1 Institute of Molecular Biology and Biotechnology (IMBB-FORTH), Vassilika
Vouton, 71110 Iraklio Crete, Greece
2 Laboratoire de Biologie du Développement, Université Paris 7, 7
quai St Bernard, 75005 Paris, France
3 Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, One
Cyclotron Road, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: averof{at}imbb.forth.gr)
Accepted 28 August 2003
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SUMMARY |
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Key words: Hox genes, Crustaceans, Body plans, Evolution
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Introduction |
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The adult body of Artemia consists of a head, eleven `thoracic' segments, two genital segments, six post-genital segments and a telson (Fig. 1B). Like many other crustaceans, Artemia hatches as a nauplius larva, which consists only of the anterior head segments, a growth-zone and the telson. Most body segments, including the thoracic, genital and post-genital segments, are generated sequentially from the growth zone during the course of larval development (Fig. 1A-D).
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In all arthropods that have been studied to date, with the exception of
Artemia (including insects, myriapods and spider)
(Delorenzi and Bienz, 1990;
Kelsh et al., 1993
;
Peterson et al., 1999
;
Hughes and Kaufman, 2002c
;
Damen and Tautz, 1999
), the
AbdB expression domain marks the most posterior segments of the body.
Posterior to this domain lie only the anal structures (thought to derive from
the non-segmental telson) and the ectodermal cells that invaginate to give
rise to the hindgut. In Artemia, the post-genital region lies between
the AbdB-expressing genital segments and the anal structures, and
consists of six well-formed segments with a characteristic morphology that
distinguishes them clearly from the other segments. The post-genital segments
are morphologically similar to each other; they have a relatively elongated
cylindrical shape, characteristic musculature, lack all trace of appendages
and do not contain any ganglia of the central nervous system (see
Schrehardt, 1987
;
Criel, 1991
)
(Fig. 1F). Engrailed is
expressed in the posterior part of these segments
(Fig. 1E). The observation that
all the known Hox genes are expressed anterior to these segments poses
interesting questions concerning their origin and identity.
Nothing is currently known about the genes that specify the identity of the
post-genital segments in Artemia. Already known developmental genes
could play a role, or new genes (perhaps new Hox genes) may have evolved to
fulfil this function. Likely candidates are the homologues of the
homeobox-containing genes caudal (Cad/Cdx) and
even-skipped (Eve/Evx), and of the zinc-finger transcription
factor spalt (Sal), that are known to play an important role
in the specification of posterior body regions in Drosophila, in
C. elegans and/or in vertebrates. Cad/Cdx genes are closely
related to Hox genes and are involved in posterior patterning in diverse
animals like Drosophila, C. elegans and chordates
(Macdonald and Struhl, 1986;
Moreno and Morata, 1999
;
Hunter and Kenyon, 1996
;
Edgar et al., 2001
;
Brooke et al., 1998
;
Meyer and Gruss, 1993
;
Subramanian et al., 1995
;
Chawengsaksophak et al., 1997
;
Marom et al., 1997
;
Epstein et al., 1997
;
van den Akker et al., 2002
).
In Drosophila, Cad has been shown to act like a homeotic gene to
specify the identity of the anal structures and hindgut of the adult
(Moreno and Morata, 1999
).
Similarly, Eve/Evx genes are closely related to the Hox genes and
have been implicated in the development of posterior structures in C.
elegans and in chordates (Ahringer,
1996
; Ferrier et al.,
2001
; Ruiz i Altaba and
Melton, 1989
; Bastian and
Gruss, 1990
; Beck and Slack,
1999
). In Drosophila, no clear role in posterior
patterning has been found for Eve, but the gene is expressed
specifically in posterior parts of the body and this expression is conserved
among divergent arthropods (Frasch et al.,
1987
; Moreno and Morata,
1999
; Patel et al.,
1994
; Hughes and Kaufman,
2002a
). Finally, Sal is a conserved zinc-finger
transcription factor that is required for the specification of anterior and
posterior structures during early embryogenesis in Drosophila; in
particular, Sal is thought to cooperate with Hox genes to define the
identity of the posterior genital and anal regions
(Jurgens, 1988
;
Kuhnlein et al., 1994
).
