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First published online December 7, 2008
doi: 10.1242/10.1242/dev.030270
University of Chicago, Department of Organismal Biology and Anatomy, CLSC 921B, 920 E. 58th Street, Chicago, IL 60637, USA.
* Author for correspondence (e-mail: uschmid{at}uchicago.edu)
Accepted 23 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Fate-map, Developmental constraint, Evolutionary development (EvoDevo), Bicoid, Episyrphus
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), but the
mechanism is not conserved across flies (Diptera). One of the key genes in the
Drosophila model, bicoid, appears to be confined to higher
dipterans (Cyclorrhapha) (Lemke et al.,
2008; Stauber et al.,
2002; Zdobnov et al.,
2002). Comparative studies in dipterans are particularly suitable
to explore how and why this gene evolved and should lead to a better
understanding of factors that shape or constrain mechanisms of AP axis
specification in evolution. One suspect is the extra-embryonic serosa anlage,
which differs between species in size and position relative to the dimensions
of the egg (Roth, 2004).
Previous work suggests a correlation between the occurrence of bicoid
and a mid-dorsal rather than anterior or anterodorsal position of the (amnio-)
serosa anlage (Schmidt-Ott,
2005). However, we recently discovered an anterodorsal serosa
anlage in the cyclorrhaphan hover fly Episyrphus balteatus
(Syrphidae) (Fig. 1)
(Rafiqi et al., 2008), which
seems to contradict this correlation and prompts the question how this species
specifies its AP axis.
In Drosophila, a long-germ insect with an embryonic rudiment that
extends from the anterior to the posterior tip of the egg
(Davis and Patel, 2002;
Tautz et al., 1994), the
amnioserosa anlage is confined to a narrow strip of mid-dorsal blastoderm
(Hartenstein, 1993). AP
polarity of the Drosophila embryo stems in part from symmetrical
signaling processes at both poles of the egg, which are mediated by the
receptor tyrosine kinase Torso, but is determined by the asymmetric
distributions of maternal of bicoid and nanos mRNAs, which
are localized at opposite poles of the egg (reviewed by
St Johnston and Nüsslein-Volhard,
1992). The bicoid protein is expressed in an
anterior-to-posterior gradient and specifies the anterior body plan
(Driever and Nüsslein-Volhard,
1988a; Driever and
Nüsslein-Volhard, 1988b;
Driever et al., 1990). It
functions predominantly as a transcription factor and regulates the expression
of direct targets such as orthodenticle or hunchback in a
spatially restricted manner (see Berman et
al., 2002; Ochoa-Espinosa et
al., 2005; Schroeder et al.,
2004; Segal et al.,
2008). Bicoid activates orthodenticle only in a narrow
anterior cap but activates hunchback throughout the anterior half of
the blastoderm (Driever and
Nüsslein-Volhard, 1989;
Finkelstein and Perrimon,
1990; Gao and Finkelstein,
1998; Gao et al.,
1996; Struhl et al.,
1989). In addition to its role as a transcriptional regulator,
Bicoid directly represses the translation of ubiquitous maternal
caudal transcripts (see Cho et
al., 2005), which would otherwise interfere with proper head
development (Mlodzik et al.,
1990; Niessing et al.,
1999). The nanos protein is expressed in a
posterior-to-anterior gradient and is essential to suppress the posterior
translation of ubiquitous maternal hunchback transcripts, which would
interfere with abdominal patterning
(Tautz, 1988). This process is
mediated by Nanos-response-elements (NREs) in the 3' untranslated region
(UTR) of hunchback mRNA (Murata
and Wharton, 1995; Sonoda and
Wharton, 1999; Sonoda and
Wharton, 2001) (reviewed by
Vardy and Orr-Weaver, 2007;
Wharton and Struhl, 1991). As
Nanos is not crucially required in other segmentation mechanisms
(Hülskamp et al., 1989;
Irish et al., 1989;
Struhl, 1989), bicoid
is the only essential determinant of AP polarity in the Drosophila
embryo. In many other cyclorrhaphan flies, this fundamental role of
bicoid is probably conserved, because in Megaselia, a basal
cyclorrhaphan taxon, suppression of bicoid results in a mirror image
duplication of the posterior abdomen, i.e. the loss of global AP polarity
(Lemke et al., 2008;
Stauber et al., 2000).
