1 Department of Biology, University of Western Ontario, London, Ontario N6A 5B7,
Canada
2 Southern Crop Protection and Food Research Center, Agriculture and Agri-Food
Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada
* Current address: Biochemistry Department, University of Otago, PO Box 56,
Dunedin, New Zealand
Author for correspondence (e-mail:
mgrbic{at}uwo.ca)
Accepted 6 August 2002
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SUMMARY |
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Key words: Pair-rule, Chelicerate, Evolution, Parasegment, Segmentation, runt, pax3/7
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INTRODUCTION |
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Studies of Drosophila development have provided details of the
genetic interactions that underlie the segmentation of this insect. The
anterior/posterior patterning process in Drosophila is initiated by
the localized deployment of maternal proteins that trigger downstream genetic
hierarchies, including the gap, pair-rule and segment-polarity classes of
genes (St Johnston and
Nüsslein-Volhard, 1992). Gradients of maternal transcription
factors activate gap genes in non-periodic domains. Overlapping domains of gap
genes activate pair-rule gene expression in domains that represent the first
signs of segmentation. Pair-rule genes act as intermediates between the
non-periodic expression of gap genes and the segmentally repeated expression
of segment polarity genes. Drosophila embryos mutant for pair-rule
genes exhibit pattern defects that affect adjacent segments in different ways.
These genes thus regulate patterning with a dual segment, rather than
segmental, periodicity. This system of defining repeated territories that
undergo further subdivision (re-segmentation) led to the recent hypothesis
that arthropod segments form by subdivision of primary segments (eosegments)
into terminal segments (merosegments)
(Minelli, 2001
). This scenario
places the pair-rule mechanism at the crux of arthropod segmentation, implying
that some form of pair-rule `logic' is shared by all arthropod groups.
Pair-rule genes were initially isolated in a Drosophila mutant
screen for pattern formation genes
(Nüsslein-Volhard and Wieschaus,
1980). The original mutant screen isolated seven genes that
exhibit a pair-rule phenotype including hairy (h), runt (run),
even-skipped (eve), fushi tarazu (ftz), odd-paired (opa), odd-skipped (odd),
paired (prd) and sloppy paired (slp). Subsequently, additional
genes have been isolated that produce pair-rule phenotypes when mutated
(Tang et al., 2001
;
Baumgartner et al., 1994
;
Levine et al., 1994
). These
genes come to be expressed in a canonical `pair-rule' fashion, in seven
stripes of cells that run across the embryo, associated with every second
segment. In addition to the `pair-rule' expression domains, many of these
genes show secondary, segmental expression.
Studies of engrailed protein expression in insects, crustaceans
(Patel et al., 1989) and
chelicerates (Telford and Thomas,
1998
) have implied that the segment polarity gene network is
probably conserved across arthropods. This observation is supported by the
expression of wingless in insects
(Dearden and Akam, 2001
;
Nagy and Carroll, 1994
) and
crustaceans (Nulsen and Nagy,
1999
). Computer modelling of the molecular interactions in the
segment polarity network (von Dassow et
al., 2000
) have implied it is robust to changes in its activation
conditions, possibly explaining its evolutionary conservation. Conservation of
the pair-rule cascade has been more controversial, but recent studies provide
evidence that it may be conserved in insects. Amongst holometabolous insects,
evidence for pair-rule patterning has been found in coleopterans
(Brown et al., 1994
;
Brown et al., 1997
;
Patel et al., 1994
;
Schroder et al., 2000
),
dipterans (Rohr et al., 1999
),
lepidopterans (Kraft and Jackle,
1994
) and hymenopterans (Binner
and Sander, 1997
; Grbi
and Strand, 1998
). The only exceptions appear to be two derived
parasitic wasps, that do not express a homologue of the Even-skipped protein
in a diagnostic pair-rule pattern
(Grbi
et al., 1996
;
Grbi
and Strand,
1998
).
Among hemimetabolous insects, the expression patterns of pair-rule genes
have been examined in grasshopper, earwig, cricket and cockroach. In the
earwig, cricket and cockroach, an Eve homologous protein is not expressed in a
pattern consistent with pair-rule function, though it is expressed in
segmental stripes (Corley et al.,
1999; Davis and Patel,
1999
) raising the possibility that eve was expressed
segmentally in the ancestors of insects. In grasshoppers neither ftz
nor eve are expressed in stripes
(Dawes et al., 1994
;
Patel et al., 1992
). These
findings led to the proposal that pair-rule patterning may have evolved only
in holometabolous insects (French,
1996
). Recent studies, however, have demonstrated that a Pax group
III gene (PgIII), pairberry 1 (pby 1), is expressed in the
grasshopper in a pattern consistent with a pair-rule function, indicating a
pair-rule mechanism does function in the segmentation of this insect
(Davis et al., 2001
).
