RESEARCH ARTICLE 3813
Development 137, 3813-3821 (2010) doi:10.1242/dev.054213
© 2010. Published by The Company of Biologists Ltd
Novel modes of localization and function of nanos in the
wasp Nasonia
Jeremy A. Lynch*,‡ and Claude Desplan
SUMMARY
Abdominal patterning in Drosophila requires the function of nanos (nos) to prevent translation of hunchback (hb) mRNA in the
posterior of the embryo. nos function is restricted to the posterior by the translational repression of mRNA that is not
incorporated into the posteriorly localized germ plasm during oogenesis. The wasp Nasonia vitripennis (Nv) undergoes a long
germ mode of development very similar to Drosophila, although the molecular patterning mechanisms employed in these two
organisms have diverged significantly, reflecting the independent evolution of this mode of development. Here, we report that
although Nv nanos (Nv-nos) has a conserved function in embryonic patterning through translational repression of hb, the timing
and mechanisms of this repression are significantly delayed in the wasp compared with the fly. This delay in Nv-nos function
appears to be related to the dynamic behavior of the germ plasm in Nasonia, as well as to the maternal provision of Nv-Hb
protein during oogenesis. Unlike in flies, there appears to be two functional populations of Nv-nos mRNA: one that is
concentrated in the oosome and is taken up into the pole cells before evidence of Nv-hb repression is observed; another that
forms a gradient at the posterior and plays a role in Nv-hb translational repression. Altogether, our results show that, although
the embryonic patterning function of nos orthologs is broadly conserved, the mechanisms employed to achieve this function are
distinct.
INTRODUCTION
Two maternally localized mRNAs, bicoid (bcd) and nanos (nos)
are crucial for patterning the anterior and posterior of the
Drosophila embryo, respectively (Struhl, 1989). The patterning
mechanisms employed by these two factors are quite distinct. bcd
mRNA is localized to the anterior pole of the oocyte during
oogenesis (Berleth et al., 1988) and is translated from this localized
source after the activation of the egg. Its protein product diffuses
through the embryo, forming an anterior-to-posterior concentration
gradient of a transcription factor that activates target genes in a
concentration-dependent manner (Driever and Nusslein-Volhard,
1988a; Driever and Nusslein-Volhard, 1988b; Driever et al., 1989;
Struhl et al., 1989). Similar to bcd, nos mRNA is localized during
oogenesis, but at the posterior. Nos protein is produced from this
localized source and diffuses through the embryo, resulting in a
posterior to anterior concentration gradient (Wang and Lehmann,
1991). Although Bicoid acts primarily as an instructive
transcription factor that activates anterior genes, including zygotic
hunchback (hb), Nos has a permissive function that allows
posterior patterning by translationally repressing ubiquitous
maternal hb mRNA (Tautz, 1988; Hulskamp et al., 1989; Irish et
al., 1989; Struhl, 1989), which contains a Nos response element
(NRE) in its 3⬘ UTR (Wharton and Struhl, 1991).
Center for Developmental Genetics, Department of Biology, New York University,
New York, NY 10003, USA.
*Author for correspondence ([email protected])
‡
Present address: Institut für Entwicklungsbiologie, Universität zu Köln, Zülpicher
Straße 47 b, 50674 Köln, Germany
Accepted 6 September 2010
Mutations in nos result in ectopic production of Hb protein at the
posterior of the embryo, leading to the complete loss of abdominal
segmentation (Lehmann and Nusslein-Volhard, 1991). This
phenotype can be mimicked by a hb transcript that is unresponsive
to Nos repression (Struhl, 1989). Even more dramatically, when
loss of nos is combined with the absence of maternal hb
expression, normal segmentation of the embryo is restored
(Hulskamp et al., 1989; Irish et al., 1989; Struhl, 1989), showing
that repressing hb translation is the only patterning function of nos.
Localized maternal nos mRNA is also a component of the germ
plasm, a structure that contains the determinants for germline
formation that will be included in the germ cells as soon as they
form at the posterior of the syncytial embryo. Formation and
localization of the germ plasm is crucial for proper function of Nos
in patterning the embryonic posterior, as mutants in genes
responsible for assembling the germ plasm, such as oskar, vasa or
tudor, all result in loss of nos mRNA localization (and thus loss of
translation), and produce segmentation defects similar to those
observed in nos mutations (Ephrussi et al., 1991; Lehmann and
Nusslein-Volhard, 1991).
Although nos mRNA is highly concentrated in the posterior
germ plasm, this localized population represents only 4% of the
total nos mRNA, while the remainder is found diffusely
throughout the embryo (Bergsten and Gavis, 1999). However,
unlocalized nos is specifically repressed by Smaug and its cofactors (Smibert et al., 1996; Dahanukar et al., 1999; Smibert et
al., 1999), resulting in a Nos protein gradient whose only source
is the germ plasm localized mRNA. The translational repression
of unlocalized nos is crucial in Drosophila because ectopic Nos
protein prevents anterior translation of the NRE, which contains
hb and bcd mRNAs, resulting in severe anterior patterning
defects (Simpson-Brose et al., 1994; Wharton and Struhl, 1991;
Gavis and Lehmann, 1992).
DEVELOPMENT
KEY WORDS: Nasonia, Nanos, Evo-devo, Patterning, Germ plasm
nos is present throughout the metazoa, including the most basal
lineages (Mochizuki et al., 2000), with broadly conserved functions
in the germline (Tsuda et al., 2003). Within the insects, some
degree of maternal localization of nos has been observed in all
species so far examined (Curtis et al., 1995; Lall et al., 2003;
Goltsev et al., 2004; Chang et al., 2006; Dearden, 2006), indicating
that the posterior patterning function of nos may be conserved.