Cad, Eve and Sal are the only genes with a well-described
role in defining the identity of posterior structures, besides Hox genes.
We take the first steps towards characterising the post-genital region of Artemia, by examining the expression of genes that could play an important role in specifying the identity of posterior parts of the body. First, we examine in detail the expression of AbdB, the most posterior-acting of known Hox genes, to see whether its expression extends into the post-genital region at any developmental stage. Second, we ask whether the post-genital segments could be related to the posterior genital or anal structures that express Cad, Eve and Sal in Drosophila and in other species. We describe the isolation of homologues of these genes from Artemia and examine their expression. Finally, we describe a screen to isolate previously unidentified Hox genes that could be expressed in these segments.
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Materials and methods |
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Preparation of Artemia genomic DNA and first strand
cDNA
Genomic DNA was isolated as described previously
(Averof and Akam, 1993). For
the production of first strand cDNA,
100 µg of material were
homogenised and poly-A mRNA was purified using Dynabeads (Dynal). The eluted
mRNA was treated with DNase I (DNA free kit, Ambion) to remove traces of
genomic DNA. The RACE-polyT primer (GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT) was
used for first strand cDNA synthesis using the Superscript II kit (GibcoBRL),
following the manufacturer's instructions. First strand cDNAs were then
treated with 2 units RNaseH for 30 minutes at 37°C, followed by
inactivation of the enzyme for 15 minutes at 75°C.
Cloning of Artemia Cad, Eve and Sal homologues
AfCad
Specific primers were designed based on a short fragment of the
AfCad homeobox isolated by PCR (details available on request) and on
a similar short sequence kindly provided by G. Balavoine and M. Akam. These
primers were used to recover large fragments of AfCad cDNAs by nested 3'
and 5' RACE, and by PCR on a phage cDNA library. A radioactive probe
prepared from one of these larger fragments was used to screen a cDNA library
prepared from unhatched cysts (Escalante
and Sastre, 1993). Three independent phage clones were recovered,
containing full-length cDNAs of AfCad.
AfEve
Degenerate primers were designed to target conserved parts of the Eve
homeodomain:
EveF1(TAFTREQ), CGGGATCCACIGCITT(T/C)ACI(A/C)GIGA(A/G)CA; EveR1(MKDKRQR), GGAATTCC(T/G)(T/C)TGIC(T/G)(T/C)TT(A/G)TC(T/C)TTCAT.
These primers were used for PCR on first strand cDNA prepared from
posterior regions of Artemia larvae, and a short fragment of
AfEve was recovered. Based on the sequence of this fragment, specific
forward primers were designed and used for nested 3' RACE carried out on
the same cDNA pool. A 710 bp fragment was recovered, corresponding to the
3' part of the AfEve cDNA.
AfSal
Degenerate primers were designed to target conserved parts of the
zinc-finger 2 region of Sal:
SalF3(HTGERPF), GGAATTCA(T/C)ACIGGIGA(A/G)(C/A)GICCITT; SalR3(CPICQKK), GCTCTAGATT(T/C)T(G/T)ITG(A/G)CAIA(T/C)IGG(A/G)CA.
These primers were used for PCR on Artemia genomic DNA and a short
fragment of AfSal was recovered. Based on the sequence of this
fragment, specific primers were designed for nested inverse PCR. A 390 bp
fragment was obtained corresponding to the zinc-finger 2 region of
AfSal.
In situ hybridisation
DIG-labeled antisense RNA probes were prepared using the Megascript T3 or
T7 kits (Ambion). In situ hybridisation was carried out on stage L1-L3
Artemia larvae, as described previously
(Gibert et al., 2000;
Mitchell and Crews, 2002
).
Production of cross-reacting antibody against AbdB
A polypeptide containing 61 amino acids of the Drosophila AbdB
homeodomain and 10 additional C-terminal residues, was expressed and purified
from E. coli, using the expression vector pABD-B HD72
(Ekker et al., 1994). A mouse
was immunised intraperitoneally with 20-60 µg of protein in complete Ribi
adjuvant, six times over a period of 10 weeks. The serum was tested for
crossreactivity in a number of species, including Drosophila virilis,
Schistocerca americana and Artemia franciscana, and was found to
recognise AbdB proteins in these species. The serum was used for
immunochemical stainings at 1:1000 dilution.
