number of CM
cells counted in the embryos stained for F-actin and DNA (see above).
At.dpp transcripts were probably expressed in all the CM cells. At
late stage 6, when the cumulus disappeared, the spot of At.dpp
expression was not detected (not shown). At later stages, At.dpp
began to be expressed in developing limb buds
(Fig. 4G), and the expression
persisted at the extending limbs (Fig.
4H). This At.dpp expression resembles that of
dpp homologs in insect limbs
(Sanchez-Salazar et al., 1996;
Niwa et al., 2000
;
Dearden and Akam, 2001
),
suggesting a conserved function of dpp in limb bud formation between
the insects and spiders.
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DISCUSSION |
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We showed that the CM cells, but not germ disc epithelial cells, expressed
At.dpp transcripts (Fig.
4E,F). Two lines of evidence suggest that the germ disc cells
receive the Dpp signals from the CM cells. First, the nuclei of germ disc
cells near the CM cells represented graded levels of pMad
(Fig. 5). In
Drosophila and vertebrates, phosphorylation and nuclear translocation
of Mad/Smad proteins are shown to be caused by the activated specific
receptors for Dpp/BMP ligands (Raftery and
Sutherland, 1999). During migration, the CM cells were always
centered in the pMad-positive circular region
(Fig. 5), implying that the
appearance of pMad in the germ disc epithelium depended on the CM cells.
Second, the germ disc epithelial cells directly contacted the CM cells by
using cytoneme-like projections, which were extended from the basal side of
the epithelial cells onto the surface of the CM cells
(Fig. 2B). In
Drosophila larval imaginal discs, the cytonemes, which are projected
from cells receiving signals, are thought to be the place for cell-cell
communication (Morata and Basler,
1999
; Ramírez-Weber and
Kornberg, 1999
;
Ramírez-Weber and Kornberg,
2000
). Similar to this case, the germ disc epithelial cells were
likely to use cytoneme-like projections to receive the Dpp signals from the CM
cells. Taken together, we suggest that the cumulus is a place for
mesechymal-epithelial cell interactions that are involved in the pattern
formation of the spider embryo.
Nuclear pMad/Smad drives transcription of specific downstream genes
(Raftery and Sutherland, 1999;
Rushlow et al., 2001
). The
expression of At.fkh in an area of the germ disc associated with the
migrating CM cells (Fig. 6E,F)
appeared to overlap that of pMad (Fig.
5). This suggested that At.fkh is a potential downstream
target gene for the Dpp-Mad signaling cascade. The At.fkh-expressing
cells at the cumulus, spreading over the yolk during stage 6 and 7
(Fig. 6G,H), appeared to
contribute to extra-embryonic tissue.
Similarities and differences in body axis formation of the
Drosophila and spider embryos
Expression patterns of spider homologs of Drosophila
region-specific genes, shown in this study, offer molecular clues for
determining homologous domains in the early spider and Drosophila
embryos (Fig. 8). In the
Drosophila cellular blastoderm, otd is expressed at a region
close to the anterior end (Finkelstein and
Perrimon, 1990), whereas At.otd was expressed at a
peripheral region of the germ disc encircled along the equator of the spider
egg (Fig. 7A,B). Of these
At.otd-expressing cells, cells around the cumulus might lack the
At.otd expression (see above), but the remaining
At.otd-expressing cells likely migrated circumferentially during
stage 6 (Fig. 7C,F; see Movie 1
at
http://dev.biologists.org/supplemental/)
and settled to the anterior region of the germ band
(Fig. 7D). Drosophila
cad is expressed at a region close to the posterior end in the cellular
blastoderm (Macdonald and Struhl,
1986
), whereas At.cad was expressed in the caudal lobe
(Fig. 7G,H), which was derived
from the central area of the germ disc (see Movie 1 at
http://dev.biologists.org/supplemental/).