Furthermore, the 3⬘ UTRs of both Tribolium hb and
orthodenticle-1 (otd1) mRNAs contain NREs. Both genes show
evidence of translational repression from the posterior. The resulting
protein gradients appear to be crucial for patterning the anterior
segments of the embryo (Wolff et al., 1995; Schroder, 2003),
although the true roles of these gradients in patterning the early
Tribolium embryo have recently been questioned (Marques-Souza
et al., 2008; Kotkamp et al., 2010). NREs have also been found in
the 3⬘UTRs of hb and otd1 in the wasp Nasonia vitripennis (Nv),
and both of these genes show evidence of translational repression
from the posterior (Pultz et al., 2005; Lynch et al., 2006b).
Like Drosophila, Nasonia undergoes a mode of long germ
embryogenesis where the embryonic anlage occupies the entire
anterior-posterior extent of the egg fate map and all segments form
at roughly the same time. The role of nos in patterning the Nasonia
embryo is of particular interest because, although Nasonia
embryogenesis appears morphologically very similar to that of
Drosophila, it uses molecular mechanisms for embryonic
patterning that are quite distinct (da Fonseca et al., 2009). For
example, mRNA for Nasonia orthodenticle 1 (Nv-otd1) is localized
to both the anterior and posterior poles, and is required to pattern
segments in both of these regions (Lynch et al., 2006b). Maternally
localized giant (Nv-gt) mRNA at the anterior plays a permissive
role in allowing anterior patterning (Brent et al., 2007), while
posteriorly localized caudal (Nv-cad) mRNA obviates the need for
translational control to restrict its function from the anterior
(Olesnicky et al., 2006).
With the availability of parental RNAi (pRNAi) (Lynch and
Desplan, 2006) and a sequenced genome (Werren et al., 2010),
Nasonia is an ideal model for detailed comparative analyses of
gene function and regulation within insects. Taking advantage of
these tools, we investigated the mode of posterior localization,
embryonic patterning function and translational regulation of Nvnos. We found that although the translational repressive function of
Nos function is conserved, the localization of its mRNA and its
genetic regulation appear to be unique to Nasonia.
MATERIALS AND METHODS
Identification and cloning of Nasonia genes
A fragment of Nv-nos was isolated from genomic DNA using degenerate
primers designed using the CODEHOP (Rose et al., 1998). This fragment
was extended by RACE PCR using the SMART RACE kit from Clontech.
dsRNA production and injection
Wasp rearing, embryo collection and fixation, and RNAi experiments were
performed as described previously (Lynch and Desplan, 2006). Previous
experiments using mock injections with water or gfp dsRNA showed no
effects on embryonic development (Lynch et al., 2006b). Nasonia orthologs
of other genes were identified by BLAST and have the following Accession
Numbers: Nv-smaug, XM_001602088; Nv-vasa, XM_001603906; Nvpumilio, XM_001607039.
Fluorescent in situ hybridization and immunohistochemistry
Antisense probes and dsRNAs were generated with the T7 Maxiscript or
Megascript Kit (Ambion), respectively. Concentration of dsRNA injected
for each gene was as follows: Nv-nos, 3 mg/ml; Nv-vas, 1 mg/ml; Nvsmaug, 1 mg/ml; Nv-pumilio, 0.1 mg/ml.
Development 137 (22)
In situ hybridization experiments were performed using a protocol
adapted from vertebrates (Brent et al., 2003). For two-color in situs,
digoxigen-labelled otd1 and fluorescein-labeled nos probes were added
simultaneously and were detected using anti-digoxigen::POD antibody
(1:200, Roche) coupled with AlexaFluor 488 tyramide detection
(Invitrogen), and anti-fluorescein::AP antibody (1:2500, Roche) coupled
with HNPP Fluorescent detection (Roche), respectively. Hb protein
expression was detected using an anti-Nv-Hb antibody (Pultz et al., 2005)
at a dilution of 1:1000 and a goat-anti-rabbit Alexafluor-488 (Molecular
probes)-conjugated secondary antibody at a concentration of 1:750.
Accessory nuclei were detected using anti-nuclear pore antibody (Sigma)
at a concentration of 1:2000 combined with AlexaFluor 488 TSA kit
(Invitrogen). DNA staining was carried out by incubating embryos or
ovaries in a 1 g/ml solution of 4⬘-6-Diamidino-2-phenylindole (DAPI).
RESULTS
Patterns of Nv-nos and otd1 ovarian localization
Nv-nos is expressed maternally in the ovary of Nasonia and its
mRNA is localized to the posterior cortex of the oocyte (Olesnicky
and Desplan, 2007). Another maternal mRNA, that of Nv-otd1,
appears to be localized early to the posterior pole in a similar
fashion, but later also shows localization to the anterior, consistent
with its function in patterning both poles of the embryo (Lynch et
al., 2006b). In order to analyze the localization of Nv-nos and
compare it with that of posterior Nv-otd1 in more detail, we have
established a two-color fluorescent in situ hybridization protocol
for Nasonia. Nv-nos mRNA is detected in the nurse cells and
oocyte in early stages, where it overlaps with Nv-otd1 expression
(Fig. 1A-A⬙). mRNA for both Nv-nos and Nv-otd1 appears initially
to accumulate strongly around the oocyte nucleus (Fig. 1A-A⬙,
topmost two follicles); then, in later follicles, Nv-nos and otd1
mRNAs move to the posterior pole of the oocyte (Fig. 1A-A⬙,
bottom-most follicle).
