1 Department of Biology, Western Washington University, Bellingham, WA 98225,
USA
2 Department of Biology, New York University, New York, NY 10003, USA
¶ Author for correspondence (e-mail: pultz{at}biol.wwu.edu)
Accepted 13 June 2005
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SUMMARY |
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Key words: Nasonia, Hunchback, Axis formation, Evolution of development, Hymenoptera, Drosophila
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Introduction |
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Anteroposterior axis formation is best understood in Drosophila,
where early embryogenesis takes place extremely rapidly and depends heavily on
maternal input (St. Johnston and
Nüsslein-Volhard, 1992;
Rivera-Pomar and Jäckle,
1996
). The bicoid homeodomain morphogen is provided
maternally as mRNA localized to the anterior of the oocyte
(Berleth et al., 1988
). Bicoid
synergizes with Hunchback, a zinc-finger protein, in controlling anterior
development (Simpson-Brose et al.,
1994
). hunchback, a gap gene, transcriptionally controls
other gap genes, as well as pair-rule and homeotic genes
(Pankratz and Jäckle,
1993
; Simpson-Brose et al.,
1994
; Tautz and Sommer,
1995
; Casares and
Sánchez-Herrero, 1995
;
Fujioka et al., 1999
;
Shimell et al., 2000
;
Wu et al., 2001
;
Clyde et al., 2003
).
hunchback, in contrast to bicoid, is provided maternally as
unlocalized mRNA, and is expressed zygotically under the control of
bicoid and other transcriptional regulators
(Bender et al., 1988
;
Schröder et al., 1988
;
Tautz, 1988
;
Margolis et al., 1995
).
Although not essential, maternal hunchback does control some
head-determining functions in wild-type Drosophila, as embryos
lacking all maternal and zygotic products have a larger anterior gap than
those lacking only zygotic hunchback
(Lehmann and Nüsslein-Volhard,
1987
). Maternal hunchback must be translationally
repressed by nanos for normal posterior development
(Hülskamp et al., 1989
;
Irish et al., 1989
;
Struhl, 1989
).
To better understand the evolution of anteroposterior patterning, we have
taken advantage of the haplo-diploid genetic system of the wasp Nasonia
vitripennis to screen for mutations affecting cuticular morphology. In
haplo-diploids, fertilized eggs develop as diploid females while unfertilized
eggs develop as haploid males, facilitating a screen of the genome for
recessive zygotic mutations (Pultz and
Leaf, 2003). We identified about one-fourth to one-third of the
genes required to pattern the Nasonia embryo, including
representatives of gap, pair-rule and Polycomb-group genes with varying
degrees of functional similarity to known Drosophila genes
(Pultz et al., 2000
). Three
zygotic mutations caused extensive disruptions of early patterning, more
severe than the defects caused by any known zygotic mutation in
Drosophila. One of the Nasonia mutations, originally named
headless, deletes all of the head except the most anterior labral
segment, as well as thoracic and posterior abdominal segments
(Fig. 1A)
(Pultz et al., 1999
).
The headless mutant phenotype suggested a similarity to
Drosophila hunchback. Zygotic loss of hunchback in
Drosophila causes a gap deletion from the posterior labial segment
the posterior border of the head through the thoracic
segments, and also affects posterior abdominal segments
(Fig. 1A)
(Bender et al., 1987
;
Lehmann and Nüsslein-Volhard,
1987
). We hypothesized that the headless mutant phenotype
is caused by a mutation in Nasonia hunchback, and that zygotic
hunchback plays a more extensive role in embryonic patterning in
Nasonia than in Drosophila
(Pultz et al., 1999
).
