squid

DEVELOPMENTAL BIOLOGY

Embryo

All ovarian transcripts are present in 3-hour embryos, showing that they are maternally inherited (Kelley, 1993).

Adult

The Drosophila hrp40 proteins are abundant nuclear pre-mRNA-binding proteins, similar to the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins of vertebrates. Hrp40 is encoded by the squid gene, which is required for dorsoventral axis formation during oogenesis. Eggs and embryos from homozygous squid mothers are severely dorsalized, and complete deletion of the squid gene results in lethality. The expression and localization of hrp40 were examined in wild-type and squid mutant ovaries. Using a monoclonal antibody specific for hrp40, the same isoforms of hrp40 are detected in both wild-type and squid ovaries, but the amount of hrp40 is reduced in squid ovaries. Furthermore, immunolocalization of hrp40 in wild-type egg chambers shows that hrp40 is present in the nurse cells, oocyte, and follicle cells. In contrast, in squid mutant egg chambers, hrp40 is absent from the germ-line-derived nurse cells and oocyte, but it is detected in the somatic follicle cells. The absence of hrp40 from the germ-line-derived cells of developing egg chambers is likely to lead to the striking dorsalized phenotype of squid eggs. In addition, dramatic stage-specific changes in the cellular localization of hrp40 are seen; the protein found in the nurse cell nuclei during early stages of oogenesis migrates to the cytoplasm at later stages. These findings reveal dynamic patterns of expression and localization of hnRNP proteins during development and provide evidence for an essential role for hnRNP proteins (Matunis, 1994).

Given the striking functional differences (rescue of germ-line and somatic function, as well as GRK mRNA and protein distribution) of the individual Sqd isoforms, it was of interest to determine whether there were any differences in their subcellular distributions. Both SqdB and SqdS are detected in the nuclei of the germ-line-derived nurse cells. In addition, SqdS also accumulates in the oocyte nucleus, whereas SqdB does not. Interestingly, examination of SqdA females reveals that this isoform is undetectable in the germ line. Given that SqdA efficiently rescues the D-V patterning defect of sqd¹ mutants, it seemed unlikely that it is not expressed in the germ line. Therefore, Sqd mRNA expression was studied in SqdA females using isoform-specific probes and it was found that the SqdA transcript is readily detectable in the germ line of ovaries from SqdA females. It seems, therefore, likely that the intracellular distribution of SqdA is predominantly cytoplasmic and thus, too diffuse to be visualized. Consistent with this possibility, the Sqd protein expression in the somatic follicle cells in SqdA egg chambers appears much more diffuse and cytoplasmic than in SqdS egg chambers (Norvell, 1999).

Effects of Mutation or Deletion

Females mutant for the newly identified squid gene are sterile and lay eggs that display only dorsal structures. Many eggs have two enlarged respiratory appendages located on the sides rather than flanking the dorsal midline. The resulting embryos are also dorsalized even if fertilized by wild-type sperm. The gene acts midway through oogenesis at about the time the dorsoventral (D/V) axis is established within the growing egg chamber. The sqd gene encodes at least three distinct proteins generated by alternative RNA processing that are members of a well-characterized family of RNA-binding proteins. 3.5-, 2.3-, 1.7- and 1.5-kb mRNAS are dramatically reduced in abundance in squid mutant embryos. At least one Sqd isoform is essential in somatic tissues. The ventralizing mutations gurken (grk), torpedo (tor), and cornichon are all epistatic to (downstream of) sqd. The data indicate that the dorsalized phenotype seen in sqd eggs requires the expression of wild-type grk, cni and tor. Strong alleles of grk and tor can act as dominant suppressors of sqd dorsalization. A model of D/V axis formation is presented postulating that squid is needed to organize a concentration gradient of a morphogen originating in the germinal vesicle (Kelly, 1993).