Unlike Nv-nos, Nv-otd1 has a second phase of localization at the
anterior pole of the oocyte. This anterior accumulation is correlated
with the abrupt transfer of the remaining Nv-otd1 mRNA from the
nurse cells (Fig. 1B⬘, lower follicle). These events are in turn
correlated with the onset of nurse cell dumping in the wasp, where
the nurse cells begin to shrink as their contents are transferred to
the oocyte (J.A.L., personal observation). At this stage, localization
of Nv-nos at the posterior pole becomes tighter and takes on a
discoid shape (Fig. 1B).
In the nurse cells, Nv-otd1 expression is restricted to the nurse
cells proximal to the oocyte, and appears to be concentrated in
particles (Fig. 1A⬘,B⬘), whereas nos mRNA is expressed more
broadly throughout the nurse cells, and does not appear to be
incorporated into particles (Fig. 1A,B).
Genetic regulation of Nv-nos localization in the
ovary
We next asked whether genes required in Drosophila for posterior
localization of nos are also required in Nasonia. We have
previously shown that RNAi against a Nasonia ortholog of tudor,
although causing oocyte polarity defects, does not affect
localization of Nv-nos mRNA (Olesnicky and Desplan, 2007). We
therefore wondered whether vasa, another crucial germ plasm
component that is absolutely required for posterior nos localization
in Drosophila (Lehmann and Nusslein-Volhard, 1991; Wang et al.,
1994) is involved in Nv-nos localization. When Nv-vasa is
knocked down by RNAi, Nv-nos localization to the posterior is not
abolished (Fig. 1C), although in later follicles, the localization
becomes less tight at the posterior cortex (compare Fig. 1D with
1B). Strikingly, however, Nv-otd1 mRNA, which is initially
DEVELOPMENT
3814 RESEARCH ARTICLE
Fig. 1. Localization of Nv-nos and otd1 mRNA in the ovary. Anterior
is upwards in all panels. Red, Nv-nos mRNA; green, Nv-otd1; blue, DAPI;
nc, nurse cells; on, oocyte nucleus; fc, follicle cells; oc, oocyte. Vertical
scale bars in A⬙,B⬙,C⬙,D⬙: 100m. (A-A⬙) Young egg chambers, showing
the early dynamics of Nv-nos and Nv-otd1 localization. (B-B⬙) Late egg
chambers, showing the later dynamics of localization and transfer of
mRNAs from the nurse cells. (C-C⬙) Young Nv-vasa pRNAi egg chambers,
showing the dynamics of Nv-otd1 delocalization from the posterior.
(D-D⬙) Late Nv-vas pRNAi egg chambers showing the final delocalization
of Nv-otd1 mRNA from the posterior.
localized normally at the posterior (Fig. 1C⬘, upper follicle),
begins to delocalize, spreads throughout the oocyte (Fig. 1C⬘,
middle follicle), and is eventually strongly depleted at the
posterior pole (Fig. 1C⬘, bottom follicle, and Fig. 1D⬘) while
increasing anteriorly.
Embryonic Nv-nos expression
In the embryo, Nv-nos mRNA is associated with the oosome, a
structure found in many Hymenoptera (Bilinski and Jaglarz, 1999;
Grbic, 2003) that is analogous to the germ plasm in Drosophila.
The Nasonia oosome exhibits a dynamic behavior in preblastoderm embryonic stages. In freshly laid embryos, Nv-nos and
the oosome are associated with the ventral posterior cortex (Fig.
2A). During the first few nuclear cleavages (cycles 1-4), the
oosome detaches from the cortex, becomes spheroid and moves
anteriorly in the interior cytoplasm, and can sometimes be observed
near the middle of the embryo (Fig. 2B). The observed position of
the oosome along the AP axis is quite variable in this stage (the
extreme case shown in Fig. 2B represents about 8% of all embryos
at this stage), but it is not clear whether this is due to the inherent
variability of the migration, or to different amounts of time spent
by the oosome at particular positions along the axis. The oosome
later returns to the posterior pole during the next few cleavages
(cycles 4-6) (Fig. 2C).
The formation of pole cells in Nasonia is initiated by a large
protrusion from the posterior pole (Bull, 1982) (Fig. 2D,E) that
eventually buds from the embryo and rapidly begins dividing. This
initial bud often contains multiple nuclei (Bull, 1982) (data not
shown). The oosome, with its associated Nv-nos mRNA, is
extruded into this initial bud (Fig. 2D,E). Nv-nos mRNA not
RESEARCH ARTICLE 3815
Fig. 2. Dynamics of Nv-nos mRNA localization in the oosome
during early embryonic development. Anterior leftwards in all panels.
Blue, DAPI (DNA); red, Nv-nos mRNA. (A)Nv-nos localization in freshly
laid egg. (B)Embryo in division cycle 3 (C) Embryo in division cycle 8.
(D)Embryo in early pole cell budding stage. (E)Embryo of similar age to
D, showing only Nv-nos mRNA (white) to illustrate more clearly the
posterior gradient. (F)Embryo at nuclear division cycle 14 (G) Early Nv-vas
pRNAi embryo. (H)Blastoderm stage Nv-vas pRNAi embryo.
associated with the oosome remains in the embryo proper, and
appears to accumulate preferentially around the somatic nuclei in
a posterior to anterior gradient at the early stages of blastoderm
formation (Fig. 2D,E). As syncytial divisions continue, the
posterior embryonic domain of Nv-nos expression begins to fade
and eventually disappears, whereas expression remains strong in
the pole cells (Fig. 2F). For the remainder of embryogenesis,
expression is seen exclusively in pole cells and prospective
germline (Fig. 2F, data not shown).