Production of antibodies against AfCad and AfEve
The full length of the available AfCad and AfEve cDNA
fragments was cloned into the BamHI/EcoRI and
BamHI/XhoI sites of the pRSETA vector (Invitrogen),
respectively, to generate His-tagged protein fusions. AfCad and AfEve proteins
were produced by transforming these constructs into BL21(pLys) cells, inducing
with IPTG, and purifying the His-tagged proteins on a Ni-NTA column (QIAGEN),
as described in the manufacturer's manual. Antibodies were raised against
these bacterially expressed and purified proteins by repeated immunisations in
rabbits; 750 µg of each protein were used to carry out eight immunisations
over a period of 8 months (carried out by Davids Biotechnologie).
The anti-AfCad serum obtained was affinity purified on an Affigel-10 column
(Biorad) carrying bacterially expressed and purified AfCad
(Harlow and Lane, 1988); the
affinity purified serum was used for immunochemical stainings at 1:100
dilution. The anti-AfEve serum was pre-absorbed overnight on acetone powder
prepared from Artemia larvae
(Harlow and Lane, 1988
) and
used at 1:1000 dilution.
Antibodies and immunochemical stainings
Production of antibodies against AbdB, AfCad and AfEve are described above.
Other antibodies used: rabbit anti-Dll
(Panganiban et al., 1995),
mouse monoclonal FP6.87 (Ubx/AbdA) (Kelsh
et al., 1994
), mouse monoclonal 4F11 (En)
(Patel et al., 1989
).
Whole-mount immunochemical staining was carried out following standard
protocols (Patel, 1994), using
sonication to break the exoskeleton of the larvae and long (4x30 minute)
washes to reduce non-specific signals. All reported stainings had nuclear
localisation and were observed reproducibly in a significant number of
larvae.
Scanning electron microscopy
Scanning electron microscopy (SEM) was carried out by adapting existing
protocols (Felgenhauer, 1987).
Specimens were fixed and kept in 4% neutralised paraformaldehyde. The samples
were then dehydrated in a graded series of alcohol and in amyl acetate before
critical-point drying. Specimens were mounted on copper stubs with silver
paint, coated with 300 A of gold in a Polaron sputtering apparatus, and
examined on a JEOL JSM 6100 scanning electron microscope at 15 kV.
Drosophila experiments
The UAS-AfCad construct was prepared by cloning the full-length
AfCad cDNA into XhoI/EcoRI sites of the pUAST
vector (Brand and Perrimon,
1993). The construct was transformed into flies and several
independent transgenic lines were obtained. Crossing these lines to MS-248
GAL4 drives expression of AfCad broadly in eye-antennal and wing
discs, causing malformations in the head and thorax of the adults. Strongest
phenotypes, including the appearance of ectopic anal plates, were obtained
when the progeny of these crosses were raised at 30°C, using at least
three independent UAS-AfCad lines. The effects of AfCad
mis-expression were also analysed in flies carrying Dll-lacZ
(Moreno and Morata, 1999
) or
Byn-lacZ (Murakami et al.,
1995
) reporters, using the MS-248 or the apterous-GAL4
drivers.
To test whether mis-expression of AfCad activates the endogenous Cad gene, immunochemical stainings were carried out using an antibody against Drosophila Cad (kindly provided by Gary Struhl). No Drosophila Cad could be detected in imaginal discs expressing AfCad, although significant expression could be seen in discs mis-expressing Drosophila Cad. We should note, however, that the sensitivity of these stainings was low (the normal expression of Cad in the genital disc was barely detectable).
PCR screen for posteriorly expressed Hox genes
The posterior part of the body, including the developing genital and
post-genital regions, was dissected from Artemia larvae at stage
L9-L10 and snap-frozen in dry ice. First-strand cDNA was prepared from this
material, as described earlier. The `universal' Hox primers HoxF1(ELEKEF)
[GGAATTCGA(A/G)CTIGA(A/G)AA(A/G)GA(A/G)TT] and HoxR1(WFQNRR)
[GCTCTAGACGICG(A/G)TTTTG(A/G)AACCA] (Averof
and Akam, 1993) were used for PCR on the first-strand cDNA
prepared from posterior regions, with an early annealing temperature of
40°C. The
130 bp products of the reaction were cloned into the
pGEMT-easy vector (Promega). Sixty independent clones were analysed by PCR
using specific Artemia AbdB and AfCad primers and/or by
sequencing.