In Drosophila, the dorsoventral asymmetry of the egg chamber depends on a dorsalizing signal that emanates from the oocyte. This signal is supplied by the TGF alpha-like gurken protein whose RNA is localized to the dorsal-anterior corner of the oocyte. Gurken protein is the potential ligand of the Drosophila EGF receptor, which is expressed in the follicular epithelium surrounding the oocyte. How changes in the dorsalizing germ-line signal affect the embryonic dorsoventral pattern are described. A reduction in strength of the germ-line signal as produced by mutations in gurken or Egfr does not change the slope of the embryonic dorsoventral morphogen gradient, but causes a splitting of the gradient ventrally. This leads to embryos with two partial dorsoventral axes. A change in distribution of the germ-line signal as caused by fs(1)K10, squid and orb mutations leads to a shift in the orientation of the embryonic dorsoventral axis relative to the anterior-posterior axis. In extreme cases, this results in embryos with a dorsoventral axis almost parallel to the anterior-posterior axis. These results imply that Gurken, unlike other localized cytoplasmic determinants, is not directly responsible for the establishment of cell fates along a body axis, but that it restricts and orients an active axis-forming process that occurs later in the follicular epithelium or in the early embryo (Roth, 1994).

The individual isoforms of Squid show striking differences in their ability to rescue the D-V patterning defects of sqd¹ mutant females. Despite the fact that the transgene is expressed in the germ line, the SqdB isoform cannot restore proper D-V patterning of the eggshell; SqdB females lay 100% strongly dorsalized eggs, which are indistinguishable from those laid by sqd¹ homozygotes. In contrast, expression of either the SqdS or SqdA isoform can rescue the dorsalized phenotype of sqd¹ mutants. Females carrying two copies of either the SqdA or SqdS transgene lay a large percentage of wild-type eggs. Neither transgene alone, however, is capable of entirely restoring the function of the wild-type gene, since SqdA or SqdS females lay a number of dorsalized and partially rescued eggs as well. Furthermore, the classification or phenotype of the partially rescued eggs laid by these females is strikingly different. SqdA females lay a number of very dorsalized eggs, whereas in contrast, SqdS females lay eggs with broad, fused dorsal appendages. It should be noted, that although SqdA females lay a number of severely dorsalized eggs, these eggs are not identical to sqd¹ or to SqdB eggs in that the SqdA eggs do not have a uniform crown of dorsal appendage material around the entire anterior circumference of the eggshell. Frequently, the SqdA eggs show a restoration of the dorsal midline fates (indicative of high levels of Grk protein). The differences in the phenotypes of partially rescued eggs are not attributable to differences in transgene expression levels, since decreasing the copy number from two to one does not affect the type of eggs observed. Interestingly, females carrying one copy of each of these transgenes (SqdS and SqdA) lay nearly all wild-type eggs (98% as compared with ~60% in females with two copies of either transgene). Therefore, the two Sqd isoforms, SqdA and SqdS, appear to have nonidentical functions in establishing proper dorsoventral patterning during oogenesis and act together in this process (Norvell, 1999).

The phenotypes of eggs laid by sqd¹ and fs(1)K10 females are similar. In both cases, GRK mRNA is mislocalized and Grk protein is produced around the entire anterior circumference of the oocyte. These two female sterile mutations, although similar, are also unique with respect to other known female sterile mutants. In most other cases in which grk mRNA is mislocalized, for example, in orb and spindleB mutant egg chambers, the unlocalized RNA is not translated efficiently. Mutations in cappuccino and spire also result in a mislocalization of grk RNA and translation of the mislocalized message, but these two mutations have more generalized effects on oocyte patterning and do not seem as specific for grk function as K10 or the germ-line forms of Sqd. In addition, early in oogenesis Grk is necessary for the establishment of anteroposterior patterning. However, eggs laid by both sqd¹ and K10 mutant mothers display no anteroposterior defects, but are abnormal along only the D-V axis. When the expression of K10 protein was analyzed in sqd¹ mutant ovaries it was found that the distribution of K10 in sqd¹ mutants is unaffected and K10 protein is detected in the oocyte nucleus of late stage egg chambers. Conversely, however, the expression of Sqd protein is affected by the K10 mutation. In K10 mutant ovaries, Sqd is present in the nurse cell nuclei but absent from the oocyte nucleus. In addition, in wild-type egg chambers Sqd protein is detected at the posterior pole at late stages and this cytoplasmic Sqd localization is unaffected in K10 egg chambers. These data indicate that Sqd protein is, in fact, expressed in K10 mutant egg chambers, but that its accumulation in the oocyte nucleus is specifically lost in the absence of K10 function (Norvell, 1999).