If pair-rule patterning is conserved in insects, is it present in more
distant groups of arthropods? The expression patterns of three pair-rule gene
homologues have been examined in the spider, Cupiennius salei
(Damen et al., 2000). In this
species, these genes are expressed during segmentation, but, owing to a lack
of segmental markers, it is difficult to interpret the patterns seen. The
expression pattern of a fushi-taratzu homologous gene has been
examined in a mite (Archegozetes longisetosus), but it is not
expressed in a pattern indicating a role in segmentation
(Telford, 2000
). To determine
if pair-rule patterning is an ancient and conserved feature of arthropod
development, we have examined the expression of two pair-rule genes in the
spider mite Tetranychus urticae. Spider mites are chelicerates, an
arthropod class that includes spiders, mites, scorpions and horseshoe crabs.
Recent phylogenetic inferences imply that chelicerates are the sister group of
myriapods, with insects and crustaceans forming a more distant clade
(Cook et al., 2001
;
Giribet et al., 2001
;
Hwang et al., 2001
). A fossil
chelicerate, dated to the middle Cambrian (520-512 MYA) has been identified,
demonstrating that the separation between the crustacean/insect clade and
chelicerates is an ancient one (Briggs and
Collins, 1988
). Thus, the great evolutionary distance between
spider mites and Drosophila implies that any developmental pathway we
find common to both species is likely to be conserved and ancestral for all
arthropods.
Here we describe the embryogenesis of T. urticae and analyse the expression of homologues of the Drosophila pair-rule genes run and prd. In T. urticae, a homologue of prd is expressed in stripes that appear first in even numbered segments, and then in odd numbered segments, implying that a pair-rule mechanism may underlie segmentation in this species. The early expression pattern of a run homologous gene, however, deviates greatly from the Drosophila pattern, being expressed in circular domains that delimit the limb primordia. These data imply that significant changes in the expression patterns of pair-rule homologous genes have evolved over 520 million years.
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MATERIALS AND METHODS |
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Embryo micro-injection
Spider mite embryos for micro-injection were individually picked off leaves
using fine forceps under a dissecting microscope and placed in a drop of
paraffin oil (Sigma) on a microscope slide. Slides were placed on a Zeiss
Axiovert microscope. Embryos were steadied with negative pressure through a
holding pipette. Holding pipettes were produced by the methods of Hogan et al.
(Hogan et al., 1986) using a
Narashige microforge. Embryos were injected with tetramethylrhodamine dextran
(3x103 Mr, anionic, lysine fixable) using
fine needles pulled from borosilicate glass on a Stutter needle puller.
Embryos were left to recover for 30 minutes and then imaged using a Zeiss
laser scanning confocal microscope.
Molecular cloning and sequence analysis
Spider mite poly(A)+ RNA was extracted using a Quickprep mRNA
purification kit (Amersham-Pharmacia Biotech). A directional cDNA library was
produced from mixed embryonic-stage poly(A)+ RNA using the Zap
Express system (Stratagene). Random colonies were picked from mass-excised
plasmid clones from this library. Plasmid DNA was extracted using a QIAprep 96
Turbo Miniprep Kit (Qiagen) on a Biomek 2000 Robot (Beckman). Clones were
sequenced from their 5' end using T3 primer. Sequencing was performed
using Big Dye chemistry (ABI) on a Perkin Elmer 377 DNA sequencer.
Spider mite DNA was extracted using a QIAquick Kit (Qiagen). Degenerate PCR
for Tu-pax3/7 was performed using the methods of Davis et al.
(Davis et al., 2001).
Tu-run degenerate PCR was performed using the following primers:
RCNRYNATGAARAAYCARGTNGC (runt 5') and MRNTTYAAYGAYYTNMGNTTYGTNGG (runt
3'). PCR products were cloned by ligation into a linearised Bluescript
vector with terminal overhanging thymine residues.
Sequences were assembled using SeqMan from the DNASTAR suite of programs
and homology assessed using translated BLAST (BlastX) searches
(Altschul et al., 1990).
Multiple alignments were created using Clustal X
(Thompson et al., 1994
), and
Maximum likelihood analysis performed using TreePuzzle
(Strimmer and von Haeseler,
1996
).
Embryo staining
Antibody staining was performed as described previously
(Patel, 1994), using an
antibody raised against Drosophila Distal-less (Dll) described by
Panganiban et al. (Panganiban et al.,
1995
).
DIG-labelled probes were produced and in situ hybridisation carried out
according to the methods of Dearden and Akam
(Dearden and Akam, 2000).
Images were collected using a Sony DXC-390P camera mounted on a Zeiss Axioplan
II microscope and processed using Photoshop (Adobe).
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RESULTS |
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Spider mite females lay a spherical, 150 µm egg with little internal morphology (Fig. 1A). Over the course of the first hour after egg laying (AEL), a central nucleus becomes visible. The egg then undergoes nine divisions, approximately one per hour, creating a blastoderm with a layer of cells surrounding a yolk filled centre (Fig. 1B-D; http://devbiol.zoo.uwo.ca/movies/smite_early_cleavages_mov.mov.). From the first division, cell membranes are visible between the nuclei.
