Howard Hughes Medical Institute, Department of Biology, Indiana University, 1001 East Third Street, Bloomington IN, 47405, USA
* Author for correspondence (e-mail: kaufman{at}bio.indiana.edu)
Accepted 18 December 2003
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SUMMARY |
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Key words: hunchback, hb, Short germband, Segmentation, Oncopeltus, Growth zone, RNAi, Parental RNAi
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Introduction |
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The fruitfly, Drosophila melanogaster exhibits long germband
segmentation. In this fly, embryogenesis begins with the first nuclear
divisions occurring without concomitant cellular divisions. Then nuclei fated
to form the blastoderm migrate to the egg periphery and persist as a syncytium
before cellularization. During the blastoderm stage, action of the
segmentation gene cascade serves to subdivide this blastoderm into smaller and
smaller regions, producing all the body segments (reviewed by
St Johnston and Nüsslein-Volhard,
1992). By the end of the blastoderm stage, fate maps show that all
of the future body regions including the head, thorax and the entire abdomen
are already represented proportionally on the Drosophila blastoderm
(Lohs-Schardin et al.,
1979
).
This is in contrast to short germband segmentation. In this type of
segmentation, nuclei fated to contribute to the embryo proper migrate to the
egg cortex and cellularize, as in Drosophila. However, it is during
the blastoderm phase where the differences between short and long germband
segmentation become apparent. In short germband segmentation, only anterior
segments typically the head and thoracic regions are
proportionally represented upon the blastoderm fate map. It is only after the
formation of the germband and during germband growth that the rest of the body
forms. The posterior segments arise from a disproportionately small region of
the posterior of the germ anlagen, termed the `growth zone'. This `growth
zone' has not yet been well characterized but is the posterior-most region of
the proliferating germband. Growth of this region results in germband
elongation during which the rest of the segments are specified. Thus, although
long germ segmentation can be thought of as successive spatial subdivision of
the early blastoderm, short germ segmentation entails both spatial and
temporal aspects, spatial during the blastoderm phase with the temporal aspect
occurring later during germband elongation (reviewed by
Davis and Patel, 2002).
How is the blastoderm of short germband insects patterned to yield only the anterior-most segments? How does the `growth zone' generate the posterior segments and how are these segments specified? Moreover, how did long germband segmentation evolve from the short form? Although much has been learned about the genetics that regulate long germband segmentation from Drosophila, we unfortunately know little about short germ segmentation. Our understanding of Drosophila segmentation can provide clues, but not answers to these questions. Therefore in order to gain insight into how the evolution from short to long occurred, and to get a better picture of insect segmentation in general, we must address our lack of understanding of short germband segmentation.
The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. For this reason, the roles of a few early developmental genes involved in anteroposterior axis specification have been the focus for understanding these differences. In Drosophila, the gap genes are responsible for the early subdivision of the blastoderm into broad regions, each of which will eventually encompass several adjacent body segments. As one of the essential differences between short and long germ segmentation lies in the early allocation of the blastoderm fate map, it seems reasonable to compare the action of the gap genes in short and long germ insects. We focus our attention on the gap gene hunchback (hb).
hunchback encodes a zinc-finger-containing transcription factor
known to be important for axial patterning in a number of insects
(Jürgens et al., 1984;
Lehmann and Nüsslein-Volhard,
1987
; Patel et al.,
2001
; Schröder,
2003
; Tautz et al.,
1987
). Drosophila embryos mutant for hunchback
show a gap phenotype, with deletions of the labial through third thoracic
segments and the eighth abdominal segment.
We have investigated the role of hunchback in an intermediate
germband insect, the milkweed bug, Oncopeltus fasciatus
(Hemiptera:Lygaeidae). We first examined embryogenesis and segmental
specification in this bug using engrailed staining. We then reported
the expression pattern of Oncopeltus hunchback (Of'hb)
during embryogenesis. A technique for parental RNAi has been previously
reported for Tribolium castaneum
(Bucher et al., 2002), and we
have adapted this technique for use in milkweed bugs in order to determine the
function of Of'hb in segmentation. We find that Of'hb is
required both for suppressing abdominal identity in the gnathal and thoracic
segments, and for proper growth and segmentation of the abdomen.