These results, together with previous observations of spider embryos
(Holm, 1940
;
Holm, 1952
;
Seitz, 1966
), strongly suggest
that the peripheral region of the germ disc corresponds to the anterior end of
the Drosophila embryo, and the central region corresponds to the
posterior end. It is possible that in the spider germ disc, the AP positional
information pre-exists as a series of concentric circles. Based on this
topology, At.fkh-expressing cells were located at the future anterior
and posterior ends of the stage 5 embryo
(Fig. 6B,C), similar to the
pattern of fkh expression in the Drosophila cellular
blastoderm (Weigel et al.,
1989
) and in the early embryo of other insects, Bombyx
and Tribolium (Kokubo et al.,
1996
; Schröder et al.,
2000
). As the fkh-expressing cells become foregut and
hindgut in these insects (Weigel et al.,
1989
; Kokubo et al.,
1996
; Schröder et al.,
2000
), the two populations of At.fkh-expressing cells are
probably fated to be gut precursors.
|
Drosophila dpp and probably other insect dpp homologs are
involved in DV patterning of the embryo. dpp is expressed in the
dorsal ectoderm during the germband extending stages, and this expression
probably contributes to pattern formation within segments, such as positioning
of the limb buds (Sanchez-Salazar et al.,
1996; Niwa et al.,
2000
; Dearden and Akam,
2001
). This role of dpp is probably conserved between the
insects and spiders, as suggested by the later expression of At.dpp
(Fig. 4G,H). At least in
Drosophila, however, the earliest function of dpp is the
specification of the DV pattern in the cellular blastoderm, not within
segments of the germband (Ferguson and
Anderson, 1992a
; Rusch and
Levine, 1996
). The most dorsal region of the cellular blastoderm,
where dpp is expressed at the strongest level, becomes
extra-embryonic tissue (the amnioserosa), and the next regions become dorsal
ectoderm in Drosophila. In the spider, the area of the germ disc that
the At.dpp-expressing CM cells have directly influenced during their
migration (Figs 1,
2,
5) develops into dorsal
structures including the extra-embryonic tissue. These may suggest a conserved
function of dpp for dorsal fate specification of the early embryo.
The expression of At.dpp in the CM cells
(Fig. 4) is probably comparable
with the dorsal expression of dpp in the Drosophila cellular
blastoderm, and might be related to the expression of dpp in an early
cell population fated to be extra-embryonic tissue in more basal insect
embryos (Sanchez-Salazar et al.,
1996
; Dearden and Akam,
2001
).
In the Drosophila blastoderm the expression of dpp, as
well as some other zygotic genes involving DV patterning, is initiated in an
asymmetric manner according to the gradient of the nuclear Dorsal protein
peaking at the most ventral region (Rusch
and Levine, 1996; Stathopoulos
and Levine, 2002
). In the spider embryo, however, the early
detectable asymmetry was in the migration of the At.dpp-expressing CM
cells rather than the expression of At.dpp itself. What mechanisms
regulate the CM cell migration? The answer to this question may be the key to
the developmental origin of the DV axis of the spider. A localized cue(s) that
attracts or repulses the CM cells might pre-exist on the germ disc.
Alternatively, the CM cells might sense only the AP positional information. In
the latter case, the direction of the cell migration is determined at the
start point randomly, or according to a local unevenness. Further molecular
investigations are needed to find the earliest asymmetries potentially present
in the germ disc and primary thickening.
Two important differences concern the Dpp signal between
Drosophila and the spider (Fig.
8). First, the Dpp signal is produced and transduced within the
surface epithelial cells in the Drosophila embryo
(Dorfman and Shilo, 2001), in
contrast to the spider embryo, in which the Dpp signal is produced by the
mesenchymal cells (the CM cells) (Fig.