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Results |
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AbdB protein is first detected during mid-late larval stages (stage L8) and
is restricted to the newly formed genital segments
(Fig. 2A); this is consistent
with the previously reported patterns of AbdB mRNA distribution
(Averof and Akam, 1995).
Expression becomes stronger in the genital segments as larval development
proceeds (Fig. 2B), and expands
anteriorly to become expressed in some cells of posterior thoracic/trunk
segments (Fig. 2C).
AbdB expression is never observed to extend posteriorly, into the
segments of the post-genital region. These results argue against a direct role
of AbdB in specifying the identity of the post-genital segments.
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Caudal (AfCad)
We isolated three full-length cDNAs and a number of smaller fragments,
which correspond to a single Artemia Cad gene (sequence Accession
Number AJ567452). Sequence comparisons with other members of the Cad/Cdx
family suggest that this gene is orthologous to Drosophila Cad and to
vertebrate Cdx genes (Fig.
3A).
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The growth zone expression domain becomes less sharp and eventually fades away at around the time when all the segments have been generated (stage L10). During later stages, AfCad is also expressed in a small number of mesodermal and ectodermal cells (data not shown) and in developing anal structures of the adult (see later).
Even-skipped (AfEve)
We cloned partial cDNA fragments of Artemia Eve, corresponding to
part of the homeobox and the 3' end of the coding sequence (sequence
Accession Number AJ567453). Sequence comparisons indicate that this gene is
orthologous to the Drosophila Eve and to the vertebrate Evx
genes (Fig. 3B).
We used whole-mount in situ hybridisation to study the expression of AfEve during early larval development (data not shown), and generated an antibody that recognises the AfEve protein to examine its expression pattern in detail, throughout larval development. AfEve is expressed in the growth zone of Artemia during early-mid larval stages, in a pattern that largely overlaps with the expression of AfCad (Fig. 4B). Additionally, AfEve expression is occasionally detected in narrow (single- or few-cell wide) stripes that appear to `split' from the anterior edge of the growth zone domain as AfEve is switched off in the intervening (`inter-stripe') cells (Fig. 4B,F). These stripes appear to be very transient and are visible only one at a time, as new segments are being generated from the growth zone.
The AfEve stripes appear before any morphological signs of segmentation become apparent. To examine the relationship between these stripes and the process of segmentation, we compared the expression of AfEve with the expression of the segmentation gene engrailed (en), using double immunochemical stainings. The AfEve stripes appear earlier than engrailed (i.e. several cell diameters posterior to the youngest engrailed stripe) and have disappeared by the time engrailed is turned on in any particular segment (Fig. 4H). We are therefore not able to determine the precise position and segmental register of these stripes.
AfEve is also expressed in cells of the developing hindgut
(Fig. 4B), in mesodermal cells
that give rise to the dorsal vessel/heart (not shown), and in a small set of
segmentally repeated cells in the central nervous system
(Fig. 4H). Similar patterns in
the central nervous system and heart have been documented in other arthropods
(Frasch et al., 1987;
Patel et al., 1992
;
Patel et al., 1994
;
Duman-Scheel and Patel, 1999
;
Hughes and Kaufman, 2002a
),
suggesting that these aspects of Eve expression are evolutionarily
conserved.
Spalt (AfSal)
We isolated several genomic fragments containing the second zinc finger of
Sal from Artemia (sequence Accession Number AJ567454).
Sequence comparisons suggest that this is orthologous to Drosophila
Sal and Salr, and to the vertebrate Sal genes
(Fig. 3C).
We used in situ hybridisation, to examine the expression of AfSal in early larvae (stage L1). Early AfSal expression is restricted to the posterior growth zone and is very similar to the expression of AfCad (data not shown). Technical difficulties with in situ hybridisation did not allow us to examine the expression of AfSal during later stages.