The blastoderm remains static for 12-14 hours with no changes in
morphology. A small swelling of blastoderm cells then appears, internally, on
one side of the egg, which we take to be the `germ disc' described for other
mite embryos (reviewed by Anderson,
1973) (Fig. 1E).
The germ disc starts as an ovoid swelling
(Fig. 1E,F), and then flattens
(Fig. 1G). Flattening of the
germ disc is quickly followed by the appearance of leg primordia on both sides
of the ventral midline (viewed from the anterior in
Fig. 1H).
Leg buds and the prosoma region of the germ band appear rapidly and simultaneously (Fig. 1I-P; http://devbiol.zoo.uwo.ca/movies/smite_legs_growth_mov.mov.) approximately two hours after the appearance of the germ disc. The two halves of the germ band are separated by a small ventral sulcus, which quickly closes (Fig. 1M). Limb buds in the chelicera-bearing segment and the germ band in the opisthosoma appear 3-4 hours after formation of the germ band. Eyes become coloured and limbs grow on the chelicera, pedipalp and first three walking leg segments, becoming jointed and hirsute by 30 hours AEL. The fourth walking leg does not extend in embryonic stages. Hexapod larvae hatch approximately 39 hours AEL. Larvae undergo two moults, during which the fourth leg extends, before becoming reproductively active.
Early T. urticae embryos do not have a syncitial phase
To determine if the initial divisions of the embryo involve cytokinesis or
are only nuclear (syncitial) we micro-injected tetramethylrhodamine dextran
into 1-, 2-, 4- and 16-cell embryos and examined its distribution using a
confocal microscope (Fig. 2). Dextran molecules of this size will not move passively through a cell membrane
(Grbi et al.,
1996
).
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Dextran injected into a 1-cell embryo diffused rapidly to fill the entire egg (data not shown) demonstrating that egg cytoplasm is not a barrier to diffusion of this molecule. Two-cell embryos injected with dextran initially showed fluorescent signal only in the injected cell. Forty minutes after injection, however, both cells were equally labelled (Fig. 2A-C). After micro-injection into 4-cell or 16-cell embryos, dextran was localised in the injected cell and no leakage to other cells was observed, even after 1-hour incubation (Fig. 2D-I). These data imply that the spider mite embryo forms partial cell membranes at the 2-cell stage, and complete ones by the 4-cell stage.
Tu-run: cloning and sequence analysis
To determine if a pair-rule gene mechanism underlies segmentation in the
two-spotted spider mite, we cloned a T. urticae homologue of
Drosophila runt. Two clones with homology to Drosophila runt
were identified in an EST screen of embryonic stage cDNA (4,000 ESTs
sequenced). These clones were found to contain, where overlapping, an
identical sequence. Further sequencing and assembly of the EST screen
sequences demonstrated that one clone contained an apparently full-length open
reading frame of a runt-like gene. This sequence has an 849 bp open
reading frame with an upstream stop codon 21 bp from the putative start
codon.
To ascertain if this clone represents the only runt homologue in the spider mite genome, PCR using degenerate primers designed to amplify the conserved runt domain was performed on spider mite genomic DNA. The single resulting PCR fragment was cloned and 18 colonies were sequenced. All colonies contained a sequence identical to a region in both runt-like clones from the EST screen, indicating that they derive from the same gene. We designate this gene Tu-run.
We performed maximum likelihood phylogenetic analysis on a multiple alignment of the most conserved region of various runt-like proteins including Tu-run (Fig. 3A). This analysis demonstrated that the Tu-run protein falls into a clade containing both Drosophila and spider (C. salei) runt-like sequences, to the exclusion of vertebrate sequences (Fig. 3C).
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Tu-run RNA is expressed in circular domains that resolve into
segmental stripes
Tu-run transcription is first detectable in blastoderm stage
embryos, at 23 hours AEL (Fig.
4). The RNA is distributed in five bilaterally paired rings of
cells (3-4 cells wide) in the ventral regions of the embryo. These rings
appear rapidly and simultaneously, and are paired across the ventral midline
of the embryo (Fig. 4A). At 25
hours AEL, as limb-buds in the prosoma (excluding the chelicera buds which
form later) become visible, the rings of Tu-run-expressing cells
surround each limb bud. As the limb-bud grows, expression becomes undetectable
in the cells of the posterior half of the ring, leaving a curved stripe of
cells expressing Tu-run just anterior to the limb-bud. As the
chelicera limb-bud becomes visible, expression is detected in a stripe of
cells directly anterior to it. Tu-run is also detected in three
rapidly forming stripes in the opisthosoma. At this stage, the embryo contains
nine stripes of Tu-run-expressing cells
(Fig. 4B). Expression is also
detected in a diffuse group of cells in the head of the embryo, anterior to
the chelicerae. By 30 hours AEL, the stripes of expression in epidermal cells
becomes undetectable and Tu-run RNA appears in segmentally repeated
groups of cells in the nervous system (Fig.