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Materials and methods |
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Northern analysis
Total RNA samples used in northern analysis were prepared as for cloning
(see above). Probes for both northern analysis and in situ hybridization were
synthesized from a 1.1 kb clone containing the 3' end of the
Of'hb ORF (Fig. 2C).
Northern probes were prepared with biotin-UTP (Enzo) using the MAXIscript kit
(Ambion). Northern blotting was performed using the NorthernMax kit (Ambion)
onto BrightStar-Plus membrane (Ambion) and detected using the BrightStar
BioDetect kit (Ambion).
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In situ hybridization
In situ probes were prepared with digoxigenin-UTP, biotin-UTP or
fluorescein-UTP (Roche) using the MAXIscript kit (Ambion). The in situ
protocol used here was based largely on that of O'Neill and Bier
(O'Neill and Bier, 1994) with
some modifications. After embryos were fixed, they were soaked for 1 hour in
RIPA detergent mix [150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,
1 mM EDTA, 50 mM EDTA, 50 mM Tris-HCl, pH 8.0]. Inclusion or omission of a
proteinase K digestion step did not seem to affect the in situ results. In
order to inactivate endogenous phosphatases, the embryos were incubated in
hybridization buffer [50% formamide, 5x saline sodium citrate (SSC), 100
µg/ml heparin, 100 µg/ml sonicated calf thymus DNA, 100 µg/ml yeast
RNA, 0.1% Tween-20] at 70°C for 30 minutes. Embryos were then
pre-hybridized at 60°C for 30 minutes before hybridization with probe at
60°C for 36 hours. Embryos were washed several times and soaked overnight
at 60°C in hybridization buffer. We found that short washes in lower-salt
solutions, first [50% formamide, 5x SSC, 0.1% Tween-20], then in [50%
formamide, 2x SSC, 0.1% Tween-20] helped reduce background. After
washing several times in PBTw, embryos were incubated in antibody
hybridization solution [PBTw, 2 mg/ml bovine serum albumin (BSA), 5% normal
sheep serum] for at least 1 hour. Alkaline phosphatase conjugated
anti-digoxigenin, anti-biotin or anti-fluorescein antibody (Roche) was added
to the embryos and allowed to rock overnight at 4°C. The next day, excess
antibody was washed off in several changes in PBTw. The final color
development step was carried out essentially as described Hauptmann and
Gerster (Hauptmann and Gerster,
2000
) except for two-color in situs where the first AP antibody
was inactivated by heating to 70°C for 30 minutes followed by additional
fixation for 2 hours before continuing with the second AP antibody.
RNAi
Template for the in vitro transcription reactions was prepared one of two
ways. Plasmids containing the insert of interest were linearized by
restriction digest, or template was prepared from a PCR where T3 and T7 phage
promoter sequences were added to the primers. Sense and anti-sense RNA was
synthesized in two separate reactions using the MEGAscript kit (Ambion). After
purification, the sense and anti-sense RNAs were mixed to a final
concentration of 2 µg/µl total RNA. The RNA was then annealed in
injection buffer (Spradling and Rubin,
1982) by heating in a thermocycler to 94°C and held at this
temperature for 3 minutes, then slowly cooled to 45°C over the course of 1
hour. Proper annealing of the RNA was confirmed on an agarose gel. Parts of
the gene to which these dsRNAs were made are shown in
Fig. 2C.
Embryonic RNAi injections were carried out as previously described
(Hughes and Kaufman, 2000). For
the parental RNAi injections, the dsRNA was loaded into a Hamilton #801
syringe with a 32 gauge point #2 needle. Virgin female Oncopeltus
were anesthetized in CO2 and injected in the abdomen between the
fourth and fifth abdominal sternites with
5 µl of dsRNA solution. This
volume was necessarily variable due to wound leakage. Injected females were
then reared individually with males and allowed to lay eggs. Eggs were allowed
to develop at 25°C and were harvested 76-80 hours after egg lay for in
situs, while embryos for morphological phenotypic analysis were allowed to
develop fully.
