orb

DEVELOPMENTAL BIOLOGY

Embryonic

Orb protein, localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. (Christerson, 1994).

Oogenesis

orb appears to be expressed only in the germline and encodes ovarian- and testis-specific transcripts. In testes, Orb RNA accumulates in the primary spermatocytes and at the caudal ends of the spermatid bundles. In ovaries, Orb transcripts display an unusual spatial pattern of accumulation in the oocyte. Preferential accumulation in the oocyte of Orb RNA is first detected in region 2 of the germarium and is dependent on Bicaudal-D and egalitarian. In stage 7 egg chambers, Orb RNA is localized posteriorly; during stages 8-10 it is localized at the anterior of the oocyte, asymmetrically along the dorsal-ventral axis. In embryos the transcripts accumulate at the posterior end and are included in the pole cells [Image] (Lantz, 1992).

The par genes, identified by their role in the establishment of anterior-posterior polarity in the Caenorhabditis elegans zygote, subsequently have been shown to regulate cellular polarity in diverse cell types by means of an evolutionarily conserved protein complex including PAR-3, PAR-6, and atypical protein kinase C (aPKC). The Drosophila homologs of par-1, par-3 (bazooka [baz]), par-6 (DmPar-6), and pkc-3 (Drosophila aPKC; DaPKC) each are known to play conserved roles in the generation of cell polarity in the germ line as well as in epithelial and neural precursor cells within the embryo. In light of this functional conservation, the potential role of baz and DaPKC in the regulation of oocyte polarity was examined. Germ-line autonomous roles have been revealed for baz and DaPKC in the establishment of initial anterior-posterior polarity within germ-line cysts and maintenance of oocyte cell fate. Germ-line clonal analyses indicate both proteins are essential for two key aspects of oocyte determination: the posterior translocation of oocyte specification factors and the posterior establishment of the microtubule organizing center within the presumptive oocyte. Baz and DaPKC colocalize to belt-like structures between germarial cyst cells. However, in contrast to their regulatory relationship in the Drosophila and C. elegans embryos, these proteins are not mutually dependent for their germ-line localization, nor is either protein specifically required for PAR-1 localization to the fusome. Therefore, whereas Baz, DaPKC, and PAR-1 are functionally conserved in establishing oocyte polarity, the regulatory relationships among these genes are not well conserved, indicating these molecules function differently in different cellular contexts (Cox, 2001).

Oocyte differentiation requires the polarized accumulation of oocyte specification factors within a single cell of the germ-line cyst. To analyze the role of baz or DaPKC in the localization of these factors, mutant germ-line clones for both genes were generated and the expression of the oocyte specification factors ORB, BIC-D, and the microtubule motor protein DHC64C were examined at early and late stages of oogenesis. In wild-type germarial cysts, both ORB and BIC-D are initially uniformly distributed among the cyst cells in region 2a, and then both molecules are targeted first to the two pro-oocytes and ultimately to the fated oocyte by late region 2a. Furthermore, whereas ORB protein initially concentrates at the anterior of the oocyte, it translocates to the posterior pole of the oocyte and condenses into a posterior crescent in region 3. In contrast, ORB fails to translocate from the anterior to a posterior crescent in both baz and DaPKC null germ-line cysts in germarial region 3 and rather remains at the anterior margin of the presumptive oocyte. An identical defect in A-P BIC-D translocation was observed in baz and DaPKC null germ-line clones in germarial region 3. The defect in the translocation of ORB and BIC-D to the posterior of the oocyte at this early stage is subsequently manifest by a failure to accumulate these proteins in later-stage oocytes (Cox, 2001).

The posterior assembly of a functional MTOC has been directly implicated in the differential segregation of oocyte specification factors within developing germ-line cysts, suggesting that the failure to translocate these factors to a posterior crescent in region 3 baz or DaPKC mutant cysts may result from a defect in microtubule reorganization within these mutant cysts. In contrast to wild-type, baz and DaPKC mutant cysts display a parallel defect in the A-P transition of the MTOC within the presumptive oocyte. These results support the conclusion that the defects observed in posterior translocation of oocyte specification factors in these mutants are likely caused, at least in part, by the observed disruption in the A-P transition of the oocyte MTOC (Cox, 2001).