4C). This expression persists until hatching.
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To understand the distribution of Tu-run RNA, we co-stained
embryos for Tu-run RNA and Distal-less protein (Dll). Dll is an
evolutionarily conserved marker for limb-bud fate
(Panganiban et al., 1997).
Both the expression pattern and function of this gene are conserved in the
spider C. salei (Schoppmeier and
Damen, 2001
). Dll protein is first detected in five paired oval
domains of cells, in ventral regions of the germ band (not shown). These
domains mark the forming limb buds. As development proceeds, a domain of
expression becomes visible in the anterior region, marking the chelicera limb
bud (Fig. 4J). As the legs
become fully formed, Dll protein is initially present in all cells of the limb
(Fig. 4K), but then becomes
restricted to a ring of cells in the proximal region of the limbs, and a broad
domain at the distal tip (Fig.
4L). In the segment containing the fourth walking leg, it is
restricted to a circular patch of cells, slightly dorsal to the proximal edge
of the other limbs, until that limb extends in larval stages. Dll protein is
also present in segmentally reiterated cells in the nervous system (asterisk
in Fig. 4F).
Dll protein expression appears slightly before Tu-run RNA. The rings of Tu-run-expressing cells abut and encircle the cells initially expressing Dll protein (Fig. 4D and G). The initial five paired rings of Tu-run expression thus mark cells surrounding, and just outside, the limb bud in the pedipalp-bearing segment and the four walking limb segments. A ring of Tu-run does not form around the chelicera limb bud, only a stripe anterior to it, as it forms later in development. Cells that express Tu-run RNA do not initially express detectable levels of Dll protein. As Dll expression initially spreads posteriorward, apparently by recruitment of cells to the limb bud, cells in the posterior of the Tu-run-expressing oval begin to express Dll. Expression of Tu-run RNA rapidly becomes undetectable in cells that express Dll protein. As the limb buds extend, Tu-run RNA becomes restricted to a stripe just anterior to, and abutting, the expression domain of Dll in the limb (Fig. 4E and H). By 30 hours AEL, Tu-run is no longer expressed in stripes of epidermal cells. Co-expression of Dll and Tu-run has not been observed (Fig. 4F and I).
Tu-pax3/7: cloning and sequence analysis
Degenerate PCR was used to amplify sequences homologous to Pax Group III
(PgIII) genes. Twenty clones were isolated containing an identical sequence
with homology to both Drosophila prd and gsb. We designate
the gene from which this sequence derives Tu-pax3/7.
We performed maximum likelihood analysis on a multiple alignment of the protein sequences containing the most conserved regions of Pax-type homeoprotein sequences (Fig. 3D). Cladograms derived from this analysis show Tu-pax3/7 to be most closely related to other PgIII genes (Fig. 3E). Phylogenetic analysis of an amino acid alignment of PgIII homologues containing the paired domain and one end of the extended homeobox motif (boxed in Fig. 3D), implies that Tu-pax3/7 forms a clade with the pax3 and pax7 genes from mouse (Fig. 3F) but not insect PgIII genes.
Stripes of cells expressing Tu-pax3/7 RNA do not form in
anterior-posterior sequence in the prosoma
Distribution of Tu-pax3/7 mRNA was determined using in situ
hybridisation to whole-mount embryos (Figs
5,
6). Tu-pax3/7 RNA is
first detected in three stripes of cells in the ventral regions of blastoderm
stage embryos (Fig. 5A,B, 19
hours AEL). The two most anterior stripes meet each other at their tips and
the posterior stripe is slightly thinner and does not meet the anterior two.
Tu-pax3/7 expression in these stripes appears quickly and
simultaneously. After examining over 1000 embryos hybridised for
Tu-pax3/7 RNA, no intact embryos were found that contained only one
or two stripes of cells expressing Tu-pax3/7 RNA.
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Very quickly after the first three stripes of Tu-pax3/7-expressing cells have appeared, a fourth stripe of cells, between the two most anterior stripes, begins to express Tu-pax3/7 RNA (Fig. 5C,D). The stripe is initially one to two cells wide, but quickly becomes as broad (3-4 cells) as the initial three stripes (Fig. 5E,F). Soon after the fourth stripe becomes visible, a fifth stripe, 1-2 cell wide, begins to express Tu-pax3/7 between the two posterior most stripes (Fig. 5G,H). Expression in this fifth stripe then widens to 3-4 cells (Fig. 5I,J). In both of the secondary stripes, a lateral focus of cells first starts expressing Tu-pax3/7, and expression spreads around the ventral surface of the embryo.
