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First published online February 9, 2006
doi: 10.1242/10.1242/dev.02260
1 Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de
Barcelona, C/Josep Samitier 1-5, 08028 Barcelona, Spain.
2 Institute of Molecular Biology and Biotechnology (IMBB), 71110 Iraklio Crete,
Greece.
Authors for correspondence (e-mail:
jcrbmc{at}cid.csic.es;
averof{at}imbb.forth.gr)
Accepted 21 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key words: Tracheae, Appendage primordia, Gills, wingless signalling, Distal-less (Dll), buttonhead (btd), trachealess (trh), ventral veinless (vvl)
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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;
Mill, 1985). In
Drosophila, as in other insects, the tracheal system is a complex
tubular network that arises from the tracheal placodes, clusters of ectodermal
cells that appear on either side of thoracic and abdominal embryonic segments.
Tracheal cells are specified by the activity of a set of `tracheal inducer
genes' that includes the transcription factors trachealess
(trh) and ventral veinless (vvl), whose expression
is controlled by genes that specify positional cues along the embryonic body
axes (Isaac and Andrew, 1996;
Wilk et al., 1996;
Boube et al., 2000). The cells
of each cluster invaginate and migrate in a stereotypic pattern to form each
of the primary tracheal branches (Manning
and Krasnow, 1993).
In recent years, many of the genes that are required for the specification
of the tracheal cells have been identified
(Ghabrial et al., 2003).
However, not much attention has been given to the evolutionary origin of these
cells. It is believed that in the common ancestors of all arthropods,
specialised parts of appendages had a major role in respiration and
osmoregulation, acting as gills (Brusca and
Brusca, 1990; Budd,
1996). Indeed, this close association between respiratory organs
and appendages is maintained currently in many crustaceans, which are the
closest living relatives of insects
(Regier and Shultz, 1997;
Boore et al., 1998;
Mallatt et al., 2004).
To investigate the origin of tracheal cells, we have asked whether these may also arise in association with the cells that give rise to appendages in a present day insect like Drosophila. We have found that indeed the tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, and that the decision between these two fates is controlled by the activity of the wingless signalling pathway. By manipulating the genetic program that controls leg specification, we have been able to show that, even in the abdomen, tracheal primordia develop in close association with cryptic appendage primordia. These results point to a close relationship between the tracheal and leg fates, and suggest some interesting similarities with the appendage-associated gills of aquatic crustaceans. To investigate these similarities further, we have cloned homologues of the tracheal inducer genes and studied their expression patterns in two divergent groups of crustaceans. We argue that crustacean gills and insect tracheae, hitherto considered to be independent systems for gas exchange, may share a number of features in their developmental origin and specification.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) is a
lacZ insertion in the trh gene. Expression of btd
and Dll was induced with the UAS/GAL4 system
(Brand and Perrimon, 1993),
using a UAS-btd line (Schock et
al., 1999) and a UAS-Dll line (Gorfinkiel and Guerrero,
1997), and a btd-Gal4 line as a driver
(Estella et al., 2003). To
induce ectopic expression of wg, we used a nullo-Gal4 (from
W. Gehring, Basel, Switzerland) and a UASwg line
(Lawrence et al., 1995). We
used ptc-GAL4 (Speicher et al.,
1994) to drive the expression of UAS-TCFDN
(van de Wetering et al., 1997)
and UAS-Arm*
(Pai et al., 1997) in
Drosophila embryos.
Drosophila immunostaining and in situ hybridisation
We used the following primary antibodies: a mAb2A12 monoclonal antibody
(1:5-1:10, from the Developmental Studies Hybridoma Bank, University of Iowa),
which recognises an epitope from the lumen of the tracheal tree, and an
antibody specific for ß-gal (Cappel, 1:2000). Embryos were stained
according to standard protocols using the Vectastain Elite ABC kit. For
immunofluorescence, we used secondary antibodies Alexa488-conjugated goat
anti-rabbit (1:200) and Alexa594-conjugated goat antimouse (1:200), both from
Molecular Probes. Whole-mount in situ hybridisation was carried out with
trh and btd anti-sense RNA probes, following the method of
Tautz and Pfeifle (Tautz and Pfeifle,
1989), with minor modifications. For immunofluorescence, we used
trh anti-sense RNA probes following the procedure described by Wilkie
and Davis (Wilkie and Davis,
1998). For antibody labelling followed by in situ hybridisation,
we followed the procedure described by Manoukian and Krause
(Manoukian and Krause, 1992).