Readers are encouraged to contact the authors directly for more detailed protocols.
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Results |
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Oncopeltus embryogenesis can be divided into two distinct phases
a blastoderm phase, which in some ways is similar to that of
Drosophila, and a germband growth phase which it shares with other
short germ insects. Oncopeltus embryogenesis begins with the first
nuclear divisions occurring synchronously within the yolk mass without
concomitant cellular divisions. After several such divisions, the resulting
cleavage nuclei migrate to the egg cortex. By fifteen hours after egg lay,
they reach the surface of the egg and after an additional two hours, the
formation of cell membranes is complete
(Fig. 1A) (Butt, 1947). At this stage,
the large ovoid blastoderm superficially appears very Drosophila
like. However, blastoderm cells of a 36- to 40-hour-old embryo are not evenly
arranged around the yolk but are concentrated in two broad lateral domains on
either side of the yolk mass (Fig.
1B) that are similar to the `lateral plates' seen in
Rhodnius (Butt, 1947
;
Mellanby, 1935
).
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Oncopeltus embryos are of the `invaginating' type, which refers to the cell movements that give rise to the germband. Shortly after the formation of the blastoderm lateral plates, the germband begins to form when the cells at the posterior end of the blastoderm dive into the center of the yolk mass. The early site of invagination is marked by a small pit at the posterior pole of the late blastoderm (arrowheads in Fig. 1B,C). The cells of the blastoderm surface migrate towards the posterior, while the leading tip of the elongating germband dives into the interior of the yolk mass, towards the anterior pole of the egg. In order to visualize these movements, it is instructive to imagine the blastoderm as an inflated balloon, with invagination occurring as if a finger is poked into the interior of the balloon. Thus, the cells on the outside of the blastoderm move towards the posterior of the egg, dive into the yolk and migrate towards the anterior of the egg. This can easily be seen by comparing Fig. 1C, where six stripes of engrailed expressing cells lie on the blastoderm surface, to Fig. 1D1 and 1D2, where the invaginating germband has pulled the two posterior stripes into the yolk, leaving the four anterior stripes on the surface of the blastoderm. As germband invagination continues, the tip of the germband eventually reaches the anterior pole of the egg and the resulting germband stage embryo ends up with its head at the posterior of the egg (the embryo does eventually right itself through later embryonic movements). As these embryonic movements can potentially lead to confusion, when discussing the blastoderm we will refer to the anteroposterior and dorsoventral axis in regards to the fate maps of the tissues.
During the germband stage, the remaining posterior body segments that were
not specified during blastoderm stage are now produced through elongation of
the posterior portion of the germband coupled with progressive anterior to
posterior segmental specification. First, the abdominal region is generated
through rearrangement and growth of the posterior growth zone and then
engrailed stripes appear one by one in an anterior to posterior
direction (Fig. 1E-H). This is
similar to other short germband insects such as Thermobia domestica,
Schistocerca americana and Tribolium castaneum
(Brown et al., 1994;
Patel et al., 1989
;
Peterson et al., 1998
). Thus,
it is clear that in Oncopeltus, as in other short and intermediate
germband insects, posterior segments arise during a secondary growth phase
during which the posterior germband undergoes great elongation with
specification of abdominal segments occurring sequentially and in an anterior
to posterior direction.
Milkweed bug hunchback gene structure
In order to clone Oncopeltus hunchback (Of'hb), we
designed degenerate primers to the conserved zinc-finger domain of known
hunchback sequences. We then performed PCR using these primers on
cDNA made from ovaries or mixed stage embryos and isolated a short initial
Of'hb clone. This clone allowed us to then design exact primers for
5' and 3' RACE and isolate fragments of Of'hb that
together total 2.1kb and encode the entire open reading frame and regions of
the 5' and 3' UTRs.