Dll protein expression becomes detectable only after all five stripes of Tu-pax3/7 are present. Dll expression appears in oval domains in each limb-bearing segment. By correlating the early expression of Dll protein with the stripes of cells expressing Tu-pax3/7 RNA, we can identify the stripes as they form. The three initial stripes of cells expressing detectable levels of Tu-pax3/7 RNA are cells that will underlie the pedipalp limb bud, the second walking leg limb bud, and the fourth walking leg limb bud. The next stripe to appear underlies the first walking leg limb bud, and the fifth stripe underlies the third walking leg limb bud. The appearance of the first five stripes of Tu-pax3/7 expression is consistent with a segmental pattern with pair-rule modulation.
The domains of Dll expression (Fig. 6A-D) overlap the anterior edge of the stripes of Tu-pax3/7-expressing cells. As the limb buds develop, Dll expression spreads posteriorwards, first overlapping the entire Tu-pax3/7 stripe, and then extending beyond it (Fig. 6E,I). Tu-pax3/7-expressing cells that come to express Dll, immediately lose detectable Tu-pax3/7 expression. As Dll expression spreads across the stripe, Tu-pax3/7 RNA is only detectable in a square block of cells, ventral to the limb bud (Fig. 6F,J).
Tu-pax3/7 RNA becomes undetectable in the epidermis by 30 hours AEL and becomes visible in the nervous system at around the same time (Fig. 6H,L). Segmentally repeated groups of cells in the central nervous system express Tu-pax3/7, appearing as a broad stripe of cells in each segment, broken at the ventral midline (Fig. 6H,L). Tu-pax3/7 is expressed in limb joints in just hatched nymphs (data not shown).
Tu-pax3/7 RNA is expressed in segmental stripes in the
opisthosoma
As Dll expression begins to spread across the stripes of cells expressing
Tu-pax3/7, a stripe of Tu-pax3/7 RNA-expressing cells
appears in the posterior of the germ band
(Fig. 6M). This 2- to 3-cell
wide stripe becomes broken at the ventral midline, and two more stripes appear
posterior to it, one after another (Fig.
6N,O). These stripes of cells mark the forming opisthosoma
segments. Without segmental markers, it is difficult to interpret the pattern
in which these stripes are forming. However, we have never seen (in over 1000
embryos) a stripe forming between two already formed ones in the opisthosoma.
This implies that Tu-pax3/7 RNA expression is not modulated in a
pair-rule manner in the opisthosoma. Stripes of cells expressing
Tu-pax3/7 RNA form in the central nervous system underlying the
opisthosoma segments (Fig. 6P)
in late embryos.
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DISCUSSION |
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T. urticae embryogenesis
Despite being the second largest group of animals, the developmental
genetics of chelicerates are poorly understood. The main obstacle for future
progress in this field is the lack of a chelicerate model organism. The
analysis of early patterning in chelicerates has proved difficult so far
because of the inaccessibility of early embryonic stages
(Damen et al., 1998;
Telford and Thomas, 1998
).
T. urticae is a good candidate organism for a model chelicerate.
T. urticae completes its embryonic development in 39 hours and its
full development from egg to adult is less than 7 days
(Rao et al., 1996
). In
contrast, the predatory spider C. salei has a nine-month development
time. T. urticae has small eggs (150 µm) that are surrounded by a
transparent chorion, allowing easy visualisation of embryonic development. Its
rapid generation time, simple diet (bean plants), and the organisation of its
genome on three chromosomes (Oliver,
1971
), also make T. urticae an ideal candidate for
genetic studies. Recent studies have further indicated that T.
urticae has a smaller genome (0.08 pg/ haploid genome) than
Drosophila (0.18 pg), or even C. elegans (0.09 pg) (T. R.
Gregory and M. G., unpublished).
Early embryogenesis in T. urticae does not include an early
syncitial phase. Chelicerates exhibit both syncitial and total cleavage
patterns (Anderson, 1973;
Hafiz, 1935
). The first nine
cleavage divisions occur over 9 hours and result in the formation of a
blastoderm surrounding a yolky interior. Germ band formation is reminiscent of
that of intermediate germ band insects. In both intermediate germ band insects
and spider mites, the anterior segments of the germ band are formed early and
almost simultaneously, and the trunk regions form later, in anterior to
posterior sequence.
Recent studies of chelicerate segmentation, using Hox genes as markers,
homologised the chelicerate prosoma with insect head segments. In this model,
the chelicera segment corresponds to the insect antennal segment, pedipalps to
intercalary segment, and walking legs to mandibular, maxillary, labial and
first thoracic segments (Damen et al.,
1998; Telford and Thomas,
1998
). These designations allow us to directly compare patterns of
Tu-pax3/7 and Tu-run expression with those of their
Drosophila homologues. For these purposes we will adopt the same
system used to number engrailed stripes (and thus parasegment boundaries) in
Drosophila (DiNardo and
O'Farrell, 1987
) (Fig.
7). Thus stripe 1 (mandibular in Drosophila), refers to
the first walking leg segment. In the prosoma, odd numbered segments are thus
the chelicera, first walking leg and third walking leg segments. Even numbered
segments bear the pedipalps, second walking leg and fourth walking leg
(Fig. 7).