Late AfCad expression marks the differentiation of the adult
anal appendages
During mid-late stages of larval development, AfCad gradually
ceases to be expressed in the growth zone (which presumably disappears after
the formation of all body segments) and starts to be expressed in the
posterior part of the telson, in the region surrounding the anus
(Fig. 5C). This expression
begins at stage L6 and coincides with the beginning of Distal-less
(Dll) expression in the same part of the telson
(Fig. 5A); it is associated
with the development of a pair of appendage-like structures, the caudal furca,
that surround the anus in the adults (Fig.
5D-F). Unlike the dynamic expression of AfCad in the
growth zone, expression in the posterior telson is relatively stable
throughout mid-late larval stages. This is reminiscent of the homeotic
function of Drosophila Cad in the primordia of the adult anal
structures, where Cad expression is also associated with the
expression of Dll (Gorfinkiel et
al., 1999; Moreno and Morata,
1999
). Dll expression is not observed in the post-genital
segments of Artemia.
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We prepared a UAS-AfCad construct and carried out the same experiments, expressing Artemia Cad in the eye-antennal and wing imaginal discs using the same GAL4 drivers. The results we obtained are strikingly similar to those observed by mis-expressing of Drosophila Cad: appearance of ectopic anal plates in the head and notum (Fig. 6A) and ectopic expression of Dll and Byn (Fig. 6B,C). The penetrance of these phenotypes was higher than that observed using Drosophila Cad (although this may depend on the particular transgenic lines that were used) and no significant activation of Drosophila Cad was detected in imaginal discs mis-expressing AfCad, suggesting that these effects are mediated directly by AfCad expression. We conclude that the Drosophila and Artemia Cad proteins behave very similarly in this in vivo assay, indicating that these proteins have inherited similar biochemical properties from their common ancestor, in their ability to bind and regulate the relevant downstream targets.
|
Previous screens to isolate Hox genes from Artemia relied on
genomic DNA as a template for PCR using degenerate primers
(Averof and Akam, 1993). We
reasoned that such screens could be strongly enriched for
posteriorly-expressed genes if, instead of genomic DNA, the starting material
was mRNA derived specifically from posterior parts of the body. We therefore
obtained larvae at around the time when the post-genital segments are being
formed (stage L9-L10), dissected the posterior part of their body (including
part of the genital region, the post-genital region and telson), and prepared
first-strand cDNA from this material. We then carried out a PCR screen on this
cDNA, using degenerate primers that are expected to recover homeobox fragments
from all classes of Hox genes and Cad (including some divergent Hox genes)
(see de Rosa et al., 1999
;
Averof and Akam, 1993
), aiming
to isolate any posteriorly expressed Hox genes that were missed by our
previous screens. Out of the 60 independent clones that we analysed, 59 turned
out to be AfCad and one was AbdB. This result confirms that
the PCR screen is highly selective for posteriorly expressed genes, and shows
that our general Hox primers are not able to detect any new Hox genes
expressed in this region.
Similarly, PCR screens using primers that target specifically AbdB/posterior class Hox genes did not yield any new genes (T.C. and M.A., unpublished). The failure to identify new posteriorly expressed Hox genes may be due to a number of reasons: such genes may not exist in Artemia, or they may be too divergent to be amplified by our Hox primers.
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Discussion |
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Possible roles of Cad and Eve in the sequential
generation of body segments
The expression pattern of AfCad and AfEve in the growth
zone does not suggest a specific, homeotic-like role of these genes in
defining the identity of the post-genital segments. Unlike homeotic genes, the
expression of these genes is not restricted to regions with a particular
segmental identity, it appears before segmentation, and it is not maintained
during the subsequent development and differentiation of segments.
Furthermore, there is no indication that these genes are expressed any
differently in the growth zone when the post-genital region is being formed
(compared with when other regions are forming), so there is no evidence that
they could play a specific role in distinguishing this region from other parts
of the body. These expression patterns, however, suggest that these genes
could play a role in segmentation that is comparable with the early functions
of Cad and Eve in Drosophila and in other
arthropods.