Of'hb is predicted to encode a 64.3 kDa protein with a total of
eight zinc-finger domains. These zinc fingers are clustered with an N-terminal
pair, a central cluster of four and a C-terminal pair
(Fig. 2C). These eight
zinc-finger domains are shared with the hunchback genes from two
grasshopper species, Locusta migratoria and Schistocerca
americana, while Drosophila melanogaster and Tribolium
castaneum hunchback each encode only a total of six zinc fingers
(Fig. 2A)
(Patel et al., 2001;
Tautz et al., 1987
). The six
fingers from the fly and beetle hunchback correspond to the central
four and the C-terminal two fingers from the milkweed bug and the
grasshoppers. Alignments of the homologous finger regions of grasshopper and
milkweed bug hunchback show that the N-terminal zinc fingers are the
most divergent.
As both the Schistocerca
(Patel et al., 2001) and
Oncopeltus hunchback encoded proteins contain eight zinc fingers,
this is likely the ancestral state. However, Tribolium and
Drosophila hb each contain only six zinc fingers, which suggests that
six fingers is the ancestral state for the holometabola
(Patel et al., 2001
;
Tautz et al., 1987
;
Wolff et al., 1995
). If this
is the case, then somewhere in the lineage leading to holometabola,
hunchback lost its two N-terminal-most fingers
(Fig. 2D). Unfortunately, as
the function of the hb encoded protein itself has been studied only
in Drosophila (which lacks these fingers), the function of these
metal-binding fingers is unknown. It would be fascinating to examine the
specific function of these ancient N-terminal fingers and see whether they can
be correlated with developmental changes that have evolved in the
holometabola.
In addition to the zinc-finger domains, three other domains were found to
be conserved. The A-Box, originally identified in Drosophila as a
region of similarity between hunchback, Kruppel and the HIV pol gene
(Tautz et al., 1987), is also
found in Oncopeltus hunchback. Additionally, sequences similar to the
Drosophila hunchback Basic Box and the C-Box are also present
(Fig. 2B)
(Hülskamp et al.,
1994
).
Of'hb transcript is maternally expressed and loaded into oocytes
Northern analysis on both maternally and zygotically derived total RNA
revealed a single band of 3.2 kb in both samples, showing that
Oncopeltus hunchback is expressed in the ovary
(Fig. 3A). As our Northern
analysis shows the presence of a larger transcript than our cloned fragments
(the 5' and 3' RACE products together total 2.1 kb), our clones
must not represent the entire Of'hb transcript.
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Oncopeltus hunchback is expressed in two broad bands in the blastoderm
Oncopeltus hunchback expression occurs in a distinct blastoderm
pattern and a separate germband pattern that reflects the distinct blastoderm
and germband phases of milkweed bug embryogenesis. In early embryos before
blastoderm formation (12 hours after oviposition at 25°C), hb
transcript accumulates homogeneously throughout the egg (not shown). Shortly
after, hb expression appears more strongly in the central region of
the blastoderm (Fig. 4A). At
20-24 hours, this central domain becomes more strongly refined
(Fig. 4B). By 24-28 hours, the
single broad domain of expression begins to contract from the poles and
resolve into two bands. The weaker, more anterior band spans 69-84% egg-length
(with 0% being the posterior) while the stronger more central band covers
40-64% egg-length (Fig. 4C). As
Oncopeltus is an intermediate germ insect, this region of the
blastoderm corresponds to different segments than what would be expected in a
long germ insect, such as Drosophila. Thus, in order to determine the
approximate segmental register of Of'hb expression on the blastoderm,
images of milkweed bug embryos separately stained for hunchback and
engrailed were juxtaposed. This allowed us to determine that the
anterior band spans the region of the blastoderm anterior to the mandibular
en stripe, while the posterior band of hunchback appears to
span the maxillary and labial segments
(Fig. 4E).