4), and is transmitted to the surface epithelial cells
(Fig. 5). Second, the
activation of the Dpp-Mad signaling pathway takes place simultaneously along
the AP axis in the Drosophila embryo
(Dorfman and Shilo, 2001
), but
took place progressively from the center to the rim of the germ disc in the
spider embryo (Figs 4,
5). These differences have
implications for evolutionary change in the mechanism governing DV axis
formation.
dpp/BMP2/4 class genes have been identified in non-chelicerate
animals, including insects and non-arthropod invertebrates, and their
expression patterns have been examined in early embryos
(Sanchez-Salazar et al., 1996;
Miya et al., 1997
;
Panopoulou et al., 1998
;
Angerer et al., 2000
;
Niwa et al., 2000
;
Darras and Nishida, 2001
;
Dearden and Akam, 2001
;
Hayward et al., 2002
).
However, of the cells expressing the dpp/BMP2/4 homologs in early
embryos, none resembles the spider CM cells in morphology and behavior. Within
the Chelicerata, the horseshoe crab
(Sekiguchi, 1973
), as well as
many spider species (Montgomery,
1909
; Holm, 1940
;
Holm, 1952
;
Seitz, 1966
), shows a cellular
thickening similar to the cumulus in early embryogenesis. Even in early
embryos of myriapod species, a cumulus-like cell mass has been reported
(Heymons, 1901
;
Sakuma and Machida, 2002
). In
these cellular structures, cells that correspond to the CM cells described in
this study might be present. Studies on dpp homologs will help to
determine whether they are homologous to the spider cumulus. To understand the
ancestral mode of DV patterning for the arthropods, the evolutionary and
developmental origin of the CM cells is an important subject to be studied.
For this purpose, crustaceans, which are suggested to be a sister group of
insects (Friedrich and Tautz,
1995
; Hwang et al.,
2001
; Giribet et al.,
2001
; Cook et al.,
2001
), are not negligible. Early patterning of crustacean embryos
at the cellular level is now being studied
(Wolff and Scholtz, 2002
;
Gerberding et al., 2002
).
Regulative development of the early spider embryo
If it is assumed that common cellular and molecular mechanisms govern the
early development of the spiders, Agelena labyrinthica and
Achaearanea tepidariorum, the organizing activity of the cumulus
demonstrated by Holm (Holm,
1952) might be explained on the basis of our findings. He removed
the cumulus resulting in ventralized embryos with the AP pattern retained to
some extent. Loss of the source of Dpp signals might account for the
ventralization. Transplanting a part of the cumulus ectopically resulted in
twin embryos. Two moving sources of Dpp signals might set up two separate
fields defined by positional values in the germ disc. Combining our findings
and Holm's experimental data, it is strongly suggested that the cumulus plays
a central role in the axial pattern formation of the spider embryo. Our
molecular evidence clearly indicates that the spider cumulus is not homologous
to the organizing center of vertebrate embryos, in which the activity of
Sog/chordin, the Dpp/BMP2/4 antagonist, is dominant
(Sasai et al., 1994
). Studies
on spider homologs of Sog/chordin are needed for a better understanding of DV
patterning in the spider embryo.
The experiments by Holm (Holm,
1952) and experiments on other spiders
(Sekiguchi, 1957
;
Seitz, 1966
;
Seitz, 1970
), indicate that
the spiders adopt more regulative development than Drosophila. Like
the spiders, the development of the horseshoe crab
(Itow et al., 1991
) and some
insects (Sander, 1976
) seems
to be regulative. Probably in these arthropod species, the axial pattern
formation largely relies on cell-cell interactions. In this study, we showed
Dpp-mediated interactions between mesenchymal and epithelial cells at the
cumulus. Probably, subsequent cell-cell interactions specify the fates of
individual cells in the germ disc. Further investigations on patterning genes
in the spider embryo will clarify the molecular basis for the regulative
development of chelicerate embryos, which may give hints about the ancestral
mode of arthropod development.
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ACKNOWLEDGMENTS |
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Footnotes |
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