In addition to its requirement in oogenesis, Sqd is also required during embryonic development. A number of lethal alleles of sqd were generated and the ability of the individual isoforms to restore viability of sqd null alleles was investigated. Again, as with the ability of the specific isoforms to function during oogenesis, the three Sqd isoforms differ in their ability to rescue the viability of a lethal sqd allelic combination. Both SqdS and SqdB are capable of rescuing the essential somatic Sqd function: expression of either of these transgenes allows recovery of 11% and 19% of the expected number of mutant sqd adults, respectively. In contrast, however, SqdA is incapable of restoring the essential somatic function of Sqd, since less than 0.2% of the expected number of sqd adults were recovered. These data further demonstrate that the individual Sqd isoforms are not functionally equivalent (Norvell, 1999).

The RNA-binding protein Squid is required for the establishment of anteroposterior polarity in the Drosophila oocyte

The heterogeneous nuclear ribonucleoprotein (hnRNP) Squid (Sqd) is a highly abundant protein that is expected to bind most cellular RNAs. Nonetheless, Sqd plays a very specific developmental role in dorsoventral (DV) axis formation during Drosophila oogenesis by localizing gurken (grk) RNA. This study reports that Sqd is also essential for anteroposterior (AP) axis formation. sqd was identified in a screen for modifiers of the Protein Kinase A (PKA) oogenesis polarity phenotype. The AP defects of sqd mutant oocytes resemble those of PKA mutants in several ways. In both cases, the cytoskeletal reorganization at mid-oogenesis, which depends on a signal from the posterior follicle cells, does not produce a correctly polarized microtubule (MT) network. This causes the posterior determinant, oskar (osk) RNA, to localize to central regions of the oocyte, where it is ectopically translated. Additionally, MT-dependent anterior movement of the oocyte nucleus and the grk-dependent specification of posterior follicle cells are unaffected in both mutants. However, in contrast to PKA mutants, sqd mutants do not retain a discrete posterior MT organizing center (MTOC) capable of supporting ectopic posterior localization of bicoid (bcd) RNA. sqd mutants also display several other phenotypes not seen in PKA mutants; these probably result from the disruption of MT polarity in earlier stages of oogenesis. Loss of Sqd does not affect polarity in follicle cells, wings or eyes, indicating a specific role in the determination of MT polarity within the germline (Steinhauer, 2005).

It is well established that the hnRNP Sqd participates in Drosophila DV axis formation. Following a screen to identify factors that interact with PKA in mid-oogenesis, Sqd was discovered to be essential also for AP axis formation. The localization of posterior factors, including osk RNA, GFP-Stau, kin-ß-gal, and Dhc, was disrupted at stages 9-10 in all sqd allelic combinations tested, was highly penetrant in strong sqd alleles, and could be rescued by expression of a Sqd cDNA transgene. These defects can be attributed to the failure of sqd mutants to establish a normally polarized MT array at mid-oogenesis. Defects were also observed in MT organization and in the localization of posterior factors, including Grk protein, in sqd mutant oocytes at stages 2-6. Despite the imperfect localization of Grk before stage 6, posterior fate appears to be specified normally in the follicle cells overlying sqd mutant oocytes. Thus, sqd mutations affect germline-specific processes required for the polarization of MTs in both early and mid-oogenesis (Steinhauer, 2005).

Loss of PKA in the germline does not affect MT polarity in early oogenesis, as judged by Orb localization in stages 2-6, but, similar to sqd mutants, it disrupts MT polarity in mid-oogenesis without discernibly altering follicle cell fate. However, despite several similarities, a significant difference was observed in the MT organization of sqd and PKA mutants at stages 8-10 (Steinhauer, 2005).

In grk and cornichon (cni ) mutants, where the posterior follicle cells do not differentiate properly, both the subsequent RNA localization defects and the failure of the oocyte nucleus to migrate from the posterior to the anterior have been attributed to defects in MT reorganization resulting from loss of a posterior follicle cell signal. In PKA and sqd mutant oocytes, the anterior migration of the oocyte nucleus, which depends on MT function, is unaffected, despite the accompanying MT defects and the highly penetrant mislocalization of osk RNA and other posterior factors. Thus, it appears that either discrete aspects of the MT organization, which direct nucleus migration, are spared in PKA and sqd mutants or the overall disruption of MT organization by loss of PKA or Sqd is simply less severe than that caused by loss of posterior follicle cell fate (Steinhauer, 2005).