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Oncopeltus hunchback germband expression
During the germband phase of embryogenesis, Oncopeltus hunchback
is expressed in a very different pattern than during blastoderm phase. If the
broad blastoderm domains persisted through germband invagination, one would
expect that in germbands, hunchback would likewise stain in a
gap-like pattern in the gnathal/prothoracic region. Interestingly, this is not
the case. From very early through very late germband stage embryos,
Of'hb is never detected in an anterior gap gene-like domain
(Fig. 5). Early germbands still
undergoing invagination show very weak hb staining in a symmetrical
chevron pattern which is segmentally reiterated and is excluded from the tip
of the germband (Fig. 5A,B).
This blocky segmental expression is most likely not a continuation of the
earlier blastoderm domain but rather represents de novo expression in this new
pattern. This expression is similar to a segmentally reiterated pattern also
seen in Musca and Tribolium and may represent mesodermal
staining as it does in Schistocerca
(Patel et al., 2001;
Sommer and Tautz, 1991
;
Wolff et al., 1995
). As the
broad bands of Of'hb expression in the blastoderm are already
beginning to weaken by late blastoderm, it is likely that expression in these
segments has already faded below detection by the time the germband completes
invagination. This is in contrast to the hunchback expression
patterns in both Schistocerca and in Tribolium, where it is
expressed in a broad gap-like domain encompassing the gnathal to anterior
first thoracic segments in early germbands
(Patel et al., 2001
;
Wolff et al., 1995
).
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Earlier, just before germband invagination, there is a small patch of hunchback expression in the posterior of the blastoderm (arrow in Fig. 4C1). As hb is expressed in the posterior growth zone during abdomen formation, these two domains may at first glance seem to represent continuous hb expression. However, this is not the case as early germbands, which are just beginning the invagination process, do not express hb at the posterior tip (Fig. 5A,B). It is only after invagination is complete and during the formation of the abdominal segments that hb expression reappears in the growth zone. Therefore, these two domains cannot represent a continuous expression of hb. Rather, the patch of expression in the `growth zone' must represent a de novo initiation of hunchback transcription.
hunchback is also expressed in a pattern that may represent the
developing nervous system. The punctate pattern in late germbands are
reminiscent of neural expression patterns and are consistent with hb
neural expression in other insects and its function in Drosophila
(Isshiki et al., 2001;
Patel et al., 2001
;
Wolff et al., 1995
). This
neural-like expression suggests that hunchback is also required for
neurogenesis in Oncopeltus.
Oncopeltus parental RNAi
Recently, it has been reported that female Tribolium pupa which
had been injected with double-stranded RNA (dsRNA) produce progeny showing an
RNAi knockdown phenotype (Bucher et al.,
2002). In order to test this method in Oncopeltus, we
injected dsRNA corresponding to a region of Oncopeltus Sex combs
reduced (Of'Scr), a homeotic gene, into the abdomens of adult
virgin females and scored their progeny for defects. These progeny had their
labial appendages transformed into a pair of appendages of mixed
leg/antennal-like identity (Fig.
6B) a phenotype identical to that already published for
Of'Scr by direct injection of dsRNA into early embryos
(Hughes and Kaufman, 2000
). We
achieved much higher penetrance and a similar range of phenotypes using
parental RNAi (pRNAi) when compared with embryonic RNAi (eRNAi). All surviving
injected females eventually produced clutches of embryos with the Scr
phenotype and these animals showed the complete range of defects as reported
earlier using embryonic RNAi. Sometimes, the first clutch laid contained
wild-type hatchlings most likely because these eggs had already
completed oogenesis and laid down their chorions that would be impenetrable to
dsRNA. These same females would later go on to lay clutches that did show the
Scr knockdown phenotype. We also found that over the span of about 3
weeks, the severity of defect would first increase, peaking around at 10 days
post-injection and then gradually decrease after that
(Fig. 6C). As pRNAi showed
identical phenotypes as eRNAi without any injection artifacts and showed the
full range of severity, this technique should prove to be a highly specific
and convenient method for studying gene function in Oncopeltus.