Non-dipteran insects must initiate embryonic patterning using different
methods from those of Drosophila. Although bicoid controls
the development of head, thorax and anterior abdomen in Drosophila,
the bicoid gene has apparently arisen only relatively recently,
within the higher Diptera (Stauber et al.,
1999; Brown et al.,
2001
; Stauber et al.,
2002
). bicoid encodes a homeodomain protein with a key
lysine at position fifty (K50) of the homeodomain, and is hypothesized to have
usurped functions originally controlled by orthodenticle, which also
encodes a K50 homeodomain protein; in addition, hunchback is
hypothesized to have played a more extensive role in patterning the anterior
of more ancestral insects (Wimmer et al.,
2000
; Lynch and Desplan,
2003a
). Parental RNA interference experiments in the beetle
Tribolium have indicated that the orthodenticle gene plays a
major role in patterning the anterior of this non-Dipteran insect; when both
Tribolium orthodenticle and Tribolium hunchback
(Wolff et al., 1995
) are
knocked down, very little remains of the segmental patterning in the
Tribolium embryo (Schröder,
2003
). The potential role of hunchback as an ancestral
morphogen has also been tested by manipulating hunchback expression
in Drosophila, where increased levels of bicoid-independent
hunchback have been shown to be capable of patterning the abdomen and
even the thorax in the absence of bicoid
(Hülskamp et al., 1990
;
Struhl et al., 1992
;
Schulz and Tautz, 1994
;
Wimmer et al., 2000
).
Studies of hunchback in representatives of more ancestral insects
have provided an intriguing perspective on the evolution of this key
regulatory gene. In the milkweed bug Oncopeltus, hunchback mRNA is
expressed both maternally and zygotically, and embryos with knocked-down
hunchback function exhibit transformations of gnathal and thoracic
segments to an abdominal identity, as well as impaired germ-band development
(Liu and Kaufman, 2004). By
contrast, in the more ancestral grasshopper, Schistocerca, hunchback
is provided maternally to the embryo as protein, rather than as mRNA, through
release from the posteriorly located oocyte nucleus, suggesting that its
function may be to distinguish embryonic from extra-embryonic cells in that
short-germ embryo (Patel et al.,
2001
). A later graded expression of Schistocerca
Hunchback is provided zygotically, consistent with a concentration-dependent
role in axial patterning. These results indicate that the ancestral
hunchback axis-determining function in insects is likely to be
zygotic, and that the expression and function of maternal hunchback
has significantly changed during insect evolution.
Here, we focus on the role of hunchback in the hymenopteran
Nasonia vitripennis. Nasonia has long-germ embryos, with a syncytial
mode of early development that is morphologically similar to
Drosophila embryogenesis although unlike Drosophila,
Nasonia does not have the highly derived condition of extremely rapid
early development. In fact, approximately three-fold more time is allocated to
early development (prior to gastrulation) in Nasonia than in
Drosophila (Fig. 1B)
(Bull, 1982;
Campos Ortega and Hartenstein,
1985
) allowing more time for the zygotic genome to control
early development. The Hymenoptera have evolved diverse embryonic
developmental strategies. These include embryos with derived holoblastic
cleavage (Grbic and Strand,
1998
), as well as several independently evolved cases of
polyembryony, in which a single fertilized egg develops into hundreds or
thousands of clonal progeny (Strand and
Grbic, 1997
; Grbic,
2000
). Syncytical long-germ development is considered to be
ancestral in the Hymenoptera (Strand and
Grbic, 1997
), so Nasonia can be considered to be a
representative of the ancestral mode of development within this clade.
We show that the severe Nasonia headless zygotic-mutant phenotype is caused by a mutation in Nasonia hunchback, and we describe the expression of Nasonia hunchback mRNA and protein. We also compare molecular mutant phenotypes of Nasonia embryos lacking zygotic hunchback to those of Drosophila embryos lacking both maternal and zygotic hunchback. We propose that the divergent mutant phenotypes for the same gene in two different species may, in large part, be due to changes in the functionally overlapping genetic regulatory network.