Alternative models for specifying AP polarity of insect embryos have been
proposed for Nasonia (a wasp) and Tribolium (a beetle). For
Tribolium, a short-germ insect with a large anterior serosa anlage
(Falciani et al., 1996), it
has been proposed that orthodenticle (Tc-otd1) and
hunchback (Tc-hb) substitute for bicoid
(Schröder, 2003). The
ubiquitous maternal mRNAs of both genes contain potential NRE sequences that
might explain their posterior repression, although at first both mRNAs are
translated throughout the blastoderm
(Schröder, 2003;
Wolff et al., 1995).
Tc-otd1 and Tc-hb function in a synergistic manner and
control the formation of all but two abdominal segments. All postoral segments
also require Tribolium caudal (Tc-cad), another maternally
expressed gene that is initially translated throughout the blastoderm but then
repressed anteriorly (Copf et al.,
2004; Schulz et al.,
1998). Thus, although the initial symmetry-breaking factors along
the AP axis of the Tribolium egg remain poorly characterized, AP
polarity of the Tribolium embryo can be explained by three maternal
gradients.
Nasonia evolved long-germ development independently of
Drosophila and develops likewise a dorsal serosa anlage that, unlike
in Drosophila, reaches almost to the anterior tip of the embryo
(Pultz et al., 2005). This species localizes maternal transcripts of
giant (Nvit gt) at the anterior pole
(Brent et al., 2007) and of
caudal (Nvit cad) at the posterior pole
(Olesnicky et al., 2006). In
addition, Nasonia embryos localize maternal transcripts of
orthodenticle (Nvit otd1) at the anterior and the posterior
pole (where translation is delayed) (Lynch
et al., 2006). Nvit otd1 and Nvit gt are
required for head development, but Nvit gt has only a permissive role
because the loss-of-function phenotype caused by Nvit gt RNA
interference (RNAi) is rescued by double RNAi against Nvit gt and the
head repressor Nvit Kr, a homolog of Krüppel
(Brent et al., 2007). Unlike
bicoid in Drosophila, Nvit otd1 has only a modest effect on
anterior hunchback (Nvit hb) expression, but like
bicoid, Nvit otd1 functions in synergism with anterior Nvit
hb in specifying head, thorax, and anterior abdomen
(Lynch et al., 2006). Nvit
cad is required for thorax and abdomen development
(Olesnicky et al., 2006;
Pultz et al., 1999). Finally,
because of NRE-like sequences in the mRNAs of Nvit otd1 and Nvit
hb, it has been suggested that AP polarity of the Nasonia embryo
also depends on a homolog of nanos
(Lynch et al., 2006).
In this article, we take advantage of the experimental amenability of Episyrphus to explore AP axis specification in a close relative of Drosophila and Megaselia that specifies an anterodorsal rather than a mid-dorsal (amnio-) serosa anlage and develop a new model for early AP axis specification in cyclorrhaphan flies.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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50% relative humidity
was 24-26 days.
Cloning procedures
Fragments of Episyrphus homologs have been obtained by PCR using
degenerate primers for hunchback
(Stauber et al., 2000),
nanos (5'-TGYGTGTTYTGYRARAAYAA/5'-GGYTTYTTNGGRCARTAYTT),
caudal (Stauber et al.,
2008) and orthodenticle
(5'-GGRTTYYCNCAAGGTATGTGGG/5'-ACCTGWACTCKWGATTCNGG). A fragment of
the Eba-otd homeodomain was also obtained with degenerate
bicoid primers (5'-TNGTNATGMGNMGNMGNMGNAC/5'-CKNCKRTTYTTR
AAC CA). cDNA was prepared from poly(A+) RNA of 0 to 5-hour-old
embryos (collected at 25°C) using the SMART RACE cDNA Amplification Kit
(Clontech). In the case of Eba-hb, we isolated three transcripts with
a common open reading frame and alternative first exons in the 5' UTR
(see Table S1 in the supplementary material) (S. Lemke, 2006, PhD thesis,
Molecular Biology Program, Georg-August-Universität, Göttingen).