Photographs were taken using Nomarski optics or a SP1 Leica confocal
microscope.
Preparation of embryonic cuticle
For the analysis of embryonic cuticle, late embryos were removed from the
chorion and vitelline membrane, and mounted in a mixture of Hoyer's medium
(van der Meer, 1977) and
lactic acid (1:1).
Artemia, Parhyale and crayfish immunostaining and in situ hybridisation
We initially cloned a fragment of vvl from Artemia, by
PCR from cDNA generated from larval RNA with degenerated oligonucleotides
designed from the Drosophila vvl sequence. An antibody against the
Vvl protein of Artemia was generated by injecting rabbits with a
His-tagged fragment of the Vvl protein [amino acids 241 to 386 of the
previously published sequence (Chavez et
al., 1999)]. The serum was then affinity purified on a nickel
column. We initially cloned a fragment of vvl and trh from
Parhyale hawaiensis by PCR from a cDNA library kindly provided by
Nipam Patel (University of California, Berkeley), with degenerated
oligonucleotides designed from the Drosophila vvl and trh
sequences. We obtained a 240 bp fragment of the Parhyale vvl gene
that we used to clone the full-length cDNA from the library. We also obtained
a Parhyale trh fragment of around 700 bp that encompasses the region
of the HLH, the PAS-1 and the PAS-2 domains (corresponding to amino acids 100
to 550 in Drosophila). In situ hybridization in Parhyale was
carried out using a protocol provided by Nipam Patel; the protocol is
available upon request. For immunostaining in crayfish embryos, we used the
4D9 monoclonal antibody for Engrailed (Patel et al., 1989) and a polyclonal
antibody for Nub/Pdm (Averof and Cohen,
1997). Immunohistochemical staining was carried out as described
by Patel (Patel, 1994).
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RESULTS AND DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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;
Isaac and Andrew, 1996;
Wilk et al., 1996)
(Fig. 1B). Conversely, the leg
precursors can be recognized as clusters of cells that express the
Distal-less (Dll) gene, on either side of each thoracic
segment; these will give rise both to the Keilin's Organs (KOs, the
rudimentary legs of the larvae) and to the three pairs of imaginal discs that
will give rise to the legs of the adult fly
(Cohen, 1993).
To investigate whether there is a direct physical association between the
leg and tracheal primordia, we examined Drosophila embryos co-stained
for the expression of trh and early markers of leg primordia.
Although Dll is one of the most commonly used markers for the leg
primordia, it is not the earliest gene required for their specification.
Instead, a couple of related and apparently redundant genes,
buttonhead (btd) and Sp1, act upstream of
Dll in the specification of these primordia
(Estella et al., 2003).
Examining the specification of tracheal cells with respect to btd
expression, we observe that tracheal cells appear in close apposition to
btd-expressing cells, from the earliest stages of their appearance
(by stage 9/early stage 10, Fig.
1E,F). Interestingly, unlike Dll, btd is initially
expressed both in the thoracic and abdominal segments, and its expression is
restricted to the thoracic segments later, under the influence of the BX-C
genes (Estella et al., 2003).
Thus, the cells of the respiratory system in Drosophila always arise
in close proximity to the cells that are fated to give rise to the legs.
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), no ectopic appendage has been reported by misexpressing
Dll in the abdomen. These observations have lead to some doubts as to
whether a leg developmental program is at all compatible with abdominal
segmental identity. As the initial expression of btd in the abdominal segments is downregulated by the BX-C genes, we reasoned that sustained expression of btd might overcome the repressive effect of the BX-C genes and force the induction of leg structures in the abdomen. To test this, we used a btd-GAL4 driver to drive btd expression, expecting that the perdurance of the GAL4/UAS system would ensure a more persistent expression of btd in its endogenous expression domain. We never obtained any sign of ectopic Dll expression or KOs in the abdominal segments, but we observed that the increased expression of btd had an effect on the KOs of the thoracic segments, which had more sensory hairs than the three normally found in wild-type KOs (Fig. 2F). Thus, on its own, btd seems unable to overcome BX-C repression of leg development.