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Materials and methods |
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To identify a Ng-specific hunchback single nucleotide polymorphism, a Ng-specific forward primer (5'-CCATCTGCGCAAGCA-3'), and a species non-specific reverse primer (5'-GCAGTCGCAGCACCT-3') were used to amplify a Ng hunchback fragment with the following PCR cycling conditions: 95°C for 30 seconds, 58°C for 60 seconds, 72°C for 60 seconds for 33 cycles.
To determine linkage, Nv headless-bearing females were crossed to
Ng males cured of Wolbachia with antibiotics, kindly
provided by Jack Werren (University of Rochester, NY, USA). The Nv
headless/Ng headless+ F1 hybrids were sorted from
their Nv headless+/Ng headless+
control sisters by assaying their embryos. Experimental and control females
were set unmated, then DNA was prepared from single surviving adult F2 males
(Gloor et al., 1993). DNA that
failed to amplify with the Ng-specific primer was shown to support
amplification with species non-specific primers.
Analysis of genomic DNA from headless mutant embryos
The deletion in Nasonia hunchback was characterized by isolating
DNA from 30-50 selected headless mutant embryos
(Gloor et al., 1993). PCR
amplification with primers at the 5' and 3' ends of the coding
region generated a product approximately 1.5 kb shorter than the wild-type
product, indicating a deletion. The mutant product was cloned and sequenced.
The precise size of the deletion was 1497 bp, consistent with the PCR
analysis. Identical sequences across the breakpoint were obtained from two
independently amplified reactions.
Collection and fixation of Nasonia embryos and ovaries
When Nasonia embryos are collected from virgin females, all
embryos are precisely staged there are no older embryos from
previously fertlized eggs as in Drosophila. Embryos after
gastrulation were fixed as described in Pultz et al.
(Pultz et al., 1999). Most of
the blastoderm embryos were shaken in heptane for 2 minutes, then an equal
volume of methanol was added and they were shaken for an additional 2-3
minutes at room temperature. Later, we found that sufficiently dry blastoderm
embryos can also be fixed in 1:1 heptane: 4% formaldehyde in 1xPBS,
improving morphology. Very early embryos (0-3 hours old) cannot be effectively
devitellinated with methanol. These were fixed for 1 hour in heptane
pre-saturated with 37% formaldehyde, then hand-peeled on double-stick tape in
1xPBS. Older hand-peeled embryos with a known expression pattern were
included as a positive control. All embryos were males, collected from virgin
mothers. To avoid cross reactivity of the anti-Nasonia hunchback
antibody with endosymbiotic bacteria, we used wild-type Nasonia
vitripennis cured of Wolbachia (a gift from Jack Werren), and we
cured the hunchbackhl stock of Wolbachia by
treating the mothers for two generations with rifampicin. Ovaries were
dissected from mothers, fixed for 10 minutes in 8% formaldehyde, dehydrated
and stored in methanol or ethanol until used for antibody staining or in situ
hybridization, respectively.
In situ hybridization
Nasonia hunchback mRNA was visualized using an anti-sense RNA
probe, as described previously (Jiang et
al., 1991). The probe, about 1100 bp in length, extended from exon
2 through the central zinc-finger region (see
Fig. 3). As a negative control,
a probe was prepared from the opposite strand of the same fragment, and was
applied to samples of all ages of embryos and tissues analyzed. No staining
was observed with the negative controls.
Anti-Nasonia hunchback antibodies
A 125-amino-acid region beginning at amino acid 79 and terminating before
the NF1 zinc finger was PCR-amplified using forward
(5'-GTTGTTGAATTCGCTGGGATAAAATCGTA-3') and reverse
(5'-GTTGATAAGCTTGGGCAGCTCGAATCC-3') primers, then cloned into the
EcoR1 and HindIII sites of pGEX-KG, producing a
GST-hunchback fusion protein. The fusion protein was isolated as described by
Leaf and Blum (Leaf and Blum,
1998) and injected into rabbits for the production of polyclonal
antiserum.