Double-stranded RNA was generated from nucleotides 198 to 1033 of the
Eba-hb open reading frame (ORF), nucleotides 65 to 671 of the
Eba-nos ORF, 162 nucleotides of the 5' UTR and adjacent
nucleotides 1 to 686 of the Eba-cad ORF, and nucleotides 250 to 987
of the Eba-otd ORF plus adjacent 67 nucleotides of 3' UTR. To
create the template for capped Eba-nos mRNA, cDNA was PCR amplified
with the primer pair
5'-CATGCCATGGGTTATCCTGACGACATGTATAGAAATAAC/5'-ACGCGTCGACTTAAGCCTTCATGTGGTGCTTGAAATAGCT,
digested with NcoI and SalI, and cloned into pSP35
(Amaya et al., 1991). The
template for capped Eba-otd mRNA was created accordingly using the
primer pair
5'-CATGCCATGGCAGCGGGCTTTTTAAAATCTGGTGAT/5'-ACGCGTCGACTACACCATATTCACATACTTGTCTTGG.
AnNcoI site within the Eba-cad ORF was deleted by generating
two overlapping PCR fragments with the primer pairs
5'-CATGCCATGGTTTCCTATTATAACTCTCTCTCATAT/5'-GGTAATTCGATTGCCATGCCCAGGGTTGAC
and
5'-GTCAACCCTGGGCATGGCAATCGAATTACC/5'-ACGCGTCGACTCACATTGACAGCGCACCTACAGAGGCGGC,
and reconstituting the full ORF from the two fragments using only terminal
primers. The product was digested with NcoI and SalI, and
cloned into pSP35. To synthesize capped mRNA with the 5' and 3'
flanking sequences of Xenopus-globin, plasmids were linearized with
EcoRI (Eba-nos, Eba-cad) or PstI
(Eba-otd), and transcribed using the SP6 mMessage mMachine Kit
(Ambion). Embryos were injected as described
(Rafiqi et al., 2008).
In situ hybridization, antibody staining, and cuticle preparation
RNA probes for histochemical detection were all labeled with digoxigenin as
we experienced background problems in pre-syncytial blastoderm embryos with
fluorescein- and biotin-labeled probes. For fluorescent detection at later
stages, the probes were labeled with fluorescein (Eba-hb) and biotin
(Eba-zen). Embryo fixation and in situ hybridization were performed
essentially as described (Kosman et al.,
2004; Tautz and Pfeifle,
1989). The Eba-hb probe comprised 163 nucleotides of
5' UTR and adjacent nucleotides 1 to 889 of the ORF, the
Eba-nos probe comprised 174 nucleotides of 5' UTR and adjacent
nucleotides 1 to 544 of the ORF, the Eba-cad probe comprised
nucleotides 94 to 1032 of the ORF, and the Eba-otd probe comprised
nucleotides 218 to 987 and adjacent 114 nucleotides of 3' UTR. Engrailed
was detected using the cross-specific monoclonal antibody 4D9 (1/10 dilution)
(Patel et al., 1989) as
primary antibody, a biotinylated horse anti-mouse (1/500 dilution; Vector
Laboratories) as secondary antibody, and alkaline phosphatase-conjugated
anti-biotin FAB-fragments (1/2000 dilution; Roche) as tertiary antibody.
Staining was carried out as described
(Schmidt-Ott and Technau,
1992) with the following modifications: embryos were fixed in a
1:1 mixture of n-heptane and 3.7% formaldehyde in PEM (0.1 M PIPES, 2 mM
MgSO4, 1 mM EGTA, pH 6.9) for 60 minutes on a shaker. Injected
embryos were postfixed for 30 minutes in 3.7% formaldehyde in PBT (0.13 M
NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4,
0.1% Tween-20) after devitellinization. Incubation with the secondary antibody
was carried out for 2 hours, incubation with the tertiary antibody for 1 hour
at room temperature. Embryos were stained in AP (0.1 mM NaCl, 0.05 M MgCl, 0.1
M Tris pH 9.5, 0.1% Tween-20) with NBT (0.08 µg/µl)/BCIP (0.04
µg/µl) overnight at 4°C. Episyrphus first instar cuticles
were mounted as described (Stern and
Sucena, 2000) with a 2:1 mixture of Hoyer's medium and lactic
acid.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). To
determine the developmental stage at which Eba-eve and Eba-h
can be used as segmentation markers, we followed the expression of these genes
in early embryos. Prior to the onset of cellularization, the expression of
both genes appeared dynamic (see Fig. S1 in the supplementary material). The
seven Eba-eve stripes of later blastoderm stages and gastrulating
embryos were complemented by weaker Eba-eve inter-stripes, and all
Eba-eve stripes were repressed along the dorsal midline
(Fig. 2A-B'). The seven
transverse Eba-h stripes were also repressed along the dorsal midline
but most of the serosa anlage of older blastoderm embryos expressed
Eba-h (Fig.