The expression of AfCad in the growth zone of Artemia is
very similar to expression patterns of Cad observed in other
arthropods that generate their trunk segments sequentially from a posterior
growth zone (`short-germ' arthropods), most notably in the beetle
Tribolium castaneum and in the locust Schistocerca gregaria
(Schulz et al., 1998;
Dearden and Akam, 2001
).
Cad expression is also localised to the growth zone, in these
species, and the most obvious difference from Artemia is that this
expression is not excluded from the posterior-most tip of the body during the
early stages of segmentation. Expression of AfCad in the growth zone
is also similar to the expression of vertebrate Cdx genes in posterior parts
of the primitive streak, prior to the formation of somites
(Meyer and Gruss, 1993
;
Marom et al., 1997
). These
similarities may reflect an ancestral role of Cad/Cdx genes during
the progressive generation of body parts (segments) from a posterior growth
zone.
Somewhat different patterns of expression are observed in
Drosophila, where segments are not generated sequentially from a
growth zone (Macdonald and Struhl,
1986). Maternal Cad mRNA is uniformly distributed in
early Drosophila embryos, but translational repression mediated by
the anterior morphogen Bicoid transforms this uniform distribution into a
gradient, with highest levels of Cad protein at the posterior end of the
embryo (Macdonald and Struhl,
1986
; Rivera-Pomar et al.,
1996
). The zygotic expression of Cad is transcriptionally
regulated and is also restricted to posterior parts of the embryo. The
combined zygotic and maternal patterns of Cad are known to regulate a
number of early segmentation and gap genes
(Macdonald and Struhl, 1986
;
Rivera-Pomar et al., 1995
).
Cad/Cdx genes are also known to directly regulate Hox genes in C.
elegans and in vertebrates (Hunter et
al., 1999
; Subramanian et al.,
1995
; Epstein et al.,
1997
; van den Akker et al.,
2002
), and may also have a similar function in some
arthropods.
In Artemia we have found no evidence for translational repression of AfCad mRNA or for a graded distribution of the AfCad protein (Fig. 4A,C,D), indicating that the mechanisms regulating Cad expression during segmentation are substantially different in Drosophila and Artemia. Nevertheless, Cad could have equivalent roles in regulating the expression of segmentation genes and Hox genes in these species. In Drosophila this regulation occurs simultaneously throughout the embryo, with different concentrations of Cad eliciting different responses on different targets along the anteroposterior axis of the embryo. In `short-germ' arthropods, like Artemia, the regulation of these targets would have to occur in a temporal sequence, as individual segments exit from the growth zone, but it is conceivable that a `temporal gradient' of Cad activity in these organisms could function in an analogous manner to the spatial concentration gradient of Cad in Drosophila: the progenitor cells that give rise to posterior parts of the body spend more time in the growth zone, and thus experience Cad expression for longer periods than the anterior progenitor cells. If target enhancers are capable of integrating Cad activity over time, the effect of this `temporal gradient' could be similar to that of a Cad concentration gradient. Thus, in arthropods like Artemia, expression of Cad in the growth zone may help to define the spatial limits for the activation of segmentation genes, in an analogous manner to the spatial gradient of Cad protein operating in early Drosophila embryos. In addition, it may help to set temporal limits required for the sequential activation of segmentation genes and Hox genes, as segments exit from the growth zone.
AfEve expression is also observed in the growth zone of
Artemia, with transient stripes of expression emerging from this
posterior domain. This is highly reminiscent of Eve/Evx expression in
short germ arthropods, like the beetle Tribolium castaneum and the
centipede Lithobius atkinsoni
(Patel et al., 1994;
Hughes and Kaufman, 2002a
).