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Tu-pax3/7 has the characteristics of an ancestral arthropod
Pax III gene
The sequence and expression pattern of Tu-pax3/7 have the
characteristics of an ancestral arthropod PgIII gene. Phylogenetic analysis of
PgIII proteins using the paired domain and a small part of the extended
homeobox domain (boxed in Fig.
3) implies that Tu-pax3/7 forms a clade with
pax3 and pax7 from mouse, to the exclusion of insect PgIII
genes. Tu-pax3/7 may thus be derived from an ancestral PgIII group
gene from before the separation of the prd and gsb genes in
Drosophila and pby1 and pby2 from
Schistocerca.
Consistent with the ancestral character of the Tu-pax3/7 sequence,
the expression pattern of Tu-pax3/7 appears to combine those of
Drosophila prd and gsb. In Drosophila the PgIII
genes prd, gsb and gsb-n are vital components of both the
pair-rule, and segment polarity cascades. Prd activates the
segment-polarity gene engrailed in odd numbered parasegments and
gsb in both odd and even numbered parasegments
(DiNardo and O'Farrell, 1987).
In Drosophila, prd is first expressed around the 13th
nuclear division, in an anterior domain. After cellularisation, this domain
splits and is joined by more posterior stripes, forming a pair-rule type
pattern of eight stripes of prd-expressing cells. These eventually
split to form segmentally iterated stripes
(Gutjahr et al., 1993a
).
Initially gsb is expressed only in odd numbered parasegments, but
expression rapidly appears in all parasegments
(Gutjahr et al., 1993b
).
gsb is also expressed in the nervous system where it activates
expression of gsb-n (Gutjahr et
al., 1993b
).
The grasshopper (Schistocerca) contains two PgIII group genes,
pby1 and pby2 (Davis et
al., 2001). Phylogenetic studies have shown these two genes to be
derived from an ancestral PgIII gene before the duplication that formed
prd and gsb. pby1 is first expressed in the embryonic
primordium in a faint posterior domain, which splits into a thoracic domain
and a gnathal arc. These arcs resolve into pair-rule stripes of cells in odd
numbered segments (i.e. mandibular, labial and second thoracic). Shortly after
the formation of the odd numbered stripes, stripes of cells in even numbered
segments begin expressing pby1 de novo (i.e. the maxillary and first
thoracic segment). In both Drosophila and Schistocerca,
PgIII genes are first expressed in broad regions of the germ band, and then
come to designate, initially, odd numbered parasegment (or putative
parasegment) boundaries.
In T. urticae, by contrast, Tu-pax3/7 is not initially
expressed in broad domains, and the stripes that form have the opposite
phasing to those in Drosophila and Schistocerca
(Fig. 7). In T. urticae,
Tu-pax3/7 is initially expressed at the center (as defined by the
placement of the limb bud) of even numbered segments (pedipalp, second and
fourth walking leg). We suggest that these domains are the equivalent of the
initial domains of prd in Drosophila and thus lie on a
potential parasegment boundary. The odd numbered stripes (first and third
walking leg) appear later, from lateral foci, in anterior to posterior
progression, expanding in width in a similar manner to that described for
gsb stripes in Drosophila
(Gutjahr et al., 1993b).
The origin of the difference in register between stripes of PgIII genes in
insects and T. urticae is unclear. It is possible that this
difference reflects the evolutionary distance between insects and
chelicerates, with PgIII stripes simply having moved during the passage of
time to pattern a different set of segments in one taxon. The difference in
register between the stripes of pby1 in the Schistocerca
abdomen and Drosophila is perhaps another example of this kind of
shift (Davis et al., 2001). It
is also possible that the difference in register may have come about because a
PgIII gene has become involved in, or regulated by, segmentation twice, once
in the lineage leading to mites, and once in the lineage leading to insects.
If PgIII genes ancestrally had a role in segmental patterning, they might
easily become modulated by pair-rule genes, and may eventually take up that
function. Indeed the expression pattern of Tu-pax3/7 is more
reminiscent of a segment-polarity gene being regulated by a pair-rule gene,
than as a pair-rule gene itself.
In later development, Tu-pax3/7 stripes are expressed in domains
shared with grasshopper pby1 and pby2, including rings in
spider mite limbs and in the nervous system (also shared with
Drosophila). The segmental expression in the CNS is consistent with
the proposal that an ancestral PgIII gene should combine functions in
segmentation and neurogenesis (Gutjahr et
al., 1993b). Even though we cannot completely exclude the
possibility that T. urticae contains another PgIII homologue,
collectively, the expression pattern of Tu-pax3/7 may represent an
ancestral pattern of PgIII genes in arthropods.
Runt domain genes in chelicerates
The Tu-run cDNA was the only run-like sequence obtained
either in our EST screen or using degenerate PCR on genomic DNA. This implies
that it may be the only runt homologue in the spider mite genome.