The normal organization of MTs in stage 8-10 oocytes is not entirely clear. In addition to MTs nucleated at the anterior cortex, MTs have been proposed to emanate from all cortical positions, with the exception of the posterior pole. This assertion is based on the observations that components associated with MT minus-ends, such as gamma-tubulin and the centrosome component Centrosomin (Cnn), can be seen along the entire oocyte cortex, and that injected bcd RNA localizes to the lateral cortices as well as the anterior, but not to the posterior pole. Hence, normal posterior localization of osk RNA may require the clearing of MTs nucleated both from a discrete posterior MTOC established before stage 6 and from dispersed cortical sites established after stage 7 (Steinhauer, 2005).

Staining with alpha-tubulin antibody following partial MT depolymerization revealed MT stubs emanating mostly from the anterior in wild-type oocytes, whereas PKA and sqd mutant oocytes retain short MTs around the entire oocyte cortex, including the posterior pole. Some PKA mutant oocytes also show an elevated posterior concentration of MTs not seen in sqd mutant oocytes. Thus, it appears that the primary MT defect in sqd mutants is the failure to eliminate cortical sites of MT nucleation beyond stage 7, whereas PKA mutants additionally retain a posterior MTOC beyond stage 6. This hypothesis can explain why ectopic bcd RNA localizes at the posterior of PKA mutant oocytes but not sqd mutant oocytes. It should, however, be noted that since classical MTOC components, such as gamma-tubulin, are present along the entire oocyte cortex at stages 9-10 even in wild-type oocytes, the inference of a discrete posterior MTOC from partial MT depolymerization experiments cannot be confirmed directly (Steinhauer, 2005).

In a proportion of sqd mutant stage 2-6 oocytes, Grk, Orb, osk RNA and MTs are distributed evenly throughout the ooplasm rather than localizing in a cap at the oocyte posterior. Although these defects do not appear to cause the subsequent AP defects by preventing posterior follicle cell specification, the possibility cannot be ruled out that the early and late polarity phenotypes are causally related in some other way. For instance, it is possible that a molecule(s) required at the posterior of the oocyte for the MT reorganization at stages 7-8 is improperly localized by stage 6 in sqd mutants, as are Grk, Orb and osk RNA. If MT rearrangements are very sensitive to the localized concentration of this hypothetical regulator, an early polarity defect of apparently low penetrance could be translated into a much more penetrant polarity phenotype at mid-oogenesis (Steinhauer, 2005).

sqd is not the only mutant to cause polarity defects in both early and mid-oogenesis. For example, defects in early polarity are caused by mutations in Armitage (Armi), a component of the RNA silencing machinery, and these defects have been proposed to be the cause of a mid-oogenesis AP polarity phenotype. However, it was found that pnt^998/12 expression is not disrupted in armi¹ homozygotes. Weak par-1 alleles also affect mid-oogenesis polarity without affecting posterior follicle cell fate, whereas strong alleles disrupt early polarity severely, causing oocyte identity to be lost. Thus, for several mutations, including sqd, armi and par-1, it is unclear whether MT organization is disrupted independently at two distinct phases of development or whether there is a causal connection between the early and later polarity phenotypes that is not evident as a failure in posterior follicle cell specification. In either case, a single molecular target might account for both the early and mid-oogenesis phenotypes (Steinhauer, 2005).

Several additional phenotypes became prevalent in older sqd ^j4B4 germline clones, rising to very high penetrances after 2 weeks. Among the varied late onset sqd phenotypes, the oocyte sometimes was mispositioned within the egg chamber, even in those egg chambers containing the normal complement of nurse cells to oocyte. This phenotype can arise in several ways, including as a result of delayed oocyte specification. A role for Sqd in oocyte specification is supported by the presence of cysts with 16 nurse cells and no oocyte in these older ovarioles. In other cases, cysts with fewer than 16 germ cells were observed, implicating Sqd in cystocyte mitosis. Both oocyte specification and the normal cystocyte divisions depend on specific arrangements of the MT cytoskeleton in the germarium. Thus, it is likely that some of these late onset sqd phenotypes, like the polarity phenotypes, are caused by a defect in regulating MT dynamics (Steinhauer, 2005).