The main difference between these species appears to be in the stability of
these stripes: the Eve stripes of Artemia appear to be very
transient, while the stripes in Lithobius and Tribolium
persist for longer, and consequently a number of stripes can be detected at
any one time. Another issue concerns the segmental periodicity of Eve
stripes. In Drosophila, Eve is expressed in alternate segments and is
well known for its role as a pair-rule segmentation gene
(Frasch et al., 1987
), but
expression with double-segment periodicity has not been observed beyond higher
insects. In Tribolium, the stripes that have just emerged from the
growth zone are broad and have a double-segment periodicity, but subsequently
each of these stripes splits into two narrower stripes with single-segment
periodicity (Patel et al.,
1994
). In the centipede, the Eve stripes have
single-segment periodicity (Hughes and
Kaufman, 2002a
). In Artemia, the transient appearance of
AfEve stripes, prior to the appearance of any morphological signs of
segmentation or engrailed expression
(Fig. 4H), does not allow us to
determine whether these stripes have single- or double-segment periodicity. In
spite of differences in the segmental periodicity of Eve stripes, the
observation that Eve is expressed in stripes associated with
segmentation in diverse arthropods (including insects, crustaceans, myriapods
and chelicerates) (Patel et al.,
1994
; Damen et al.,
2000
; Hughes and Kaufman,
2002a
) provides strong evidence for a conserved role of this gene
in the process of segment formation.
Beyond arthropods, Eve/Evx genes appear not to be involved in
segmentation, but instead play a role in the development of posterior
structures (Ahringer, 1996;
Ferrier et al., 2001
;
Ruiz i Altaba and Melton,
1989
; Bastian and Gruss,
1990
; Beck and Slack,
1999
). A role in posterior development may also exist in
arthropods, where posterior expression is a prominent feature of Eve
expression. Although no function has been assigned to Eve expression
in the anal plates and hindgut of Drosophila, conservation of this
posterior expression in Artemia and in insects
(Frasch et al., 1987
;
Moreno and Morata, 1999
;
Patel et al., 1994
) suggests
that it is likely to play a conserved role in the development of posterior
structures. Other aspects of Eve function, in the heart and in the
CNS, are also likely to be conserved
(Frasch et al., 1987
;
Patel et al., 1992
;
Patel et al., 1994
;
Duman-Scheel and Patel, 1999
;
Hughes and Kaufman,
2002a
).
Finally, the early expression of AfSal is very similar to the early expression of AfCad, encompassing the growth zone at least during the time when thoracic/trunk segments are being generated. This suggests that, like AfCad and AfEve, AfSal may have a role in the process of segment formation, but is unlikely to have a direct role in defining the identity of the post-genital segments.
Conserved role of Cad in the specification of anal
structures
Besides the dynamic expression of AfCad in the growth zone, during
later stages this gene is also expressed in the posterior part of the telson.
This expression pattern appears shortly before the anal appendages of the
adult begin to differentiate in this region, and remains relatively stable
during the development of these structures
(Fig. 5). This aspect of
AfCad expression also coincides with the onset of Dll
expression in precisely the same part of the telson, in striking parallel to
Drosophila, where Cad is associated with the expression of
Dll in the anal plates
(Gorfinkiel et al., 1999;
Moreno and Morata, 1999
).
Drosophila Cad is known to have an important homeotic-like function
in defining the identity of anal structures
(Moreno and Morata, 1999
).
These similarities, therefore, suggest that AfCad could play a
similar role in defining the identity of anal structures in Artemia.
Similar expression patterns have also been observed in other arthropods
(insects, crustaceans and chelicerates)
(Xu et al., 1994
;
Schroder et al., 2000
;
Dearden and Akam, 2001
;
Abzhanov and Kaufman, 2000b
;
Akiyama-Oda and Oda, 2003
),
suggesting that this role is likely to be ancient and phylogenetically
conserved among arthropods. The effects of mis-expressing AfCad in
Drosophila (Fig. 6)
also suggest that the functional properties of Cad proteins are likely to be
widely conserved.
Identification of a unique body region that expresses no known Hox
genes
In this work, we set out to investigate the nature of the post-genital
segments of Artemia, asking whether any of the known Hox genes or
related candidate genes could play a role in defining the identity of these
segments. The expression patterns of AbdB, Cad, Eve and Sal
homologues suggest that none of these genes are likely to have such function.
The expression patterns of AbdB and AfCad, however, provide
useful landmarks that allow us to place the post-genital segments in the
context of other regions within the body plan of Artemia.