Antibody-staining experiments
The anti-Nasonia Hunchback antibodies were used at a dilution of
1:1000 to stain Nasonia embryos. All staining patterns observed in
wild-type embryos of cellular blastoderm age and older were
verified to be absent in hunchbackhl mutant embryos. The
FP6.87 monoclonal antibody (Kelsh et al.,
1994), which recognizes conserved epitopes on both Ultrabithorax
(Ubx) and Abdominal-A (Abd-A) proteins was used at a dilution of 1:7 to stain
Nasonia and Drosophila embryos. The anti-Drosophila
hunchback guinea pig polyclonal antibody
(Kosman et al., 1998
) was used
at a dilution of 1:400. All antibodies were visualized with horseradish
peroxidase-labeled secondary antibodies and diaminobenzidine substrate, as
described by Pultz et al. (Pultz et al.,
1999
).
Drosophila crosses
To analyze maternal Hunchback expression, embryos were collected from
parents heterozygous for Df (3R) p25, which deletes the
5' end of the hunchback transcription unit and does not produce
hunchback mRNA (Bender et al.,
1988). To analyze Ubx-Abd-A expression in embryos lacking both
maternal and zygotic hunchback, we used a hbFB
FRT strain kindly provided by Ernst Wimmer (Georg-August-University
Göttingen, Germany), collecting the embryos from FLP-bearing
hbFB FRT/ovoD mothers that had been heat
shocked as larvae to induce clones homozygous for the null hunchback
mutation in their ovaries (Dang and
Perrimon, 1992
). These females were crossed to
hb14F/TM3 males, such that approximately 50% of the
offspring lacked both maternal and zygotic hunchback, whereas the
other 50% lacked only maternal hunchback. Because maternal
hunchback is not needed by Drosophila embryos in the
presence of zygotic hunchback, half of the embryos showed a wild-type
pattern of Hox gene expression. The other half, lacking both maternal and
zygotic hunchback, were severely defective. The mutant phenotypes
were confirmed using cuticle preparations.
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Results |
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To identify the headless mutation, genomic DNA from headless mutant embryos was amplified and sequenced, revealing a deletion of 1497 bp after the first 40 amino acids of the predicted protein-coding sequence (assuming that translation starts in exon 2). As shown in Fig. 3B, this deletion disrupts the reading frame for the protein-coding sequence. This most likely defines a null allele for the Nasonia hunchback gene (see Discussion), consistent with our hypothesis that the very severe headless mutant phenotype is caused by a loss of zygotic Nasonia hunchback. Consequently, we re-designated Nasonia headless (hl) as Nasonia hunchbackhl.
Does Nasonia hunchback have candidate Nanos-response elements?
The translational regulation of Drosophila hunchback is mediated
by the binding of Pumilio to Nanos Response Elements (NREs) within the
3' untranslated region (UTR), and the subsequent recruitment of Nanos
and Brain Tumor to form a quarternary complex
(Murata and Wharton, 1995;
Sonada and Wharton, 2001
;
Wang et al., 2002
).
Figure 3C shows candidate NREs
from Nasonia hunchback, which are similar to the conserved Box A and
Box B of the Drosophila hunchback NREs. The canonical
hunchback NREs have a characteristic spacing of three to four bases
between Box A and Box B. By contrast, the candidate NREs of Nasonia
hunchback have 12-16 bases separating Box A and Box B, reminiscent of the
structure of a candidate NRE found in the 3' UTR of Drosophila
cyclin B1 mRNA (Wang et al.,
2002
). In the germline, Pumilio and Nanos translationally repress
Drosophila cyclin B1 expression
(Nakahata et al., 2001
). The
presence of candidate NREs is of interest in light of the difference between
the mRNA and protein expression of Nasonia hunchback described
below.
Wild-type expression of Nasonia hunchback
To determine whether Nasonia hunchback is expressed maternally,
and how embryonic expression compares with that of other insects, we examined
Nasonia hunchback mRNA expression, and we raised an antibody against
part of Nasonia Hunchback (HB-GST,
Fig. 3). We found that
hunchback mRNA is supplied to the embryo maternally, from high-level
expression in the oocyte (Fig.