Nv-vasa is required for the assembly of the
oosome and formation of pole cells
vasa orthologs are crucial components of the germ plasm across
the metazoa (Raz, 2000), which made surprising the weak effects
of Nv-vasa pRNAi on the localization of Nv-nos to the posterior of
the oocyte, as localization of nos mRNA is dependent on the
assembly of germ plasm in Drosophila (Ephrussi et al., 1991;
Lehmann and Nusslein-Volhard, 1991). We therefore asked
whether Nv-vasa has a role in establishing the germline in the
Nasonia embryo. When Nv-vasa function is knocked down by
RNAi, the oosome is not formed, and Nv-nos is diffusely localized
at the posterior pole (Fig. 2G). Furthermore, pole cells do not form,
and Nv-nos remains in a cap-like expression domain at the
posterior after the formation of the blastoderm (Fig. 2H).
Nv-nos is required for translational repression of
Nv-hb
We next asked whether Nv-nos has a similar role in axial patterning
to its fly ortholog. In Nasonia, maternal hb (Fig. 3A) is expressed
ubiquitously as in Drosophila, but Nv-Hb protein is also present
DEVELOPMENT
nanos function in Nasonia
Fig. 3. Function and regulation of Nv-nos in the early
blastoderm. (A,B)Red, Nv-hb mRNA; (A⬘,B⬘,C-F) green, Nv-Hb protein.
(C-F)Nv-hb mRNA is expressed ubiquitously in all embryos shown, but
is omitted for clarity. (A,A⬘) Early blastoderm wild-type embryo showing
ubiquitous expression of Nv-hb mRNA (A) and Hb protein (A⬘).
(B,B⬘) Later, Nv-hb mRNA (B) is still ubiquitous, while Nv-Hb protein (B⬘)
is repressed from the posterior. (C)After Nv-nos pRNAi, posterior
repression of Nv-Hb is lost. (D)Nv-vasa RNAi also results in ectopic NvHb protein in the posterior of the embryo. (E)Expression of Nv-Hb
protein in the early embryonic stages is not affected by Nv-smaug RNAi.
(F)Loss of Nv-smaug results in repression of Nv-hb throughout the early
blastodermal embryo. (G,H)Simultaneous Nv-smaug and nos pRNAi
embryo.
early and is found ubiquitously until blastoderm formation (Pultz
et al., 2005) (Fig. 3A⬘). After pole cell and blastoderm formation,
Nv-hb mRNA is still present throughout the embryo (Fig. 3B),
while Nv-Hb protein has been cleared from nuclei in the posterior
half (Fig. 3B⬘), indicating that Nv-hb is being translationally
repressed in the posterior at this stage (Pultz et al., 2005). In
embryos where Nv-nos has been knocked down by parental RNAi,
the production of Nv-Hb prior to pole cell formation is not affected
(data not shown), but it is derepressed at the posterior of early
syncytial blastoderm stage embryos (63%, n71). In many cases
(31%, n71), ectopic Nv-Hb can be seen all the way to the
posterior pole (Fig. 3C).
Regulation of Nv-nos function requires vasa and
smaug
In Drosophila, only the small amount of nos mRNA that is localized
to the germ plasm is translated and functions in embryonic
patterning, whereas the majority is translationally repressed. We have
shown that Nv-vasa, although not required for targeting Nv-nos to
the posterior, is required to assemble it into the oosome (germ plasm)
(Fig. 2G,H). Nv-Hb protein is derepressed in the posterior of
embryos where Nv-vasa has been knocked down (Fig. 3D), despite
Development 137 (22)
the higher levels of Nv-nos mRNA observed at the posterior in these
embryos (Fig. 2G,H). This indicates that Nv-vasa function is required
for Nv-nos-mediated translational repression of Nv-hb.
In Drosophila, translational repression of unlocalized nos is
mediated by Smaug protein. Although it has not yet been possible
to directly detect Nv-Nos protein expression, the delay in the
appearance of translational repression of Nv-Hb until after
blastoderm formation suggests that Nv-nos function is blocked
during the early embryonic stages when we can observe ubiquitous
Nv-Hb protein expression. We hypothesized that Nv-smaug
performs an early general repressive function on Nv-nos, and tested
this idea by pRNAi against Nv-smaug. If our hypothesis were
correct, we would expect to see premature translational repression
of Nv-hb. This is not observed, and the distribution of Nv-Hb
remains at normal levels and ubiquitous, up to the pre-pole cell
stages, in Nv-smaug RNAi embryos (Fig. 3E). However, after
blastoderm formation, during approximately nuclear cycles 10-12
(the same time period where translational repression of posterior
Nv-hb is observed), levels of Nv-Hb protein are significantly
reduced throughout the embryo (Fig. 3F) (45%, 31), suggesting a
general increase in translation of Nv-Nos.
To further confirm that the reduction of Nv-Hb translation in Nvsmaug pRNAi is caused by derepression of Nv-nos translation, we
knocked down Nv-nos and smaug simultaneously with pRNAi. NvHb protein is expressed at the early blastoderm stages (approximately
cycles 10-12) in most of these embryos (Fig. 3G), and its expression
domain expands towards the posterior pole (Fig. 3H) (55%, n44),
similar to what is seen in single Nv-nos pRNAi embryos. These
results suggest that the effects of Nv-smaug pRNAi on Nv-Hb protein
production are carried through regulation of Nv-nos.
Maternal expression of Nv-Hb protein
One of the most striking differences between the mode of hb
translational repression between Nasonia and Drosophila is the
apparent long delay of the onset of posterior translational
repression in the wasp, which is only evident after blastoderm
formation and appears to reflect a delay in the production of NvNos protein. In the fly, hb repression is detectable soon after egg
activation (Tautz, 1988). One hypothesis to explain this delay is
that perhaps Nv-nos is not translated in the early embryo. This
would be consistent with the migratory nature of the oosome
containing Nv-nos, which makes it a poor source of positional
information in the early embryo. In the absence of an antibody
against Nv-Nos, we could not test this hypothesis directly.