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hunchback suppresses abdominal identity
The first aspect of the hunchback depletion phenotype is a
homeosis of the gnathal and thoracic regions towards abdominal identity. Class
I embryos (the weakest phenotypic class) made up 8.3% of the embryos
(Table 1). These embryos showed
strong suppression of the labium and mildly defective first thoracic legs
(Fig. 7B1,B2). The fact that
the labial segment is most strongly affected demonstrates that this segment is
at the epicenter of defect and is consistent with the strong band of
hunchback expression spanning the maxillary through anterior
prothoracic segments in the blastoderm.
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We reasoned that transformation of the gnathal and thoracic regions towards abdomen should be accompanied by expression of abdominal genes in these transformed segments. In wild-type animals, the homeotic gene abdominal-A (abd-A) is expressed in the abdomen from the second through eighth abdominal segments (Fig. 7E). Therefore, we examined the expression domains of abd-A in embryos that were depleted for hunchback. Although we did not detect abd-A expression during the blastoderm stage in hb RNAi embryos (data not shown), we did find ectopic expression of abd-A during the germband stage. In hb-depleted germband stage embryos, abd-A was ectopically expressed in the gnathal and anterior thoracic segments, coincident with suppression of appendages on these segments (Fig. 7F,G). In weaker hypomorphs, the ectopic domain spanned the posterior of the maxillary segment through the anterior of the first thoracic segment and leg (Fig. 7F). In more strongly affected RNAi embryos, the ectopic domain of abd-A was more expanded into the first and second thoracic segments and was associated with suppression of legs on these segments (Fig. 7G).
The ectopic expression of abd-A shows that hb RNAi embryos at both the morphological and molecular level show a homeosis of the labium and thorax towards abdominal identity. Moreover the hypomorphic series shows that the domain of transformation starts at the labial and first thoracic segments in weakly affected animals, expands posteriorly to include the second thoracic segment in moderately affected ones, and incorporates the third thoracic segment in strong hypomorphs, which suggests that the labial and first thoracic segments are the most sensitive to hunchback depletion.
Severe hunchback depletion results in posterior compaction
In addition to the anterior homeotic transformations described above,
hunchback depletion revealed another defect apparent in the abdomen.
Decreasing hb activity results in increased posterior compaction.
This defect is most evident through the hypomorphic series where a trend
towards smaller and more defective abdominal segments in stronger phenotypic
classes is apparent. Whereas class I embryos show a largely wild-type abdomen
(Fig. 7B1), class II embryos
show mild compaction of the abdomen (compare
Fig. 7C1 with 7A1). This
compaction increases and is often associated with segmental defects in class
III embryos (Fig. 7D1). Embryos
of stronger phenotypic classes are smaller than normal overall, and the
abdomen shows much stronger compaction (compare the hb RNAi embryos
in Fig. 8A,C2,D to the
uninjected animals in Fig.
7A1,A2). In class IV embryos, a tiny and deformed posterior leg is
often situated near the extreme posterior of the animal
(Fig. 8B,C1). As the third
thoracic segment is the last to be transformed and its leg is most resistant
to suppression, this posterior appendage is most likely the partially
suppressed remnants of the third thoracic leg. In these animals, the gnathal
and anterior thoracic segments are strongly transformed towards abdomen,
resulting in suppression of their appendages, while the third thoracic segment
is only partially transformed, leaving a remnant of the third leg. Therefore,
the posterior position of these stubby legs underscores the extreme degree of
posterior compaction in these animals. Coupled with the homeosis described
above, strongly affected animals would thus have a normal head followed by
several abdominal-like segments that consist of a transformed labium and
thorax and a tiny compacted abdomen.