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A leg-tracheal equivalence group: wingless signalling provides a genetic switch for the specification of leg versus tracheal fate
Previous results have shown that the leg primordia are specified straddling
the segmental stripes of wingless (wg) expression in the
early embryonic ectoderm (Cohen et al.,
1993), whereas tracheal cells are specified in between these
stripes (de Celis et al.,
1995). To investigate whether wg might play a role in
determining the fate of these primordia, we studied what happens when the
normal pattern of wg expression is disrupted. We find that, in
wg mutant embryos, trh and vvl from the earliest
stages of their expression are no longer restricted to separate clusters of
cells; instead larger patches of expression add up to a continuous band of
cells running along the anteroposterior axis of the embryo
(Fig. 3C)
(de Celis et al., 1995), while
btd expression is suppressed in this part of the embryonic ectoderm
(Fig. 3D)
(Estella et al., 2003).
Conversely, ubiquitous expression of wg suppresses trh
expression (Fig. 3E), while
causing an expansion of btd expression along the embryo
(Fig. 3F,L). Restricted
activation or inactivation of the wg pathway by the expression of a
constitutive form of armadillo or a dominant-negative form of
dTCF, respectively, are also able to specifically induce or repress
trh and btd expression
(Fig. 3G-J). trh/vvl
and btd seem to respond independently to wg signalling and
there is no sign of cross-regulation among them, as btd expression is
normal in trh vvl double mutants, and trh and vvl
expression is normal in mutants for a deficiency uncovering btd and
Sp1 (data not shown).
The role of wg as a repressor of the tracheal fate is further
illustrated by looking at the behaviour of transformed cells: the clusters of
cells that have lost btd expression and gained trh and
vvl expression in wg mutant embryos begin a process of
invagination that is characteristic of tracheal cells
(Fig. 3K). Furthermore, these
cells also express the dof (stumps – FlyBase) gene, a
target gene of both trh and vvl in the tracheal cells
(Boube et al., 2000) (data not
shown). Although further development of these cells is hard to ascertain
because of gross abnormalities in wg- embryos, these
results indicate that they have been specified as tracheal cells. Thus,
wg appears to act as a genetic switch that decides between two
mutually exclusive fates in this part of the embryonic ectoderm: the tracheal
fate, which is followed in the absence of wg signalling; and the leg
fate, which is followed upon activation of the wg pathway
(Fig. 3M). Given that there are
no cell lineage restrictions setting apart the cells of the tracheal and leg
primordia (Meise and Janning,
1993), these two cell populations could be considered as a single
equivalence group, with the differences in their fate controlled by the
activation state of the wg signalling pathway.
Crustacean homologues of tracheal inducer genes are expressed in appendage-associated gills
A link between respiratory organs and appendages is also found in many
primitively aquatic arthropods, like crustaceans, where gills typically
develop as distinct dorsal branches (or lobes) of appendages called epipods
(Brusca and Brusca, 1990).
Following our observations, which suggest a link between respiratory organs
and appendages in Drosophila, we decided to examine whether further
similarities could be found between insect tracheal cells and crustacean
gills. Specifically, we considered whether homologues of the tracheal inducing
genes might have a role in the development of appendage-associated gills in
crustaceans.
|
) and
generated an antibody for immunochemical staining in developing
Artemia larvae. We observe that Artemia Vvl is initially
absent from early limb buds; it becomes weakly and uniformly expressed while
the limb is developing its characteristic branching morphology, and becomes
strongly upregulated in one of the epipods as its cells begin to differentiate
(Fig. 4A,B). Uniform weak
expression persists in mature limbs, but expression levels in the epipod are
always significantly higher. The trh homologue from Artemia
has previously been studied by Mitchell and Crews
(Mitchell and Crews, 2002),
and its expression appears to be restricted to the same epipod as Vvl.
Similarly, we have cloned homologues of vvl and trh from
Parhyale hawaiensis and have studied their expression by in situ
hybridization. Both genes are specifically expressed in the epipods of
developing thoracic appendages (Fig.