4), and as an mRNA that is dispersed throughout the egg (data not
shown) and not yet translated, as no protein could be detected in 0-1 hour
embryos (data not shown).
The timing of key morphological events during embryogenesis at 28°C is summarized in Fig. 5A. Just before pole cell formation, Nasonia Hunchback is expressed ubiquitously (data not shown). An anterior to posterior gradient of Nasonia Hunchback begins to form soon after the nuclei migrate to the surface and begin dividing at the surface of the embryo (Fig. 5C). The protein is localized to nuclei. During the next two hours (at 28°C), until the beginning of cellularization, the embryos continuously express an anterior domain of Nasonia Hunchback, with a sharpening border (Fig. 5E). However, throughout this period of graded Nasonia Hunchback expression, we did not observe a parallel gradient of Nasonia hunchback mRNA expression. Rather, the embryos express a low ubiquitous level of hunchback mRNA, superimposed with a small anterior and a larger posterior domain of expression during the cell cycles just after pole cell formation (Fig. 5B). This is followed by restriction of the posterior domain to the posterior, with incipient expression at the center of the embryo (Fig. 5D). The difference between the lack of Nasonia Hunchback at the posterior and the continuous presence of the mRNA at the posterior, throughout this two-hour period, indicates that Nasonia hunchback must be translationally controlled.
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Just prior to gastrulation, a narrow stripe of Nasonia hunchback
mRNA expression appears on the dorsal side of the embryo
(Fig. 6A). Upon germ-band
extension, this dorsal expression domain appears to be associated with serosa
development (Fig. 6B-D).
Although in many primitive insects the serosa develops from the anterior, in
Nasonia the serosa, an extra-embryonic membrane, begins to develop in
the dorsal region of the embryo, then expands anteriorly and ventrally to
eventually envelop the entire embryo (Bull,
1982). Finally, Nasonia Hunchback is also expressed in a
patterned subset of cells in the central nervous system, most strongly during
the period of head involution (Fig.
6E,F).
We investigated whether Nasonia hunchback function is needed for serosa formation, by comparing living wild-type and hunchbackhl embryos (data not shown). We found that the serosa still forms apparently normally in the mutant embryos, indicating that although zygotic Nasonia Hunchback is expressed relatively early and strongly in this tissue, it is not necessary for its morphological determination.
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How late does maternal Hunchback persist?
Why does a lack of zygotic hunchback result in more severe
consequences in Nasonia than in Drosophila, despite graded
maternal Hunchback expression in both species? We hypothesized that because of
the longer period of early development in Nasonia
(Fig. 1) maternal Hunchback
does not overlap temporally with zygotic Hunchback to the same extent that it
does in Drosophila. To test this hypothesis, we examined Hunchback in
Nasonia hunchbackhl mutant embryos, and compared them with
Drosophila embryos lacking zygotic Hunchback, during the period when
maternal Hunchback is decaying. Specifically, we examined whether residual
maternal Hunchback is detected near the onset of cellularization, when both
Nasonia and Drosophila embryos begin to express Hunchback
zygotically in a posterior cap (in addition to the anterior domain).
In a tightly staged collection of male Nasonia embryos from hunchbackhl/+ virgins, we observed 34 embryos expressing Hunchback in the anterior and incipient posterior caps (Fig. 7A), while 31 sibling embryos had no detectable Hunchback expression (Fig. 7B). In a control experiment, all of 50 Nasonia wild-type embryos of a similar age clearly showed the zygotic Hunchback expression pattern. These results show that in Nasonia embryos, maternal Hunchback does not persist into the period of posterior cap expression, but our characterization of maternal Hunchback in earlier embryos (see the previous section above) indicates that it is weakly expressed just prior to that time.