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The most severe class of animals comprised 11.3% of the total
(Table 1) and the
interpretation of this class is based somewhat on an extrapolation of the
hypomorphic series. These embryos are extremely small and are found buried in
the yolk mass. In normal embryogenesis, the germband undergoes dorsal closure
to encircle the yolk so in the wild-type case, the yolk mass ends up inside
the embryo. In class V embryos, it appears instead that the embryo develops
without enveloping the bulk of the yolk and thus much of the egg space is
filled with unused yolk. Upon closer examination of these embryos it seems
that some internal organs may have actually developed on the outside of the
embryo, similar to the `everted' embryos described by Sander
(Sander, 1976). In this class
of animals, only the anterior-most structures are clearly identifiable. Eyes
and antenna develop, but the rest of the body is highly reduced and composed
of fewer and smaller segments (Fig.
8E). It is important to note that although this body region is
small, it is apparent that some segmentation has occurred. In a putative class
V germband, segmentation of the anterior is apparent, with a few anterior
segmental grooves clearly forming (arrows in
Fig. 8H). This region of
somewhat normal segmentation is followed by a highly defective region.
abd-A in situs show that aside from the head and mandibular segments,
the entire trunk expresses abd-A. Therefore, in this extreme class of
hb depletion, posterior segmentation is highly defective and all of
the post-maxillary body has adopted an abdominal fate. This phenotypic class
may represent the most severe combination of the two aspects of
hunchback depletion where the labial and thoracic segments are
completely transformed towards abdominal identity and the abdomen has been
severely compacted. This results in an animal in which the head is followed by
a small number of segments, all of which have abdominal identity.
Our complete model including both aspects of hunchback function in Oncopeltus is presented in Fig. 9. We propose that as Of'hb function is depleted, first the labium and then the thorax is transformed towards abdominal identity. A second requirement is evident in the abdomen. In increasingly severe hb depletions, growth and segmentation in the abdomen becomes increasingly defective resulting in posterior compaction. In addition, apparent is a lack of clear segmental defects in the gnathal and thoracic regions, even in strongly affected animals. Therefore, in the most severe cases, a normal head is followed by a few segments of abdominal identity. Although this would at first glance appear to mimic the hunchback gap phenotype in Drosophila and Tribolium, we conclude that the strongest phenotype is due to the combination of gnathal and thoracic transformation towards abdomen coupled with severe posterior compaction.
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Discussion |
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Oncopeltus hunchback expression and function in the labium and thorax
Oncopeltus hunchback expression and function reflect the biphasic
nature of milkweed bug embryogenesis. hb is expressed in two broad
stripes during the blastoderm stage. The stronger band spans the posterior
maxillary, labial and anterior first thoracic segments. As hb is not
expressed in these segments during the germband stage (except in the mesoderm
and a neural-like domain), we attribute its region-specifying function in
these segments to its expression domain in the blastoderm. This
abdomen-repression function in the labial and thoracic segments may occur
either through direct suppression of abd-A in the anterior, or may
occur indirectly through regulation of a downstream gene responsible for
specifying abdominal regional identity. As the ectopic domain of
abd-A in hb RNAi animals was not detected at the blastoderm
stage but only later during the germband stage and the region of homeosis in
hb RNAi animals is much larger than the hb blastoderm
expression domain, it is most likely that hb indirectly regulates
abd-A. Indeed, normal abd-A expression in the abdomen
appears long after hb expression in the growth zone has already
faded. Our observation that hunchback in milkweed bugs serves to
repress abdominal identity is not without precedence. In Drosophila,
certain hypomorphic alleles of hunchback, class V alleles, also
produce homeotic transformations of the gnathal or thoracic segments
(Jürgens et al., 1984;
Lehmann and Nüsslein-Volhard,
1987
). However, unlike Oncopeltus, these transformations
are superimposed on a deletion phenotype.