An alternative hypothesis is that the Nv-Hb protein observed in
the early embryo is provided maternally, i.e. is derived from
mRNA translated during oogenesis. If this hypothesis were correct,
then the issue of whether Nv-Nos is produced in the early stages
would be irrelevant, as Nv-Nos protein should not affect the levels
of Nv-Hb protein already produced. We examined Nasonia ovaries
using an antibody against Nv-Hb, and found that, indeed, Nv-Hb is
found in the oocyte nucleus, as well as in the posterior nurse cells
starting at very early stages of oogenesis (Fig. 4A).
Interestingly, the pattern of Nv-Hb accumulation changes as
follicles mature: Nv-Hb is no longer restricted to the oocyte nucleus
and Nv-Hb-bearing particles become visible initially around the
nuclear periphery, and are later distributed throughout the oocyte
(Fig. 4B). The behavior of these particles appears similar to that of
structures called accessory nuclei that were recently described in
Nasonia. These structures result from a budding of membrane from
the oocyte nucleus and have been found to be important for
distributing components of centrosomes around the cortex of the
DEVELOPMENT
3816 RESEARCH ARTICLE
Fig. 4. Nv-Hb protein is expressed in the ovary. (A,B)Red, Nv-Hb
protein; blue; DAPI. (C,C⬘) Red, Nv-Hb protein; green, nuclear pore.
(A)Nv-Hb protein (red) is expressed maternally, initially in the nurse cells
and in the oocyte nucleus. (B)At later stages, Nv-Hb protein is taken in
to particles away from the oocyte nucleus, and is distributed
throughout the oocyte. (C)Nv-Hb protein (red) is contained within the
accessory nuclei [marked with nuclear pore antibody (green)].
(C⬘)Enlarged view of boxed region in C.
oocyte where they will be crucial for fertilization and the activation
of embryonic development (Ferree et al., 2006). To determine
whether Nv-Hb protein is contained within accessory nuclei, we
stained ovaries simultaneously with an antibody against nuclear
pore components, and with anti-Nv-Hb. The particles of Nv-Hb
appear to be surrounded by nuclear membrane, indicating that
maternal Nv-Hb is contained within accessory nuclei, which are
distributed throughout the oocyte at the late stages of oogenesis
(Fig. 4C,C⬘) and are presumably the source of Nv-Hb protein
observed in the early embryo.
Nv-nos and embryonic patterning
In Drosophila, larvae resulting from embryos depleted of nos
function lack all abdominal segments, which results from ectopic
posterior Hb (Hülskamp et al., 1990). We sought to understand
RESEARCH ARTICLE 3817
the role of Nv-nos in patterning the embryo by examining the
effect of loss- or gain-of-function Nos through pRNAi against
Nv-nos or its regulators (Nv-vas and smaug) on the expression of
the gap genes Nv-giant (Nv-gt; Fig. 5A) and Nv-Krüppel (Nv-Kr)
(Fig. 5B), and the cuticular structures of first instar larvae (Fig.
5C). Nv-gt is expressed in two gap domains, one at the anterior
and one at the posterior (Fig. 5A) (Brent et al., 2007). In Nv-nos
pRNAi embryos, the anterior domain of Nv-gt is unaffected,
whereas the posterior domain is shifted and expands posteriorly
(Fig. 5D).
Similar to Drosophila, Nv-Kr is expressed in a band towards the
middle of the embryo, just posterior to the edge of the zygotic hb
domain (Fig. 5B) (Brent et al., 2007). After Nv-nos pRNAi, the
domain of Nv-Kr expands significantly towards the posterior, while
the anterior boundary appears unaffected (Fig. 5E), which is similar
to the behavior of Kr in Drosophila mutant for nos (Cinnamon et
al., 2004).
In the late blastoderm stages (nuclear cycles 13-14), Nv-hb is
expressed in a broad anterior domain covering the anterior 50% of
the embryo, and a narrower domain at the extreme posterior (Fig.
5A) (Pultz et al., 2005). In Nv-nos pRNAi, the anterior domain of
zygotic Nv-Hb protein (and mRNA; data not shown) expression
does not appear to change (Fig. 5D,E). This pattern indicates that
the posterior border is set independently of the extent of
translational repression of the ubiquitous early maternal hb mRNA
by Nos. However, the posterior domain of Nv-Hb (and Nv-hb
mRNA, data not shown) is usually lost completely (Fig. 5D,E),
again reflecting a shift in the posterior fate map.
The patterning effects of knocking down Nv-nos can also be seen
in first instar larval cuticles. As with other genes knocked down by
pRNAi in Nasonia, variable phenotypes are observed (Lynch and
Desplan, 2006). In the most severe cases, up to seven of the
posterior abdominal segments are lost in Nv-nos pRNAi embryos
(Fig. 5F). In many cases, the remaining segments are also highly
compressed, with very thin denticle belts visible on each remaining
segment (Fig. 5F). Although the loss of posterior segments can be
explained by the observed misregulation of posterior gap genes
(Fig. 5D,E), the origin of the compression phenotype, which is not
observed in flies, is not clear.
Fig. 5. Nv-nos function in embryonic
patterning. (A)Wild-type Nv-gt mRNA
expression (red) with Nv-Hb protein (green).
(B)Wild-type expression of Nv-kr mRNA (red)
and Nv-Hb protein (green). (C)Wild-type first
instar larval cuticle. Green arrow indicates
spiracle on second thoracic segment, red
arrows indicate the first three abdominal
segment spiracles (also in F and I). (D)Nv-nos
pRNAi embryo showing posterior shift of the
posterior Nv-gt domain. (E)Nv-nos pRNAi
embryo showing expansion of Nv-kr domain
posteriorly and loss of the posterior Nv-Hb
domain at the posterior. (F)A severely
affected Nv-nos pRNAi embryo. (G)Nv-vasa
pRNAi results in loss of the posterior Nv-gt
and Nv-Hb domains. (H)Nv-kr expression
expands to the posterior pole in Nv-vasa
pRNAi. (I)The majority of abdominal
segments are lost in Nv-vasa pRNAi. (J)Nvsmaug causes a compression of the Nv-gt and
Nv-Hb anterior domains, while the Nv-kr (K)
domain is shifted significantly towards the
anterior.