4C-E). Besides epipods, the Artemia trh and vvl
homologues are also expressed in the larval salt gland, an organ with
osmoregulatory functions during early larval stages of Artemia
development (Chavez et al.,
1999; Mitchell and Crews,
2002).
Implications for the origin of insect tracheal systems
What is the significance of the two Drosophila tracheal inducer
genes being specifically expressed in crustacean epipods/gills? One
possibility is that the expression of these two genes was acquired
independently in insect tracheae and in crustacean gills. Alternatively,
tracheal systems and gills may have inherited these expression patterns from a
common evolutionary precursor, perhaps a respiratory/osmoregulatory structure
that was already present in the common ancestors of crustaceans and
insects.
The latter possibility is considered unlikely by conventional views,
because of the structural differences between gills and tracheae (external
versus internal organs, discrete segmental organs versus fused network of
tubes), and the difficulty to conceive a smooth transition between these
structures. Yet, analogous transformations have occurred during arthropod
evolution: tracheae can be organized as large interconnected networks or as
isolated entities in each segment (as in some apterygote insects),
invagination of external respiratory structures is well documented among
groups that have made the transition from aquatic to terrestrial environments
(terrestrial crustaceans, spiders and scorpions), and conversely evagination
of respiratory surfaces is common in animals that have returned to an aquatic
environment (tracheal gills or blood gills in aquatic insect larvae)
(Snodgrass, 1935;
Mill, 1985;
Brusca and Brusca, 1990). A
very similar (but independent) evolutionary transition is, in fact, thought to
have occurred in arachnids, where gills have been internalised to give rise to
book lungs, and these in turn have been modified to give rise to tracheae in
some groups of spiders (Lankester,
1885; Purcell,
1910; Damen et al.,
2002). Thus, a relationship between insect tracheae and crustacean
gills is plausible.
A particular type of epipod/gill has also been proposed as the origin of
insect wings (Wigglesworth,
1976; Kukalova-Peck,
1983), a hypothesis that has received support from the specific
expression of the pdm/nubbin (nub) and apterous
(ap) genes – that have wing-specific functions in
Drosophila – in a crustacean epipod
(Averof and Cohen, 1997). In
fact, the Artemia nub and ap homologues are expressed in the
same epipod as trh and vvl, raising questions as to the
specific relationship of this epipod with either tracheae or wings. A
resolution to this conundrum becomes apparent when one considers the different
types of epipods/gills found in aquatic arthropods, and their relative
positions with respect to other parts of the appendage.
|
; Patel et al.,
1989a; Basler and Struhl,
1994; Damen,
2002). Different types of epipods/gills, however, differ in their
position with respect to this boundary. For example, in the thoracic
appendages of the crayfish, some epipods develop spanning the AP boundary
[visualized by engrailed (en) expression running across the
epipod], whereas others develop exclusively from anterior cells (with no
en expression; Fig.
4F). Given that wing primordia comprise cells from both the
anterior and posterior compartments, wings probably derived from structures
that were straddling the AP boundary. Conversely, given that tracheal
primordia arise exclusively from cells of the anterior compartment (anterior
to en and even wg-expressing cells)
(de Celis et al., 1995), it
seems probable that tracheal cells evolved from a population of cells that was
located in the anterior compartment. In this respect, it is interesting to
note that the former type of epipods express nub, whereas the latter
do not (Fig. 4G).
In summary, we would like to suggest that the ancestors of arthropods had
specific areas on the surface of their body that were specialized for
osmoregulation and gas exchange. Homologues of trh and vvl
were probably expressed in all of these cells and played a role in their
specification, differentiation or function. Some of these structures were
probably associated with appendages, in the form of epipods/gills or other
types of respiratory surfaces. A particular type of gill, straddling the AP
compartment boundary, is likely to have given rise to wings
(Averof and Cohen, 1997),
whereas respiratory surfaces arising from anterior cells only may have given
rise to the tracheal system of insects. Confirmation of this hypothetical
scenario may ultimately come from the discovery of new fossils, capturing
intermediate states in the transition of insects from an aquatic to a
terrestrial lifestyle.
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ACKNOWLEDGMENTS |
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Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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