As in flies, hunchback RNAi depletion in the red flour beetle,
Tribolium castaneum, also produces the gnathal and thoracic gap
phenotype (Schröder,
2003). In this light, it is interesting that Oncopeltus
hunchback knockdowns do not show the canonical gap phenotype but rather a
transformation. This may reflect either incomplete RNAi knockdown of the
Of'hb gene product or may reflect differences in anterior-posterior
patterning in milkweed bugs. It is possible that if further depletion were
possible, the anterior homeotic phenotype would be replaced by deletion of
those segments. However, this would be in contrast to the case in
Drosophila as weak alleles of hunchback in flies are not
associated with transformations but rather yield small deletions while
stronger alleles serve to increase the size of the deleted region
(Lehmann and Nüsslein-Volhard,
1987
). The Oncopeltus hunchback phenotype reported here
seems to reflect a strong depletion of the hunchback gene product
because of the large domain of the homeotic defect from the labium to the
third thoracic segment in the severe phenotypic classes. Transformation of the
third thoracic segment is indicative of strong hb RNAi yet in these
same animals, the labial segment is present and without segmental defects. If
deletion of the labial segment were merely an issue of sensitivity, we would
expect that animals with transformed third thoracic segments also show
segmental defects in the labial segment. Therefore, if hunchback has
a gap function in this animal, its requirement must be minimal.
We have shown that hunchback transcript is provided maternally in
Oncopeltus and it is a formal possibility that the protein is as
well. If this were the case, it is possible that maternal hunchback
serves to specify the presence of the gnathal and thoracic segments, while
zygotic activity functions to suppress abdominal identity in these regions.
Although maternal loading of hunchback-encoded protein has not been
reported in either flies or beetles, the protein is provided maternally in
grasshoppers. In grasshoppers however, axial patterning by hunchback
appears to be performed entirely by zygotic function whereas maternal
hunchback activity in this animal may serve to distinguish embryonic
from extra-embryonic cells (Patel et al.,
2001).
Oncopeltus hunchback expression and function in the growth zone
In strongly affected Oncopeltus hunchback RNAi animals, the
abdomen is severely compacted and segmentation is defective.
hunchback function in the developing germband probably reflects its
expression in the posterior `growth zone'. Therefore we propose that
Of'hb is required for proper growth and segmentation of the posterior
germband. At this time we can only speculate on the nature of this
requirement. It may be that Of'hb is directly involved in the
generation of segments as the posterior germband grows. However, it may also
be that Of'hb is merely required for posterior elongation of the
germband while the actual patterning of segments occurs relatively
independently of growth. Thus, the segmental defects seen in developing
germbands may be a consequence of improper elongation. Alternatively,
hunchback may be required for proper functioning of the growth zone
itself.
hunchback has also been examined in Schistocerca and
Tribolium, two other short germband insects. Schistocerca
hunchback is not expressed continuously in the growth zone as it is in
both milkweed bugs and beetles but rather arises in abdominal patches
corresponding to the A4/A5 and A7-A9 segments
(Patel et al., 2001).
Therefore the continuous expression in the growth zone may represent a derived
pattern in the insects. As noted, Tribolium and Oncopeltus
hunchback are expressed in identical patterns in the growth zone.
However, it has not yet been reported that hb RNAi in
Tribolium leads to posterior compaction, rather the phenotype
reported is the canonical hunchback gap phenotype. With such
disparate expression and functional data from these three insects, it is
difficult to determine the ancestral function of hunchback in the
insect growth zone and it is clearly imperative that wider taxonomic sampling
needs to be done.
This brings us to examinations of the very nature of the insect growth zone
itself. Almost nothing is known about how this special region of the germband
develops and ultimately gives rise to the posterior segments. There are no
overt morphological features that distinguish it. However, the growth zone
must be special as several segmentation genes such as even-skipped,
caudal and hunchback are expressed there
(Dearden and Akam, 2001;
Patel et al., 1994
;
Wolff et al., 1995
) (P.Z.L.,
unpublished). Given that some form of short germband development is ancestral
in insects, we must understand this mode of development in order to understand
the evolutionary transition from short to long germ segmentation. Moreover, as
other arthropods undergo embryogenesis in a manner similar to insect short
germband segmentation, functional studies in Oncopeltus and other
short germband insects may in fact shed light on all the
arthropods.
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ACKNOWLEDGMENTS |
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