DEVELOPMENT
nanos function in Nasonia
3818 RESEARCH ARTICLE
Development 137 (22)
Fig. 6. Effects of Nv-nos and Nv-pumilio pRNAi
on oogenesis. Blue, DAPI; red, Nv-nos mRNA;
green, Nv-otd1 mRNA; g, germarium. (A)Complete
wild-type ovariole. (B)Nv-nos pRNAi ovariole
showing severely depleted germarium and one
maturing egg chamber. (C)Nv-nos pRNAi ovariole
with short germarium, and egg chamber with
reversed polarity (A, anterior; P, posterior) (D,E) Nvpumilio pRNAi leads to severe reduction in egg
chamber production, similar to Nv-nos pRNAi.
Green staining on surface of pRNAi ovarioles is
peritoneal sheath, which is difficult to remove in
severely affected ovarioles. Vertical scale bars:
100 m.
with Nv-Hb. Alternatively, or in parallel, localized maternal Nv-gt
mRNA (Brent et al., 2007) could be responsible for preventing the
expansion of the Nv-Kr domain all the way to the anterior pole.
Nv-smaug, like its Drosophila ortholog (Dahanukar et al., 1999),
appears to have functions not directly related to repression of Nvnos, as pRNAi against this gene causes defects in blastodermal
mitoses (see Fig. S1 in the supplementary material), and prevents
the production of larval cuticle. Smaug has recently been shown to
be crucial for mediating the maternal-to-zygotic transition in
Drosophila, and many of its additional targets are involved in
regulating the cell cycle (Benoit et al., 2009). Our results with Nvsmaug indicate that many of the targets of Smaug regulation may
have been conserved across holometabolan insect phylogeny.
Conserved function of Nv-nos and Nv-pumilio in
maintaining the germline
In Drosophila, nos and its partner pumilio, also have crucial roles
in maintaining the germline stem cells, a requirement for
continuous production of egg chambers (Forbes and Lehman,
1998; Wang and Lin, 2004). A similar role seems to be conserved
in other animal groups (Draper et al., 2007). This function is
conserved Nasonia, where egg chamber production becomes
strongly reduced over time (Fig. 6B) compared with wild type (Fig.
6A), and defects in the polarity of these truncated ovarioles are also
observed (Fig. 6C) after Nv-nos pRNAi. Similar effects are
observed with Nv-pumilio pRNAi (Fig. 6D,E), although this gene
is much more sensitive to pRNAi knockdown, as no conditions
were found between those that gave complete sterility and those
where only wild-type embryos were produced.
DISCUSSION
We have shown that Nv-nos has a conserved role in repressing
translation of hb in the posterior region of the embryo, and that the
loss of Nv-nos function leads to misregulation of posterior gap
genes and loss of posterior abdominal segments, similar to what is
seen in Drosophila. In addition, the regulatory logic upstream of
Nv-Nos function appears quite similar to that in the fly: Nv-Vas, a
component of the germ plasm, promotes Nv-Nos function, while
Nv-Smaug serves to restrict its production to the posterior. These
results, in combination with the basal phylogenetic position of
Nasonia among holometabolous insects (Savard et al., 2006;
Wiegmann et al., 2009) indicate that the general strategy to restrict
Nos function to the posterior was probably present in a common
ancestor of all holometabolous insects.
DEVELOPMENT
These results show that Nv-nos function is required for proper
patterning of the posterior half of the Nasonia embryo, similar to the
situation observed for Drosophila nos. Because we also see ectopic
persistence of Nv-Hb protein in the posterior during the early
blastoderm stages (Fig. 3C), it is likely that the patterning function
of Nv-Nos is mediated through its ability to translationally repress
Nv-hb mRNA. Thus, in Nv-nos pRNAi embryos, we propose that the
ectopic Nv-Hb protein changes the expression of posterior gap genes,
for example, by activating Nv-Kr expression further toward the
posterior, leading to the repression of other posterior gap genes, such
as Nv-gt. In principle, it would be possible to test whether the Nv-nos
pRNAi phenotype is mediated by Nv-hb by eliminating both factors
simultaneously. However, the fact that pRNAi for both Nv-nos and
Nv-hb leads to phenotypes of variable strengths makes the
interpretation of such an experiment difficult.
We next asked whether the loss of Nv-vasa leads to patterning
defects similar to those of Nv-nos. The posterior domain of Nv-gt
is often shifted posteriorly or lost, the posterior domain of Nv-Kr
expands posteriorly, and most of the abdominal segments are
missing in Nv-vasa pRNAi embryos. These posterior phenotypes
are often more severe than those seen with Nv-nos mRNA, which
could be due to the simultaneous loss of posterior localization of
Nv-otd1 observed in Nv-vasa pRNAi ovaries (Fig. 1D). We have
previously shown that posteriorly localized Nv-otd1 is an activator
of posterior Nv-gt and Nv-hb (Lynch et al., 2006b) and a posterior
repressor of Nv-Kr (Brent et al., 2007). In addition, other
phenotypes are sometimes observed in Nv-vas pRNAi cuticles,
including apparent dorsoventral defects, empty eggshells and, in
some cases, head defects, indicating other roles for Nv-vasa besides
allowing for Nv-nos and Nv-otd1 localization and function.