2C-D'). An eighth Eba-h stripe at the posterior end
of gastrulating embryos retained its dorsal continuity
(Fig. 2D,D').
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). This antibody allowed us to distinguish the ocular,
antennal, intercalary, mandibular, maxillar and labial segments of the head,
the three thoracic segments (T1-3) and nine abdominal segments (A1-9)
(Fig. 2E-F'). In the
clypeolabrum, Engrailed was expressed in a (weak) patch
(Fig. 2F), and in the hindgut
Engrailed was expressed in a narrow circular ring (not shown). Denticles of first instar cuticles provided unique markers for each of the three thoracic segments T1, T2 and T3, the first abdominal segment A1, abdominal segments A2-7 and abdominal segment A8 (Fig. 3A-C). The most posterior cuticle markers were a pair of `Filzkörper'. These structures line the inner wall of the posterior spiracles and are probably an A8 derivative. The cephalopharyngeal skeleton, and the `antennomaxillary complex' (including the antenna and the maxillary sense organ) provided cuticular markers for the head region. Within the cephalopharyngeal skeleton we distinguished an anterior `median tooth' (presumably a clypeolabral derivative), a pair of mouthhooks, a medioventral `H-piece', as well as bilateral `cephalopharyngeal plates', `neck clasps' and `Lateralgräten' (Fig. 3D-H).
Expression of hunchback, nanos, caudal and orthodenticle homologs in Episyrphus
Current phylogenies place Episyrphus close to the monophyletic
higher Cyclorrhapha (Schizophora)
(Grimaldi and Engel, 2005;
Yeates and Wiegmann, 2005),
i.e. within the clade that uses bicoid mRNA as anterior determinant
(Fig. 1; see Fig. S2 in the
supplementary material) but we have not been able to identify any
bicoid-like gene in this species. We performed PCR on cDNA and
genomic DNA templates of Episyrphus using various sets of degenerate
PCR primers spanning conserved regions of bicoid inside and outside
the homeobox. These experiments yielded homeobox fragments that were
homologous to zerknüllt
(Rafiqi et al., 2008) and
orthodenticle (Michael Stauber and U.S.-O., unpublished). Because of
the conserved genomic position of bicoid immediately upstream of its
paralogous sister gene zerknüllt
(Brown et al., 2001;
Negre et al., 2005), we also
sequenced 79 kb of the Episyrphus zerknüllt
(Eba-zen) locus, including 60 kb upstream of this gene but this
approach did not yield a bicoid-like sequence either (A. M. Rafiqi,
J. Raedts, O. Schön, H. Blöcker and U.S.-O., unpublished).
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75% EL
(Fig. 4B). At the onset of
cellularization, the posterior boundary had sharpened and was positioned at
about 50% EL. Older blastoderm embryos also expressed Eba-hb at the
posterior pole (Fig. 4C) and
along the dorsal midline (Fig.
4D-F). At the onset of gastrulation, the dorsal domain coincided
exactly with the serosa anlage (see Fig. S4C-E' in the supplementary
material) but in slightly older embryos this domain appeared to be centered on
the boundary region between prospective serosa and amnion
(Fig. 4G). Eba-hb was
also expressed in the central nervous system and in yolk nuclei
(Fig. 4H). Eba-nos transcripts were detected throughout early embryos and were enriched in the posterior pole plasm (Fig. 4I). Somatic transcripts disappeared during cellularization but the germ cells continued to express Eba-nos in older embryos (Fig. 4J-L).