Despite this, Tu-run is significantly different in its sequence and
expression from other run-like genes. The most obvious sequence
difference is in the VWRPY motif at the carboxyl terminus of the protein. This
motif is conserved in all arthropod and vertebrate runt homologues
examined except spider mites, where the sequence is modified to LWRPF, and
C. elegans. In Drosophila, this motif mediates interaction
between run-like proteins and Groucho, a transcriptional co-repressor
(Aronson et al., 1997). While
the changes in sequence of this motif in Tu-run are conservative,
they may affect the interaction of Tu-run with Groucho (a spider mite
homologue of the groucho gene has been identified in our EST screen).
The lack of apparent pair-rule expression of this protein in T.
urticae is also significantly different from run expression in
Drosophila (Kania et al.,
1990
) and Manduca sexta
(Kraft and Jackle, 1994
). The
later expression of this gene in spider mites is, however, consistent with the
role of run in segmentation, cell fate specification in the nervous
system in Drosophila, and with the expression of a run
homologue in the opisthosoma of the spider C. salei
(Damen et al., 2000
).
Early expression of Tu-run may be involved in limb
specification
The earliest expression of Tu-run is in oval domains in each
prosoma segment (excluding the chelicera segment). This expression precedes
the morphological differentiation of limbs. Dll expression, however, appears
before Tu-run rings, implying that Tu-run does not play a
role in the specification of the initial limb primordia but rather in
delimiting their outer perimeters. Cells expressing Tu-run do not
initially express Dll, implying that they are not initially included in the
limb primordia. As the limb extends posteriorwards, however, cells posterior
to the initial primordia that once expressed Tu-run, lose this
expression, and express Dll instead. These cells are incorporated into the
limb bud. Expression of Tu-run thus does not preclude limb bud cell
fate.
Expression domains surrounding the limb primordia have not been observed in Drosophila. Drosophila limbs form from imaginal discs, a derived mode of limb specification peculiar to holometabolous insects. In most other arthropods, the appendages develop as an outgrowth of the body wall. run homologues have not been isolated from non-holometabolous insects, nor has the expression of the run homologues discovered in the spider (C. salei) been examined during limb primordium specification. It is thus not possible to determine if the limb bud-associated expression of Tu-run represents a conserved pathway found in other arthropods, but missing from holometabolous insects, or a derived gene expression pattern specific to mites or chelicerates.
The relative timing of the expression of Drosophila run and
prd is also not conserved in spider mites. In Drosophila,
run is expressed earlier than prd and modulates prd
expression (Gutjahr et al.,
1993a). Such changes are not surprising given the differences in
early run expression and 520 million years of independent
evolution.
Different mechanisms pattern the prosoma relative to the
opisthosoma
As mentioned previously, spider mite embryogenesis resembles that of
intermediate germ band insects. Both the morphological development and the
expression patterns of Tu-run and Tu-pax3/7 imply that the
prosoma is patterned by a mechanism that differs from that of the opisthosoma.
While the pattern of Tu-pax3/7 is pair-rule modulated in the prosoma
of the spider mite, no evidence for pair-rule patterning of the opisthosoma
exists. In this tissue, stripes of both Tu-pax3/7 and Tu-run
appear to form one by one, in an anterior to posterior progression. In the
absence of segmental markers we presume that the opisthosomal stripes
represent segmental repeats. This assumption is based on the fact that the
stripes display the same width and inter-stripe spacing as those in the
prosoma.
This pattern of segmentation gene expression is consistent with the
expression of the spider homologues of the segment polarity genes
engrailed, wingless and cubitus interruptus
(Damen, 2002). In this species
expression of these genes first appears as stripes simultaneously formed in
all the prosomal segments, followed by the appearance of individual stripes,
in anterior to posterior sequence, in the opisthosoma. This observation
supports the notion that two mechanisms exist to segment the chelicerate germ
band. In the prosoma, a mechanism exists that deploys pax3/7
expression in a pair-rule-like manner and leads to all segments expressing
segment polarity genes simultaneously. In the opisthosoma, pax3/7 is
not regulated in a pair-rule like manner but, like segment polarity gene
expression, appears in anterior to posterior sequence.
Differences in the patterning of different body domains have also been
shown in grasshoppers, where several genes are expressed differently during
segmentation of the gnathum and thorax, as compared to the abdomen
(Davis et al., 2001;
Dearden and Akam, 2001
;
French, 2001
). The gnathum and
thorax of the grasshopper are first demarcated by the expression of the
hunchback gene (Patel et al.,
2001
). Within this domain pby1 stripes appear in a
pattern that reflects pair-rule modulation, with secondary stripes forming de
novo (Davis et al., 2001
).
Early stripes of wingless (wg) also appear in this region
with the mandibular wg stripe appearing first, followed by the
simultaneous formation of all the thoracic stripes
(Dearden and Akam, 2001
).