Nv-smaug pRNAi appears to lead to an increase in Nv-Nos
function and thus reduced Nv-hb translation in early blastoderm
embryos (Fig. 3E). Late embryos lacking Nv-smaug function show
dramatic anterior fate map shifts. The zygotic Nv-Hb domain is
reduced to a small region at the extreme anterior (Fig. 5J,K), but is
never completely lost. The anterior Nv-gt domain also shows a
reduction and shift of its expression, but is also never completely
lost. Nv-Kr shows a dramatic anterior expansion, the extent of
which is correlated with the reduction of the Nv-Hb domain.
The maintenance of anterior caps of zygotic Nv-gt and Nv-Hb
expression indicates that a small amount of anterior patterning
capacity remains after Nv-smaug pRNAi, and the resulting reduction
of Nv-Hb at the early blastoderm stage. This could arise as a result
of residual patterning capacity of Nv-Otd1 in the absence of synergy
Despite these similarities, there are a number of unique
properties of the Nasonia system, including its mode of localization
during oogenesis, the time at which acts during embryogenesis, and
the nature of the functional mRNA population that patterns the
embryo.
Evolution of Nv-nos localization mechanisms and
germ plasm assembly
In Drosophila, posterior localization of nos mRNA occurs very late
in oogenesis, relying on both the streaming movements of the
oocyte cytoplasm (Forrest and Gavis, 2003), and the earlier
assembly of the pole plasm by factors, including Osk, Vas and Tud
(Wang et al., 1994; Bergsten and Gavis, 1999). By contrast, Nv-nos
mRNA is localized posteriorly from a very early stage (Fig. 1A)
and this early localization does not appear to depend on the
assembly of the germ plasm, as knockdown of Nv-vas does not
disrupt targeting of Nv-nos to the posterior pole (Fig. 1C,D). In
addition, the timing of Nv-nos localization is not consistent with
cytoplasmic streaming playing a crucial role, as this process only
occurs at the latest stages of oogenesis in the fly. Thus, the mode
of early localization of Nv-nos appears to depend on molecular
factors and cellular processes that differ from those in the fly.
Nv-vas pRNAi leads to looser posterior localization of Nv-nos
mRNA and posterior Nv-otd1 mRNA becomes delocalized and
eventually moves to the anterior pole (Fig. 1C⬙,D⬙). As this
loosened localization at late stages of oogenesis correlates with the
loss of organized germ plasm (oosome) in freshly laid eggs, NvVas, although not required to recruit Nv-nos and Nv-otd1 mRNA to
the posterior, might be required for its tight posterior anchoring and
proper integration into the germ plasm late in oogenesis. This
anchoring is particularly crucial for Nv-otd1, probably because, in
the absence of the anchoring function of Nv-Vas, Nv-otd1 mRNA
follows its normal anterior localization at late stages of oogenesis
(Fig. 1C⬘,D⬘), a process that depends on microtubule polarity in
Nasonia (Olesnicky and Desplan, 2007).
The timing of Nv-nos function in the embryo is
significantly delayed compared with Drosophila
nos
In Drosophila, Nos-dependent translational repression of maternal
hb mRNA is evident almost immediately after egg activation. By
contrast, the Nv-Nos dependent translational repression of Nv-hb is
not detected until well after the migration of the syncytial nuclei to
the embryo surface and the formation of the pole cells (Fig. 3A,B).
In addition, the effects of derepression of Nv-nos translation after
Nv-smaug pRNAi are also delayed until this later time point (Fig.
3E,F).
This indicates that the delay in clearing Nv-Hb protein from the
posterior of the wasp embryo is due to the presence of maternally
provided Nv-Hb in the oocyte (Fig. 4), which persists in the early
embryo (Fig. 3A⬘) and is apparently degraded only after the
formation of the syncytial blastoderm. This is in contrast to
Drosophila, where maternal Hb protein has not been detected.
These observations raise the question: what is the significance of
maternal Nv-Hb protein and the consequent delay in the formation
of the Nv-Hb gradient?
The maternal provision of Hb protein is conserved in at least one
other insect, the locust Schistocerca (Patel et al., 2001).
Furthermore, in both Schistocerca and Nasonia, maternal Hb
protein is initially highly concentrated in the oocyte nucleus, then
enters the cytoplasm at later stages, which suggests a conserved
mode of maternal Hb function in these organisms. Nv-Hb seems to
RESEARCH ARTICLE 3819
have an important role in oogenesis as Nv-hb pRNAi causes a
severe reduction in egg production (Lynch et al., 2006a). In
Schistocerca, it was proposed that the maternally provided Hb
protein plays a role in specifying the region of the egg that will
give rise to the embryonic primordium (Patel et al., 2001). In any
case, as Hb protein is not observed in Drosophila oocytes, the
requirement for maternal Hb protein appears to have been lost in
the fly.
From these observations, we propose that the loss of maternal
Hb in Drosophila is related to one of the remarkable features of this
organism: its extremely rapid early development [approximately
four times faster than Nasonia (Pultz and Leaf, 2003)]. As there is
no population of maternal Hb protein in need of specific
degradation in Drosophila, exclusion of Hb protein from the
posterior pole by translational repression can begin directly after
egg laying. This could then allow for more rapid progression of
syncytial nuclear divisions by obviating the danger of any ectopic
Hb protein at the posterior persisting into the crucial embryonic
patterning stages and disrupting abdominal patterning.
An additional interesting feature of maternal Nv-Hb is that it is
taken up into the accessory nuclei. Accessory nuclei have been
proposed to perform a number of functions in hymenopteran
oocytes (Bilinski and Kloc, 2002; Jaglarz et al., 2005), and have
recently been shown to distribute centrosomal components
throughout the oocyte and embryo (Ferree et al., 2006). However,
a potential role in embryonic patterning had until now not been
proposed. Thus, further analysis of Nv-Hb function, particularly
during oogenesis, may provide insights into additional ancestral
functions of Hb in insects, and possibly into functions of the
accessory nuclei among the Hymenoptera.