Effects of Mutation or Deletion

Strong orb alleles arrest egg development at the time of nurse cell-oocyte cyst formation; weak alleles disrupt formation of the anterior-posterior and dorsoventral axes within the oocyte during late oogenesis. (McKearin, 1994)

Strong orb alleles arrest oogenesis at an early stage, prior to egg chamber formation, whereas females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of Oskar and Gurken mRNAs within the oocyte during the later stages of oogenesis. In addition, double heterozygous combinations of orb and grk or orb and top/DER alleles reveal that mutations in these genes interact genetically, suggesting that they participate in a common pathway (Christerson, 1994).

A change in distribution of the germ-line dorsalizing 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 which occurs later in the follicular epithelium or in the early embryo (Roth, 1994).

Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions, transgenic flies were made expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation (Pare, 2000).

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, the role of the orb gene in the Drosophila gurken (grk)-epidermal growth factor receptor (Egfr) signaling pathway has been examined. During the pre-vitellogenic stages of oogenesis, the grk-Egfr signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, the grk-Egfr pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. orb functions in both the early and late grk-Egfr signaling pathways, and in each case is required for the localized expression of Grk protein. orb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10) (Chang, 2001).

orb activity is required for early grk-Egfr signaling, since abnormalities in Grk expression are observed in orb³⁴³ and orb³⁰³ ovaries. In the presumed Orb protein null, orb³⁴³, Grk is not detected. In orb³⁰³, Grk expression parallels the aberrant pattern of Orb³⁰³ protein accumulation. In newly formed 16-cell cysts, all germ cells have high levels of the Orb³⁰³ protein. These germ cells also express much higher than normal levels of Grk protein. In older pseudo-egg chambers, both Orb³⁰³ and Grk disappear. These findings argue that the Orb³⁰³ protein inappropriately activates translation of GRK mRNA, and that the mutant Orb protein must be present to sustain Grk expression. Later in oogenesis, after the oocyte nucleus moves from the posterior of the oocyte to the dorsal anterior corner, the grk-Egfr pathway is used to signal dorsal identity to the follicle cells above the oocyte. At this stage orb is required for the proper expression not only of Grk but also of K(10) (Chang, 2001).

How does orb function in regulating translation and localization? Orb homologs in other organisms, the CPEB proteins, interact with elements in the 3' UTRs of masked mRNAs, and activate their translation by a mechanism that is thought to involve polyA addition. Since the translational function of the CPEB proteins is conserved in animals as diverse as clams and mice, it would be reasonable to suppose that the role of the orb gene in the Drosophila grk-Egfr signaling pathway also involves translational activation. Accordingly, the defects in the expression of both Grk and K(10) proteins would arise because wild type orb activity is required to properly regulate the translation of GRK and K(10) mRNAs. In the case of K(10), it seems possible that Orb protein might act directly on the mRNA: (1) K(10) mRNA is associated with Orb protein in an immunoprecipitable complex and (2) K(10) mRNA is mislocalized in orb mutant ovaries (Chang, 2001).

Since translational activation by CPEB proteins in other systems has been tied to polyadenylation, an obvious question is whether the polyA tails of K(10) mRNA are affected in orb mutants. Unfortunately, experiments aimed at testing this point have been inconclusive. Using an anchored-dT RT-PCR procedure, it was found that K(10) mRNA isolated from the strong loss-of-function orb mutant, orb³⁴³, had shorter poly(A) tails than wild type. However, the possibility cannot be excluded that the short poly (A) tails in this mutant arise because K(10) mRNA is targeted for deadenylation in the absence of translation. For orb^mel, the average poly(A) length appeared, at most, to be only marginally shorter than wild type. Of course, since K(10) protein is expressed normally in pre-vitellogenic stages in this mutant, the presence of mRNAs with extended poly(A) tails is not altogether surprising. Further studies will be required to determine whether the mechanism used to promote the translation of K(10) mRNA depends upon polyA addition as is thought to be the case in other organisms (Chang, 2001).

In contrast to K(10), GRK mRNA was not found in Orb immunoprecipitates. Although there are many reasons why an Orb protein:GRK mRNA complex might not be detected, this result forces consideration of the possibility that orb acts on GRK only indirectly. In this case, other mechanisms would have to be proposed to account for the defects in both the localization and translation of GRK mRNA that are observed in orb mutants (Chang, 2001).