In sqd ^j4B4 germline clones, the accumulation of Osk protein was noticed in the cytoplasm of stage 9-10 oocytes. A similar observation was reported for mutations in another hnRNP A/B family member, Hrp48 (Hrb27C - FlyBase) (Yano, 2004). Normally, Osk protein accrues only at the posterior cortex, and translation of osk RNA is presumably repressed elsewhere. Therefore, loss of sqd may cause de-repression of osk translation. However, no ectopic osk translation was seen in stage 6-8 sqd mutant oocytes, detected either with Osk antibody or with an osk translation reporter, in contrast to the premature expression observed with the osk translation reporter in hrp48 mutants (Yano, 2004) or with similar reporters lacking specific repressor elements. Thus, although the idea that sqd is directly involved in translational regulation of osk cannot be dismissed, an alternative hypothesis is proposed for the ectopic Osk protein accumulation in sqd mutants. Since most of the posterior components examined were mislocalized to the center of sqd^j4B4 oocytes, it is believed that the primary AP defect in sqd mutants is that the MT plus-ends are focused incorrectly at the center of the oocyte. Hence, all the necessary components for osk translation may be localized together, and it is hypothesized that the osk translation machinery is assembled and activated in the middle of the sqd mutants as it normally is at the posterior of wild-type oocytes at stage 9 (Steinhauer, 2005).

A low penetrance of ectopic Osk protein was detected in PKA mutants. The scenario outlined above could be true for PKA mutants as well. Regardless of the mechanism, it is clear from this result that PKA is not absolutely required for Osk translation, although it may enhance osk translation (Steinhauer, 2005).

Although sqd was identified in a screen for modifiers of PKA in oocyte polarity, retesting with various alleles indicated that there is not a strong genetic interaction between the two loci. Both Sqd and PKA act in mid-oogenesis to reorganize the oocyte MTs in response to a normal posterior follicle cell signal, but specific MT defects differ between the two mutants. Thus, they probably have different targets and mechanisms in this complex process (Steinhauer, 2005).

The hnRNP Sqd is an RNA-binding protein. Another hnRNP of the same family, Hrp48, is also required for MT reorganization at mid-oogenesis (Yano, 2004). Sqd and Hrp48 bind each other in vitro, cooperate in grk RNA localization and have similar localization patterns throughout oogenesis. Thus, one might expect these two proteins to act together in MT reorganization. Although no strong genetic interaction was detected between sqd and hrp48 in AP polarity, it is speculated that they are collectively necessary for the localization and translation of one or a small number of specific RNA molecules required for MT repolarization at mid-oogenesis (Steinhauer, 2005).

hnRNPs normally participate in the processing of many RNAs, but their generic functions may be partially redundant, so that, for example, in sqd mutants, continued cell viability is not impaired despite the presence of a strong AP polarity defect. The ability to induce large, persistent somatic cell clones for sqd ^j4B4 without causing any polarity or other phenotypes supports this idea. Follicle cell polarity was also normal in PKA mutant clones. Thus, the disruptions in MT polarity that were observed for both PKA and sqd mutants represent specialized functions of these proteins in germline cells (Steinhauer, 2005).

Squid is required for efficient posterior localization of oskar mRNA during Drosophila oogenesis

The nuclear-cytoplasmic shuttling heterogeneous nuclear RNA-binding protein (hnRNP) Squid is required during Drosophila melanogaster oogenesis, where it plays a critical role in the regulation of the TGFalpha-like molecule Gurken (Grk). Three Sqd isoforms have been described, SqdA, S and B, and two of these, SqdA and SqdS, differentially function in grk mRNA nuclear export, cytoplasmic transport and translational control during oogenesis. Sqd is also required for the regulation of oskar mRNA, functioning in the cytoplasmic localization of the osk transcript. In oocytes from sqd females, osk mRNA is not efficiently localized to the posterior pole, but rather accumulates at the anterior cortex. Furthermore, anterior patterning defects observed in embryos from sqd females expressing only the SqdS protein isoform suggest that Sqd may also play a role in the translational regulation of the mislocalized osk mRNA. These findings provide additional support for models of mRNA regulation in which cytoplasmic events, such as localization and translational regulation, are coupled. These results also place Sqd among an emerging class of proteins, including such other members as Bruno (Bru) and Hrb27C/Hrp48, that function in multiple aspects of both grk and osk mRNA regulation during Drosophila oogenesis (Norvell, 2005).