Eba-cad mRNA was detected in the nurse cells and the oocyte and of ovarian follicles (see Fig. S4B in the supplementary material) and was evenly distributed in early embryos (Fig. 4M). At the syncytial blastoderm stage, the anterior embryo (0%-20% EL) was cleared of Eba-cad transcripts, whereas strong zygotic expression was observed in the remaining blastoderm except in the pole cells (Fig. 4N,O). In subsequent blastoderm stages, Eba-cad expression was gradually reduced to a posterior ring, which persisted through gastrulation as a ring closing about the proctodeum (Fig. 4P-R). Other tissues did not express Eba-cad until germband retraction, at which stage a new expression domain was visible in the posterior midgut (Fig. 4S,S').
Eba-otd mRNA was not detected until the onset of blastoderm cellularization. At this stage, Eba-otd was expressed in a cap spanning the anterior pole (Fig. 4T). Slightly older embryos expressed Eba-otd in two lateral anterior patches but not along the dorsal midline (Fig. 4U-V'). During germband extension, Eba-otd was also expressed along the ventral midline and in segmental neuroblasts of the gnathocephalic, thoracic and abdominal segments (Fig. 4W-X').
Anterior pattern formation depends on two distinct localized factors that function upstream of Eba-hb, Eba-otd and Eba-cad
The posteriorly enriched maternal mRNA of Eba-nos raises the issue
of whether this gene serves as a determinant of AP polarity in the
Episyrphus embryo. Furthermore, putative NREs in the 3' UTRs of
Eba-hb and Eba-otd (Fig.
5A) raise the issue of whether the Nanos-dependent regulation of
Eba-hb and Eba-otd is important for embryonic development
despite the absence of maternal transcripts of these genes in early embryos.
To address these issues we induced Eba-nos RNAi in very early embryos
but these experiments did not perturb the process of segmentation. Resulting
cuticles were indistinguishable from wild type (n=63; data not shown)
and the majority of the larvae hatched, even when double-stranded RNA was
injected within the first 15 minutes of development, i.e. prior to the first
nuclear division cycle. We verified efficient degradation of Eba-nos
transcripts following RNAi by in situ hybridization with an Eba-nos
probe and detected Eba-nos transcripts in only one of 48 embryos.
These results suggest that Eba-nos mRNA in the embryo is not
essential for segmentation.
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To determine whether ectopic Eba-nos causes abdominal phenotypes due to interference with a factor that is produced in the anterior or the posterior embryo, we also examined cuticles from embryos that had been injected with Eba-nos mRNA at the posterior pole. These cuticles were mostly indistinguishable from wild type (70%, n=87) and many of the larvae hatched. Some cuticles exhibited defects in T3 and/or A1 (24%), which might best be explained by translational repression of Eba-hb (see below). In other parts of the embryo, suppression of markers was observed only sporadically (6%) (see Fig. S5C in the supplementary material). In particular, posterior Eba-nos mRNA injection caused defects posterior to A1 much less frequently than anterior Eba-nos mRNA injection. As a negative control we injected dsRed mRNA at the anterior or the posterior pole. These embryos developed a cuticle that was indistinguishable from wild type and mostly hatched. Together, these results suggest that ectopic Eba-nos activity targets mRNA that is localized to the anterior embryo.
To test whether the maximal Eba-nos gain-of-function phenotype can be explained by Nanos-dependent translational repression of Eba-otd and Eba-hb, we compared this phenotype with the RNAi phenotypes of Eba-hb and Eba-otd. Eba-hb RNAi cuticles exhibited wild-type denticles in abdominal segments A2-6 (100%, n=37) (Fig. 6A). T1-T3 denticles were absent, and A1 denticles were reduced (25%) or absent (75%). The denticle fields of A7 and A8 were reduced, and could not be distinguished from each other, whereas the Filzkörper were lost or reduced and spread apart. The median tooth was arc shaped, presumably because the clypeolabrum failed to involute, and the cephalopharyngeal skeleton appeared shortened and reduced (Fig. 6B). In particular, the H-piece was missing. However, the antennae, maxillary sense organs, mouth hooks and neck clasps were present (Fig. 6C,D). Eba-hb RNAi embryos lacked Engrailed expression in the labial and thoracic segments, and in the A1 epidermis (except for an Engrailed expressing cell in the anterior dorsal compartment that is characteristic for abdominal segments A1-7) (Fig. 6E,E').