Two populations of Nv-nos mRNA with distinct
functions and fates in the Nasonia embryo
In Drosophila, only the nos mRNA incorporated into the posterior
polar granules is translated in the early embryo. In addition, as the
polar granules are completely incorporated into the pole cells, only
the protein produced from this mRNA population prior to pole cell
formation has a role in embryonic patterning. A very different
pattern is observed in Nasonia: while a large portion of Nv-nos
mRNA is taken up into the pole cells, a significant amount remains
in the embryo proper, forming a posterior to anterior gradient (Fig.
2D,E). As Nv-Nos dependent translational repression of Nv-hb
mRNA is not evident until well after the pole cells have budded,
and rather coincides with the formation of a posterior-to-anterior
gradient of the non-oosomal mRNA population, we propose that it
is this gradient of Nv-nos mRNA that carries out the patterning
function.
It is not yet clear how the distinction arises between mRNAs
localized to the oosome and those that will remain in the posterior
pole cytoplasm. We propose that the oosome is a dynamic
structure, and that mRNA and protein shuttle between it and the
surrounding the posterior cytoplasm. In this case, the mRNA
molecules that will remain in the embryo once the oosome buds
would not be pre-determined. Rather, the mRNAs that remain in
the embryo to perform the posterior patterning function would be
those that just happened by chance to be outside of the oosome at
the time of pole cell budding.
Consistent with the second hypothesis, Nv-vas seems to be
required for the regulation of both populations of Nv-nos mRNA.
It is required for the assembly of the oosome, and the production
of pole cells. Furthermore, Nv-vas pRNAi gives an Nv-nos-like
phenotype, despite the presence of high levels of Nv-nos mRNA
DEVELOPMENT
nanos function in Nasonia
remaining at the posterior, implying that another function for NvVas is to promote translation of Nv-nos mRNA. In addition,
because all mRNAs known so far from Nasonia that are localized
and maintained in the oosome and pole cells also show the same
perinuclear gradient in the early blastoderm stages as seen for Nvnos (J.A.L., unpublished), the posterior pole cytoplasm appears to
have a similar molecular composition to the oosome, indicating a
common origin for both populations of mRNA.
Classical embryological manipulations of the Ichneumonid wasp
Pimpla turionellae (Achtelig and Krause, 1971) fit well with our
proposed distinction in function of the two populations of Nv-nos
mRNA. Two distinct cytoplasmic regions could be discriminated
at the posterior pole of this wasp embryo: the oosome, and the
posterior pole plasm, which surrounds the oosome. Complete
removal of the oosome prevented the formation of pole cells, but
had little effect on posterior abdominal segment formation, as long
as the posterior pole plasm was not disturbed. Furthermore,
ligations of the egg resulted in increasing loss of abdominal
segments from the posterior, but these losses could be rescued by
translocation of posterior cytoplasm anterior to the restriction point.
The position of the oosome itself in regard to the ligations did not
matter (Achtelig and Krause, 1971). This indicates that the
posterior cytoplasm in Pimpla contains a factor with patterning
capabilities similar to those of Nos. Indeed, we propose that Pimpla
nos mRNA, like Nv-nos, also has dual, functionally distinct,
aspects of localization.
nos expression in the honeybee Apis mellifera, is somewhat
different from what is seen in Nasonia. In Apis, there is no oosome,
and no pole cells are formed, which is unlike Nasonia, Pimpla and
other hymenopterans (Dearden, 2006). However, Apis nos mRNA
is loosely localized at the posterior during oogenesis, and forms a
posterior to anterior gradient of mRNA in the early embryo
(Dearden, 2006). We speculate that this gradient of Apis nos is the
equivalent to the gradient formed by Nv-nos after pole cell
formation. If this were the case, we would expect Apis Nos to have
a posterior patterning function and Apis Vasa to be required for this
function to be performed.
Conclusion
In summary, we have shown that Nasonia nos has a function in
embryonic patterning that is conserved with its Drosophila
ortholog. This patterning function appears to be carried out through
the regulation of hb translation in the posterior of the embryo in
both species. On the other hand, the mechanisms of mRNA
localization and translational regulation used to establish the
gradient of Nos activity seem to diverge significantly between
Drosophila and Nasonia. Thus, the conserved patterning function
of Nos probably reflects a shared ancestral condition that cannot
easily be displaced, whereas the divergent means of localization
and function of nos probably reflect divergent evolutionary
pressures on the mechanisms of germ plasm formation and
maternal provisioning experienced by the separate lineages leading
to Nasonia and Drosophila.
Acknowledgements
We thank Daniel Jackson and Ava Brent for performing early Nv-nos in situs,
and Arzu Celik for providing the basis of the two-color FISH technique. We
thank Mary Anne Pultz and David Leaf for providing the Nv-Hb antibody. We
thank Ava Brent, Eugenia Olesnicky, Veit Riechmann, Siegfried Roth, Brent
Wells and Zhenqing Chen for helpful comments on this article. We thank the
Desplan and Roth labs for their constant support. This work was supported by
grants from NIH GM-64864 to C.D. J.A.L. was supported by NIH Training
Grant T32 HD007520, by a Dean’s Dissertation Fellowship from NYU, by NIH
Development 137 (22)
Fellowship F32 GM078832 and by the SFB 680 at the University of Cologne.
J.A.L. designed and carried out experiments. J.A.L. and C.D. wrote the paper.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.054213/-/DC1
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DEVELOPMENT
nanos function in Nasonia