A feedback loop between Wolbachia and the Drosophila gurken mRNP complex influences Wolbachia titer

Although much is known about interactions between bacterial endosymbionts and their hosts, little is known concerning the host factors that influence endosymbiont titer. Wolbachia bacterial endosymbionts are globally dispersed throughout most insect species and are the causative agent in filarial nematode-mediated disease. gurken (grk), a host gene encoding a crucial axis determinant, has a cumulative, dosage-sensitive impact on Wolbachia growth and proliferation during Drosophila oogenesis. This effect appears to be mediated by grk mRNA and its protein-binding partners Squid and Hrp48/Hrb27C, implicating the grk mRNA-protein (mRNP) complex as a rate-limiting host factor controlling Wolbachia titer. Furthermore, highly infected flies exhibit defects that match those occurring with disruption of grk mRNPs, such as nurse cell chromatin disruptions and malformation of chorionic appendages. These findings suggest a feedback loop in which Wolbachia interaction with the grk mRNP affects both Wolbachia titer and grk mRNP function (Serbus, 2011).

The major findings of this study are that host grk has a cumulative, dosage-sensitive impact on Wolbachia titer. This impact does not appear to be related to the Grk protein, invoking a role for the grk mRNP. Accordingly, Wolbachia exhibit association with a grk mRNP protein, and disrupting known protein constituents of the grk mRNP affects Wolbachia titer analogously to grk disruptions. Highly infected flies also have defects analogous to grk mRNP disruptions, including defects in nurse cell chromatin structure and dorsal appendage formation. These findings suggest that Wolbachia interaction with the grk mRNP has a significant impact on both Wolbachia titer and grk mRNP function (Serbus, 2011).

One of the surprising outcomes from this study is the microtubule-independent impact of grk on Wolbachia titer. Disruptions of microtubules and cytoplasmic dynein have been shown to disrupt Wolbachia distribution and density in oogenesis. One interpretation of this study is that Wolbachia are transported along microtubules into the oocyte where Wolbachia replicate preferentially at the oocyte anterior end. A role for grk in regulating Wolbachia titer initially appeared consistent with that scenario. Grk signaling is crucial for proper microtubule orientation in oogenesis, and grk mRNPs are known to be transported by the microtubule-based motor, cytoplasmic dynein. Thus grk could be argued to directly or indirectly affect Wolbachia transport toward a replication-promoting area of the oocyte. However, the results of this study indicate that the impact of grk on Wolbachia titer is independent of these models. grk has a comparable repressive effect on Wolbachia titer in both nurse cells and the oocyte throughout oogenesis, although grk is primarily known to affect oocyte microtubules. The effects of grk on Wolbachia titer are detected before any known influence of grk on microtubules in oogenesis. Furthermore, colcemid tests indicate that the impact of grk on Wolbachia titer is largely independent of microtubules in both nurse cells and the oocyte. This indicates that grk affects Wolbachia titer primarily through a different mechanism (Serbus, 2011).

A microtubule-independent role for grk could be explained by a previously unrecognized function for grk mRNA or Grk protein. An initial genetic tests did not differentiate between these possibilities because the grk mutants used disrupt both mRNA and protein, and the grk overexpression tests should elevate both mRNA and protein loads. However, this issue is addressed by using well-established mutations in translational repressors sqd and hrb27C, which encode components of the grk mRNP complex that repress grk translation. The reduction in Wolbachia titer seen in sqd and hrb27C mutants ultimately suggest that Wolbachia density does not correlate with Grk protein availability in the cytoplasm. An alternative possibility is that the grk mRNP complex has a function in regulating Wolbachia titer. The appearance of Wolbachia associated with GFP-Sqd in fixed samples and live imaging are also consistent with a possible interaction between Wolbachia and mRNP components (Serbus, 2011).

One of the issues raised by this study is specificity of the grk effect because Sqd and Hrb27C are hnRNP proteins that are not exclusive to the grk mRNP complex. Thus, it is also possible that distinct mRNPs with a protein composition similar to grk mRNPs also contribute to Wolbachia titer control. However osk mRNP complexes are thought to share many components with grk mRNPs, including Sqd and Hrb27C, yet genetic disruptions of osk that reduce both mRNA and protein load did not induce a striking reduction in Wolbachia titer in a preliminary screen. Although this study does not rule out a role for other mRNPs, it suggests that the grk-related effects on Wolbachia titer are not necessarily a general property of host mRNPs (Serbus, 2011).

One of the remaining questions is how a grk mRNP exerts an influence on Wolbachia titer. Electron microscopy evidence shows no indication of a mortality-based effect. The significant increase in Wolbachia doublets detected in grk mutant nurse cells might be informative, however. Perhaps grk mutants prevent final abscission of the Wolbachia membrane during binary fission. Another interpretation is that upon completion of binary fission, the Wolbachia daughter cells remain trapped within the single original host vacuole, subjecting the bacteria to competition for limited nutrient resources. Either scenario would be consistent with a role for host grk in promoting Wolbachia growth and proliferation. Future studies are needed to address how directly this defect might be attributable to the grk mRNP complex (Serbus, 2011).