In Eba-otd RNAi cuticles, the cephalopharyngeal skeleton was strongly reduced and the antennae were missing but the mouth hooks, neck clasps and maxillary sense organs were present (Fig. 6F-I). In addition, the ventral denticle fields were interrupted along the midline (compare Fig. 3C with Fig. 6F). In strong RNAi phenotypes, all thoracic and abdominal segments exhibited this defect. Less severe phenotypes showed this defects only in segments posterior to T1 or T2. In about 50% of the specimens, we noticed a cuticular irregularity between the ventral denticle fields of A4 and A5. Eba-otd RNAi embryos consistently lacked Engrailed markers of the ocular and antennal segments (Fig. 6J-K). The intercalary segment and the clypeolabrum were shifted anterodorsally and the stomodeum opened to the dorsal side (Fig. 6J').
Double RNAi against Eba-hb and Eba-otd resulted in cuticles with an additive phenotype. All segments posterior to A1 could be identified in all but one cuticle (98%), and an arc-shaped median tooth could be unambiguously identified in 38% of the cuticles (n=64) (Fig. 6L). A single cuticle lacked segmentation and displayed a strongly reduced cephalopharyngeal skeleton. In summary, ectopic anterior Eba-nos expression causes a much stronger phenotype than the combined loss of Eba-otd and Eba-hb activities, and must therefore repress the activity of at least one additional gene (Factor 1). Repression of this factor, however, does not lead to anterior caudal expression, and is not sufficient to suppress hunchback expression in a narrow anterior cap, suggesting the presence of a second anterior factor that does not respond to ectopic Eba-nos (Factor 2). Thus, our gain-of-function experiments with Eba-nos suggest that anterior pattern formation in Episyrphus is controlled by two independent anterior factors.
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Precise regulation of Eba-cad is required for embryonic development and segmentation
Eba-cad RNAi embryos rarely survived until the cuticle stage. The
few cuticles that we obtained exhibited a strongly reduced cephalopharyngeal
skeleton, a single field of small denticles similar to those in T1 or T2, and
sclerotized material at the posterior end (n=6)
(Fig. 7A). The presumptive
median tooth was arc shaped. Mouthhooks, neck clasps and the antennomaxillary
complex were tentatively identified (Fig.
7B-D). Engrailed expression of RNAi embryos was strongly reduced
and restricted to the head region (Fig.
7E-F'). The expression of pair-rule genes was reduced to one
(Eba-eve) (Fig. 7G) or
two anterior stripes (Eba-h) (Fig.
7H). The Eba-h stripes appeared dorsally incomplete and
all stripes were shifted towards the posterior pole by about 5%. Diffuse
Eba-h expression was observed in parts of the serosa anlage and in
posterior blastoderm, which, judging by a proctodeum-like ventral invagination
at about 65% EL, appeared to be excluded from older embryos. In summary,
Eba-cad is required for proper segmentation in parts of the
gnathocephalon and for specifying thoracic and abdominal segments.
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To test whether Eba-cad and Eba-nos are sufficient to determine overall AP polarity of the embryo, we injected mRNA of both genes together at the anterior pole, but these embryos did not survive. Double RNAi against the mRNAs of both genes resulted in cuticles that were mostly indistinguishable from Eba-cad RNAi cuticles (n=20). However, two of the cuticles differed from Eba-cad RNAi cuticles: their medioventral denticle field had a symmetry plane indicative of reversed planar polarity in the epidermis, and their posterior ends contained structures reminiscent of mouth hooks and maxillary sense organs (Fig. 7K,L). These cuticles suggest that some head-inducing activity may unfold at the posterior pole of severely shortened embryos when both Eba-nos and Eba-cad are downregulated.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Niessing et al.,
1999; Niessing et al.,
2000). Ubiquitous expression of a caudal transgene in the
syncytial blastoderm also causes head involution defects and, in addition,
leads to variable fusions of adjacent segment pairs along the entire embryo
(Mlodzik et al., 1990). The
much stronger gain-of-function phenotype of caudal in
Episyrphus could reflect differences in the experimental designs that
were employed. However, loss-of-function experiments also suggest that
embryonic development in Episyrphus relies more heavily on
Eba-cad than embryonic development in Drosophila does on
caudal. In Episyrphus, Eba-cad RNAi suppresses the formation
of all but one of the seven Eba-eve stripes and severely affects or
deletes most postoral segments, whereas caudal-deficient
Drosophila embryos form four out of the seven even-skipped
stripes and show segmentation in the head, thorax and even parts of the
abdomen (Macdonald and Struhl,
1986; Olesnicky et al.,
2006). The comparatively weak dependence of AP axis specification
in Drosophila on caudal can be explained by compensatory
input from the anterior gradients of bicoid and maternal
hunchback (Hülskamp et al.,
1990). In turn, the high caudal-dependence of AP
axis-specification in Episyrphus, which is similarly observed in
species that lack the bicoid gene such as Nasonia
(Olesnicky et al., 2006),
Tribolium (Copf et al.,
2004) and the cricket Gryllus
(Shinmyo et al., 2005), might
reflect the absence of maternal hunchback and/or bicoid
activities in this species.