To examine maternal Hunchback in Drosophila, we made synchronous collections of embryos from hunchback/+ parents (see Materials and methods) and from wild-type parents, such that the youngest embryos were just beginning to express the zygotic posterior Hunchback cap (Fig. 7C). The progeny of the wild-type parents all expressed Hunchback in a strong anterior domain, as well as in the posterior cap. However, in 25% of the progeny of heterozygous parents (57/228), we found either weak staining only in anterior nuclei presumably from residual maternal expression or no detectable staining. Specifically, we observed 27 progeny of the heterozygous parents with only weak anterior staining (maternal expression only; Fig. 7D) and 30 with no staining. The 90 youngest siblings with strong anterior staining (mostly zygotic expression) showed incipient posterior-cap staining. (The remaining 81 siblings with zygotic expression were older, exhibiting either strong posterior cap staining or resolution of the posterior cap into a posterior stripe.) Because 27 is close to one-fourth of 117 (27 maternal plus 90 youngest zygotic), these results indicate that in Drosophila, maternal Hunchback perdures into the period of incipient zygotic posterior cap expression, during early cellularization. This is in contrast to Nasonia maternal Hunchback, which appears to decay before the equivalent stage of posterior cap expression during early cellularization. This timing difference may contribute to Nasonia's stronger dependence on zygotic hunchback. However, the experiments described below indicate that maternal hunchback cannot fully account for the difference in functions covered by zygotic hunchback in Nasonia and Drosophila.
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Discussion |
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The hunchbackhl mutant phenotype (hemizygous progeny of
heterozygous mothers) reveals that zygotic hunchback is essential in
Nasonia for development of almost the entire head, as well as the
thorax and the posterior abdomen. In Drosophila, when only zygotic
hunchback is removed (in homozygous progeny of heterozygous parents),
the posterior labial segment and thorax plus a small posterior
abdominal region are deleted, but underlying maternal
hunchback still patterns part of the head. When both maternal and
zygotic hunchback are removed, the anterior defects expand further
into the head (Bender et al.,
1987; Lehmann and
Nüsslein-Volhard, 1987
).
The above comparison of mutant phenotypes, the observation that more time
is allocated to early development in Nasonia, and evidence from
Schistocerca that the hunchback axial patterning function
may originally have been zygotic (Patel et
al., 2001), together suggested that hunchback might be
expressed only zygotically in Nasonia. However, we have found
hunchback mRNA in ovaries and in very early embryos, indicating that
hunchback is transcribed maternally in Nasonia, as in
Drosophila and Tribolium
(Wolff et al., 1995
), although
it is not translated maternally as in Schistocerca
(Patel et al., 2001
).
Moreover, by examining Hunchback expression in hunchbackhl
mutant embryos, we found that maternal hunchback mRNA appears to be
solely or primarily responsible for directing the synthesis of the early
anterior Hunchback domain during the first cell cycles after pole cell
formation in Nasonia embryos.
The finding that Nasonia hunchback is expressed maternally
even though zygotic hunchback controls more extensive patterning in
Nasonia than in Drosophila raises the question:
what, if any, is the function of maternal hunchback in
Nasonia? One possibility is that maternal hunchback is
necessary as a positive regulator of zygotic hunchback; for example,
in Drosophila, the parasegment 4 expression of hunchback is
under positive regulation by hunchback gene products
(Hülskamp et al., 1994;
Margolis et al., 1995
). If
autoregulation were the sole role of maternal hunchback, then
eliminating both maternal and zygotic hunchback would produce the
same defects as eliminating only the zygotic gene products. Attempts to
eliminate both maternal and zygotic hunchback function in
Nasonia with parental RNA interference have only rarely yielded
embryos with a phenotype as strong as that of hunchbackhl
mutant embryos (J.L. and C.D., unpublished). This suggests that the maternal
gene products may not control additional anteroposterior patterning
functions.