Wingless stripes in the maxillary and labial segments appear de novo
between the mandibular and first thoracic stripes. All of these stripes form
before the expression of Engrailed protein. In the Schistocerca
abdomen, by contrast, pair-rule pby1 domains form segmental stripes,
but by splitting of initially broad stripes, rather than de novo appearance of
inter-stripes. Wingless stripes form with anterior to posterior
progression, with Engrailed protein being expressed soon after each
wg stripe forms.
Conservation of limb positioning between chelicerates and
insects
The relationship between Tu-pax3/7-expressing cells and the
initial domains of Dll-expressing cells provides some evidence that the
mechanism specifying placement of the limb primordia in Drosophila
may be conserved in spider mites. In Drosophila, prd is
expressed in a stripe of cells that spans the parasegment boundary
(Gutjahr et al., 1993a). This
expression is required to activate both wg and engrailed
expression in their respective domains on either side of the parasegment
boundary (reviewed by Nasiadka and Krause,
1999
). The initial expression of Dll in Drosophila is
also regulated by the parasegment boundary. The leg imaginal discs derive from
wg-expressing cells just anterior to the parasegment boundary
(Cohen et al., 1993
).
The relative positions of the expression domains of Dll and paired
in Drosophila appears conserved in spider mites. Dll expression
domains appear in ovals centered on top of the anterior parts of the stripes
of Tu-pax3/7-expressing cells. If Tu-pax3/7 is expressed
across a putative parasegment boundary, then the limb bud is placed just
anterior to the parasegment boundary, as in Drosophila. As the limb
bud grows, apparently recruiting cells in the posterior of the
Tu-pax3/7 stripe, and beyond it, Tu-pax3/7 expression is
repressed in the majority of the stripe, but remains active in a block of
cells, ventral to the limb bud. These cells form a domain that lies just
anterior to and extends posterior of, the edge of the cells of the limb bud.
It is possible that the juxtaposition of cells expressing these two genes
represents conservation of the pathways specifying the anterior-posterior
positioning of the limbs in spider mites. These data imply that the
parasegment boundary may be conserved in spider mites, and is possibly an
ancestral feature of arthropod development. Similar observations in spiders
(Damen, 2002) also imply that
the placement of the limb and the parasegment boundary are conserved in
arthropods.
Pair-rule patterning in chelicerates?
The expression pattern of Tu-pax3/7 in the prosoma is consistent
with that expected for a gene regulated by a pair-rule like process. The
appearance of the stripes with an alternate-segment periodicity is similar to
the expression patterns of some segment polarity genes in Drosophila
that are modulated by pair-rule genes. This may provide indirect evidence for
a pair-rule mechanism acting in spider mite segmentation. Similar expression
patterns of PgIII genes in the gnathal and thoracic regions of the grasshopper
have been interpreted as evidence for a pair-rule mechanism acting during
segmentation of these areas of the grasshopper embryo
(Davis et al., 2001). In the
spider mite opisthosoma however, Tu-pax3/7 is not expressed in a
pair-rule like pattern, supporting recent findings
(Damen, 2002
) that the prosoma
and the opisthosoma are patterned differently in the spider C.
salei.
A recent model (Wilkins,
2001) proposes a scenario for the evolution of the complex set of
pair-rule genes seen in Drosophila. According to this model,
co-option of a new set of gap genes is necessary for the simultaneous
patterning of the entire embryo. Incorporation of these new gap genes in turn
requires modulation and refinement by recruiting new pair-rule genes so that
in Drosophila they form a complex regulatory hierarchy formed to
correct regulatory imbalances. Ultimately, in Drosophila, these
interactions modulate the expression of the segment-polarity cascade. This
model predicts that only a few proto-pair-rule genes would be present in
primitive arthropods, and that homologues of Drosophila pair-rule
genes might have alternative functions in basal arthropods.
The expression of Tu-pax3/7 provides the first suggestion that a
pair-rule patterning mechanism may exist outside insects. If pair-rule
patterning does act in the development of chelicerates then the evolutionary
distance between spider mites and Drosophila would suggest that
pair-rule patterning is an ancient pathway, and is probably deployed in
segmentation, of at least some of the body, in all arthropods. However,
expression of two other homologues of the Drosophila pair-rule
cascade in mites, Tu-run (described in this paper) and ftz
(Telford, 2000) suggests that
they are not involved in pair-rule patterning in chelicerates. This implies
that the upstream gene(s) that regulate pair-rule modulation of
Tu-pax3/7 could be different from `traditional' pair-rule genes
isolated in Drosophila, though they may illustrate utilization of a
`pair-rule logic'.
Confirmation that a pair-rule pathway exists will require cloning of upstream modulator(s) of Tu-pax3/7 and functional studies of genes involved in segmentation. Examining the expression patterns of pair-rule genes in other arthropod groups (such as myriapods and crustaceans), as well as close relatives of arthropods (such as onychophorans and tardigrades) will provide a better understanding of the origins of pair-rule patterning in arthropods.
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ACKNOWLEDGMENTS |
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