An association of Wolbachia with GFP-Sqd raises the possibility that grk mRNPs affect Wolbachia biochemistry or trafficking though a direct mechanism. It is also possible that the impact of grk mRNPs on Wolbachia is facilitated by intermediate factors. For example, Sqd has been shown to coimmunoprecipitate with the retinoblastoma Rb protein from Drosophila cell culture and ovarian extract. Rb is known to bind and repress E2F family transcription factors. Thus, grk mRNPs might affect Dp/E2F-based host transcriptional activation patterns that support Wolbachia trafficking and/or replication. Furthermore, Sqd binds Cup, a translational repressor that is required for localization of grk mRNA. Cup has been shown to interact with Nup154, a member of a protein family that supports nuclear pore assembly and nuclear import processes. This scenario provides another route by which grk mRNPs might affect availability of host products relevant to Wolbachia titer regulation (Serbus, 2011).

One of the additional questions raised by this study is its applicability to neglected tropical diseases. It is known that Wolbachia endosymbionts of Onchocerca volvulus, Wuchereria bancrofti and Brugia malayi contribute significantly to African river blindness and lymphatic filariasis. Because elimination of Wolbachia from these nematodes disrupts both the filarial host and manifestations of disease, any insight into Wolbachia titer control is potentially useful. The recently sequenced Brugia genome does not appear to encode a grk gene, but it has possible homologs for hrb27C and sqd, as well as for the grk mRNA-binding proteins Bruno/Aret, Imp and Orb. Thus, a speculative possibility is that Brugia cells might also harbor grk-like mRNPs that exert an influence on Wolbachia titer (Serbus, 2011).

This study raises the further question of whether intracellular pathogens could interact similarly with host mRNP components. Viruses such as hepatitis C have been shown to bind host mRNP proteins and use them to facilitate viral protein synthesis and viral replication. Although preliminary, there are some hints that pathogenic bacteria interact with host mRNPs as well. A number of genome-wide RNAi screens have been done in Drosophila tissue culture to assess the effect of host factors on Listeria, Mycobacterium, Chlamydia and Francisella infection. This work indicated that disruption of certain splicing or translation initiation factors correlated with reductions in intracellular Listeria, Chlamydia and Francisella infection levels. The datasets also indicate that disruption of the grk mRNP component hrb27C, or Brain Tumor, a suppressor of Hunchback translation, reduced Francisella and Listeria infection loads. It will be of great interest to see whether future studies find Wolbachia interactions with grk mRNP components to be representative of a generalized titerinfluencing mechanism shared by other intracellular symbionts and pathogens (Serbus, 2011).

During oogenesis, Wolbachia are not only positioned where key developmental events occur, but also rely on the same transport mechanisms as many of the morphogens that control these events. For example, early in oogenesis, anterior localization of Wolbachia occurs at the same time and position as that of the patterning events that establish the anterior- posterior axis. Later in oogenesis, both Wolbachia and host germline determinants rely on the motor protein kinesin-1 to concentrate at the posterior pole. In both of these situations, despite relying on the same transport mechanisms and occupying the same position as the host patterning molecules, Wolbachia do not interfere with these essential developmental events. This suggests that Wolbachia achieve a balance in which titer is maximized without disrupting oocyte development. Support for this comes from the finding that Wolbachia with abnormally high titer produce defects in dorsal appendage formation. Because this event relies on the Grk signaling pathway, one possibility is that this occurs as a consequence of a disruptive association of Wolbachia with grk mRNP components. This would create a selective pressure to establish more moderate Wolbachia levels within the host. Thus, the functional interaction between Wolbachia and grk provides a molecular example for how the interests of host and Wolbachia success can be achieved (Serbus, 2011).

Other symbiotic organisms have been shown to direct morphogenetic processes in the host. Vibrio fischerei induce formation of the light-producing organ in squid and Rhizobium induce root nodule formation in leguminous plants for nitrogen fixation. Perhaps the interaction between Wolbachia and grk represents a step toward the evolution of symbiosis in which Wolbachia also become integral to regulation of host morphogenesis (Serbus, 2011).

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date revised: 15 March 2014

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