Nasonia hunchback mRNA expression at the posterior
In contrast to the largely conserved expression of Nasonia
hunchback protein (see below), the expression of Nasonia
hunchback mRNA differs from that of Drosophila. In
Drosophila, hunchback maternal mRNA at the posterior of the embryo
degrades as the mRNA is being translationally controlled by Nanos, generating
a gradient in both the mRNA and the protein expression
(Bender et al., 1988;
Schröder et al., 1988
;
Tautz, 1988
). Similar
posterior degradation of the maternal mRNA has also been observed in the
housefly Musca domestica (Sommer
and Tautz, 1991
). By contrast, throughout the two-hour period when
Nasonia embryos are expressing an anterior Hunchback domain (from
just after the nuclei arrive at the surface until the onset of
cellularization), there is a substantial domain of hunchback mRNA at
the posterior end of the embryo. The absence of Hunchback at the posterior of
the embryos indicates that Nasonia hunchback is under translational
control, presumably by Nanos. Schistocerca hunchback also appears to
be translationally controlled at the posterior of the embryo
(Lall et al., 2003
).
Nasonia hunchback does not have a canonical NRE such as is found in
Schistocerca, Locusta, Tribolium and Drosophila hunchback
mRNAs; however, Nasonia hunchback does have candidate NREs that are
similar in structure to the Drosophila melanogaster cyclin B1 NRE,
which is translationally regulated by Pumilio and Nanos in the germline
(Nakahata et al., 2001
).
Comparative timing and expression of Nasonia and Drosophila Hunchback
Because Nasonia and Drosophila differ in the extent of
essential zygotic hunchback function and in the timeline for early
development, we compared the overall timing, as well as pattern of Hunchback
expression in the wild-type embryos. Our comparative observations of wild-type
Hunchback expression in Drosophila (not shown, see Materials and
methods) and Nasonia indicate that the dynamics of expression largely
correlate with the same morphological markers during blastoderm development,
rather than with absolute developmental time. In both Nasonia and
Drosophila, just before pole cell formation, Hunchback is expressed
ubiquitously, then this expression is replaced by an anteroposterior gradient
with a progressively sharpening border as the cells begin dividing at the
surface of the embryo. Very early in the process of cellularization, in both
Nasonia and Drosophila embryos, a posterior cap of Hunchback
begins to be expressed in addition to the anterior domain of expression. In
both organisms, during the cellular blastoderm period, the posterior cap
resolves into a posterior stripe and expression retracts from the anterior.
However, the expression patterns of Hunchback in Nasonia and
Drosophila are not entirely identical. The anterior domain of
Hunchback persists into the early stages of germ band extension in
Drosophila but not in Nasonia, and at gastrulation, a dorsal
stripe is expressed in Nasonia that is not present in
Drosophila.
Dorsal Nasonia Hunchback expression appears to be associated with
development of the serosa. Expression in extraembryonic membranes is an aspect
of Hunchback expression that has been described for other insects, including
Schistocerca and Drosophila
(Patel et al., 2001), as well
as the mosquito Anopheles gambiae
(Goltsev et al., 2004
). We
find that despite zygotic expression in the serosa, Nasonia zygotic
hunchback function is not necessary for serosa formation. The dorsal
expression domain suggests that zygotic hunchback might be positively
regulated in Nasonia by the zerknüllt (zen)
homeobox gene. In Drosophila, zen is dorsally expressed during
blastoderm development and is required for development of the amnioserosa
(Wakimoto et al., 1984
;
Doyle et al., 1986
).
zen is also expressed in the anterior and dorsal regions that give
rise to extraembryonic membranes in other insects
(Falciani et al., 1996
;
Stauber et al., 2002
); in
Tribolium, anterior expression of zen1 has also been shown
to specify serosa cell fates, differentiating them from those of the more
posterior germ rudiment (van der Zee et
al., 2005
). Nasonia Hunchback is also strongly expressed
in the nervous system, approximately during the period of head involution, in
a pattern that appears to be similar to that observed in the nervous system of
other insects (Woff et al., 1995; Rohr et
al., 1999
; Patel et al.,
2001
).