AbdB, the most posterior acting of all Hox genes that have been
identified in arthropods, is expressed specifically in the two genital
segments of Artemia, supporting the notion that these segments may be
related to the AbdB-expressing genital segments of insects
(Fig. 7). Consistently, all the
other Hox genes are expressed anterior to this region and never extend beyond
the posterior boundary of AbdB expression
(Hughes and Kaufman, 2002b).
AbdB is not expressed even transiently in the
post-genital segments, suggesting that these segments bear no direct
relationship to the genital segments.
|
In all arthropods where the expression of the relevant genes is known, AbdB and Cad are expressed in abutting domains that define the most posterior parts of the body (Fig. 7). Uniquely, in Artemia, a series of six post-genital segments have become intercalated between these AbdB and Cad domains. These segments do not express either of these genes or any other known Hox-related gene. Thus, we consider that the post-genital segments of Artemia constitute a unique body region that bears no relationship with any of the regions that have been previously characterised by Hox gene expression in other arthropods (Fig. 7). Certainly, we can find no counterpart for this region in insects, where the role of Hox genes has been studied in most detail.
Origin and affinities of the post-genital region
The identification of the post-genital segments as a unique body region
that is not specified by any of the known Hox genes, raises a number of
questions. First, we know nothing about how the identity of these segments is
specified. Are there new, yet unidentified, Hox genes that have adopted this
role, or are these segments able to develop without any input from Hox genes?
A precedent for the latter is the specification of the antennal segment of
insects, where no Hox genes appear to have a direct role
(Struhl, 1982;
Stuart et al., 1991
).
Another question relates to the origin of this region. Is it a novelty that
appeared in Artemia and its closest relatives, or could it be an
ancient feature that is shared with other arthropod groups? Anostracan
crustaceans, to which Artemia belongs, all share the same general
body plan consisting of the head, a series of limb-bearing thoracic/trunk
segments, the genital segments, the limb-less post-genital segments and the
telson/anus. Yet the homologues of the post-genital segments are more
difficult to trace in other groups of branchiopod crustaceans
(Brusca and Brusca, 1990),
like the Conchostraca and Notostraca, which have a series of thoracic-like
limb-bearing segments posterior to their genital segments, followed by a
smaller number of limb-less segments; or the Cladocera, which have such a
modified and reduced segmental pattern that it is difficult to identify any
counterparts of the post-genital segments.
In spite of the differences observed among branchiopods, however, there is
evidence to suggest that the post-genital region could have an ancient origin,
and may not be strictly restricted to anostracan crustaceans. One line of
evidence comes from fossils: the body plan of Lepidocaris
(Scourfield, 1926) shows
striking similarities to the body plan of present-day anostracans, suggesting
that the origin of the post-genital segments can be traced at least back to
the early Devonian (
390 million years before present). Similarly, a
number of other groups of crustaceans, including copepods and cephalocarids
(thought to be among the earliest-branching groups of crustaceans)
(Brusca and Brusca, 1990
), as
well as some controversial fossils whose phylogenetic/taxonomic status is not
well resolved (e.g. McNamara and Trewin,
1993
; Hou and Bergstrom,
1997
; Walossek and Muller,
1998
), have a segmental organisation that is comparable with that
of anostracans, showing differences mainly in the number of segments found in
their thoracic/trunk, genital and post-genital regions
(Fig. 7). These similarities
raise the possibility that the limb-less post-genital region may be an ancient
feature shared by a number of divergent arthropod groups.
Our study is the first to propose the existence of a distinct segmental
identity, that is not dependent on any of the known Hox genes, between the
domains of AbdB and Cad expression in Artemia.
Similar studies of Hox gene expression in other arthropods have not identified
a comparable body region in major groups like the insects, myriapods,
chelicerates and malacostracan crustaceans
(Damen et al., 1998;
Telford and Thomas, 1998
;
Abzhanov and Kaufman, 2000a
;
Hughes and Kaufman, 2002c
;
Hughes and Kaufman, 2002b
),
but a number of phylogenetically interesting groups, like copepods and
cephalocarids, have not yet been examined. Extending studies of developmental
gene expression to diverse groups may be a key to understanding the origin of
evolutionary innovations, like the post-genital segments, which contribute to
the enormous morphological diversity of arthropod body plans.
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ACKNOWLEDGMENTS |
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