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;
Simpson-Brose et al., 1994).
This effect is due to the translational repression of maternal bicoid
and hunchback mRNAs, which control all aspects of anterior
development (see Introduction). Both genes contain functionally important
NREs, although in wild-type embryos Nanos appears to be irrelevant for the
regulation of bicoid (Gamberi et
al., 2002; Wharton and Struhl,
1991). In Episyrphus, we did not observe any trace of
abdominal development at the anterior pole after ectopic expression of
Eba-nos, although the activity was high enough to completely suppress
the formation of all but the most posterior segments (A6-A8). This phenotype
would be expected if at least two independent factors determine anterior
development in Episyrphus, only one of which is targeted by ectopic
anterior Eba-nos activity, whereas the second factor prevents the
formation of ectopic posterior structures. We propose that the first factor
(Factor 1) consists of an anteriorly enriched NRE-containing mRNA that encodes
a protein for the early zygotic activation of Eba-otd and
Eba-hb, and that the second factor (Factor 2), which is not repressed
by ectopic Eba-nos activity, mediates the repression of
Eba-cad and part of the anterior Eba-hb activation
(Fig. 8). Factor 2 appears to
function independently of the terminal system, as neither Eba-cad nor
Eba-hb display altered anterior expression domains following RNAi
against the putative torso homolog of Episyrphus (S.L. and
U.S.-O., unpublished). Candidate genes for Factor 1 could possibly be
identified by searching for NRE-containing sequences in an early embryonic
Episyrphus EST database. In summary, AP polarity of the Episyrphus embryo appears to be determined by two distinct factors at the anterior pole. We cannot exclude that one of these factors shares homology with bicoid, but in any case our model differs significantly from AP axis specification in Drosophila, where a single protein, Bicoid, activates orthodenticle and hunchback, and represses caudal. Furthermore, the Episyrphus model differs from the Nasonia model in that the transcripts of Eba-otd and Eba-gt (the putative Episyrphus ortholog of giant; S.L., unpublished data) are of zygotic origin and not localized.
Primitive features of Episyrphus development
Episyrphus shares various traits of early embryonic development
with non-cyclorrhaphan rather than other cyclorrhaphan flies. It features an
anterodorsal serosa anlage, strong influence of caudal on the AP
axis, a (nearly) ubiquitous early zygotic activation of hunchback, as
well as hunchback expression in the serosa anlage, which has been
reported for non-cyclorrhaphan insects
(Goltsev et al., 2004; Pultz
et al., 2005; Rohr et al.,
1999; Wolff et al.,
1995) and is absent in Drosophila, Musca and
Megaselia (Sommer and Tautz,
1991; Stauber et al.,
2000; Tautz and Pfeifle,
1989). During late embryonic development, Engrailed expression in
the hindgut of Episyrphus embryos is narrow and ring-shaped (S.L. and
U.S.-O., unpublished data) similar to some non-cyclorrhaphan insects, whereas
Engrailed expression in the hindgut of other cyclorrhaphans is much broader
and restricted to the dorsal half
(Schmidt-Ott et al., 1994).
Based on the primitive features of Episyrphus development, we
speculate that the ancestral cyclorrhaphan mechanism of AP axis specification
was retained in the Episyrphus lineage. The restriction of the serosa
anlage to dorsal blastoderm in response to increased Eba-otd activity
might therefore indicate the evolutionary mechanism that altered the position
of the serosa anlage.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/117/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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