Because maternal hunchback is partially redundant with zygotic
hunchback in Drosophila
(Lehmann and Nüsslein-Volhard,
1987), we also examined the timing of the maternal component
relative to the zygotic component in Nasonia and Drosophila.
We found that maternal Hunchback expression in Drosophila appears to
overlap with a slightly later zygotic phase of expression than in
Nasonia. This timing difference may contribute to Nasonia's
greater reliance on zygotic hunchback. However, the strikingly
different essential roles of hunchback in Nasonia and
Drosophila call for further explanation.
Are phenotypic differences revealing changes in functionally overlapping gene functions?
We have considered several possible explanations to account for the
observation that the zygotic hunchback loss-of-function phenotype is
more severe in Nasonia than in Drosophila. As discussed
above, we first hypothesized that Nasonia lacks maternally provided
hunchback function, but this explanation was ruled out, as
Nasonia does have strong maternal Hunchback expression. Second, we
found that more limited perdurance of maternal Hunchback during the blastoderm
stage of Nasonia may contribute to the differential function. Third,
we consider here that Nasonia Hunchback might also regulate more
downstream genes, either by DNA-binding or protein-protein interactions, than
Drosophila Hunchback. In this regard, it is notable that
Nasonia Hunchback has an N-terminal zinc finger (NF-1) that is
lacking in Drosophila. However, the function of NF-1 is not
understood, and N-terminal zinc fingers of Hunchback have been independently
discarded in number of insect taxa including Hymenoptera (Apis) and Orthoptera
(Cricket). Finally, Nasonia and Drosophila may differ in the
degree to which other genes are redundant or synergistic with Hunchback
function.
Our analysis of Hox gene expression in Drosophila embryos
indicated that even when both maternal and zygotic hunchback products
are removed, the defects are not as extensive as the zygotic defects of
Nasonia hunchbackhl. Consistently, cuticular analyses of
Drosophila embryos lacking both maternal and zygotic
hunchback show that the deleted region extends forward only through
the maxillary segment (E. Wimmer, personal communication); however, all
gnathal plus at least two pregnathal segments are deleted in Nasonia
hunchbackhl (Pultz et al.,
1999). This raises the question of whether the absence of a
bicoid gene in Nasonia could potentially be responsible for
the extent of the defects observed with a loss of zygotic Nasonia
hunchback.
When the dose of maternal bicoid was reduced by half in Drosophila embryos that also lacked all maternal and zygotic hunchback (E. Wimmer and C.D., unpublished), the array of head segments deleted (all except the labrum) was identical to the region deleted in Nasonia hunchbackhl mutant embryos. Importantly, these `headless' Drosophila mutant embryos can be rescued by a single zygotic hunchback+ allele, indicating that although zygotic Drosophila hunchback is not usually needed to pattern multiple head segments, it is sufficient to do so (in the context of a remaining half dose of bicoid expression). In this comparison, Drosophila hunchback appears to be functionally similar to Nasonia hunchback in the range of segments that it can pattern, although this was not originally obvious from single-mutant analyses.
The roles of genes with overlapping functions, such as orthodenticle and bicoid, have changed during the course of evolution as hunchback has continued to control anterior development. Our finding that hunchback is responsible for controlling more of the anterior development in Nasonia than in Drosophila may indicate that the Hunchback protein has changed its interactions with downstream regulatory genes. Alternatively, the evolution of overlapping gene functions may be sufficient to account for the changing responsibilities of hunchback during the evolution of insect embryos.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Program in Neurobiology and Behavior, University of
Washington, Seattle, WA 98105, USA
Present address: Program in Molecular and Cellular Biology, University of
Washington, Seattle, WA 98105, USA
Present address: Division of Biological Sciences, University of California
San Diego, San Diego, CA 92093, USA
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