It seems possible that the mislocalization of GRK mRNA in the weak hypomorphic orb^mel mutant could arise, at least in part, because the expression of K(10) protein is greatly reduced in stage 8-10 orb^mel chambers. However, since the localization defects in orb^mel are more severe than those seen in K(10) mutants, orb may regulate some other factor in addition to K(10) that helps direct the proper localization of GRK mRNA. An obvious candidate is sqd. Although no alterations in Sqd protein expression could be detected in orb^mel chambers, it should be noted that only one of the three Sqd isoforms seems to be involved in GRK mRNA localization. Consequently, any effects on the expression of this specific isoform could be obscured by the other isoforms (Chang, 2001).

Why is GRK mRNA not properly translated in orb mutant ovaries? Orb protein could be required for the expression of factors that activate translation of GRK mRNA. In orb³⁰³ this factor(s) could be prematurely produced throughout the cyst, leading to the very high levels of unlocalized Grk seen in this mutant. As K(10) and sqd do not seem to function in the localization or translation of GRK mRNA at the posterior of the oocyte in pre-vitellogenic stages, the orb regulatory target(s) early in oogenesis could be different from that used later in DV signaling. Another possibility is that orb regulates the expression of a signal(s) that coordinates the activation of GRK mRNA translation with other events in oogenesis. This function is suggested by the fact that CPEB activity in other organisms helps govern progression through oogenesis and by the finding that grk expression in the DV pathway is sensitive to check points that monitor progression through meiosis. In this case, signals crucial for translation of GRK mRNA might not be produced in the absence of orb activity (Chang, 2001).

The epistatic relationship between orb^mel and K(10) is rather surprising. Since orb is required for the localization and translation of GRK mRNA, it is expected that orb^mel would be epistatic to K(10). However, contrary to this expectation, eggs produced by K(10);orb^mel double mutant females have the dorsalized egg shell phenotype that is characteristic of K(10) mutations, rather than the ventralized phenotype of orb^mel. This result implies that the loss of K(10) function rescues the orb^mel defect in GRK mRNA translation (but not the localization defect). Interestingly, a similar epistatic relationship is found for K(10) and mutations in the spindle (spn) genes. Mutants in the spn genes resemble orb in that GRK mRNA is mislocalized in a K(10)-like pattern but is not properly translated, giving ventralized eggs. Moreover, the defects in GRK mRNA translation in spn mutants can also be rescued by mutations in K(10) and double mutant females produce dorsalized eggs. To explain these findings, it has been postulated that the function of the spn genes is to alleviate K(10)-dependent repression of GRK mRNA translation (Chang, 2001 and references therein).

Although orb could have a similar role in alleviating K(10)-dependent repression of GRK, an alternative (or additional) explanation for the epistatic relationship between orb^mel and K(10) is that K(10) negatively regulates Orb protein expression. This possibility is suggested by the finding that the amount of Orb protein in vitellogenic chambers from the double mutant is close to that seen at equivalent stages in wild-type ovaries. The restoration of near wild-type levels of Orb protein in these orb^mel;K(10) chambers would in turn be expected to produce a concomitant increase in Grk expression, giving the observed gain-of-function phenotype (Chang, 2001).

Complicating the conclusion that K(10) negatively regulates Orb expression is the finding that K(10) protein does not properly accumulate in the oocyte nucleus of vitellogenic orb^mel chambers. One might have expected that this reduction in the level of K(10) protein would alleviate the K(10)-dependent repression of Orb protein expression, leading to an increased accumulation of Orb protein in the orb^mel mutant and a dorsalized (not ventralized) DV phenotype. However, it does not. One explanation for this paradox is that orb^mel is wild type for K(10), whereas this is not the case in the double mutant. In addition, there are no apparent defects in K(10) expression in pre-vitellogenic orb^mel chambers. It is possible that there is sufficient residual K(10) protein remaining at later stages to effectively repress orb, or that K(10) repression of orb is linked to a process that occurs before the time when the accumulation of K(10) protein drops below some critical threshold value in the orb^mel chambers. In this context, it is interesting to note that the most severe defects in both ORB mRNA localization and Orb protein expression in orb^mel occur after the reorganization of the cytoskeleton and the concomitant movement of the oocyte nucleus from the posterior to the anterior of the oocyte. This marks a shift in the localization of orb mRNA and the site of Orb protein synthesis from the posterior of the oocyte to the anterior. Since the expression of K(10) protein before this time is normal in orb^mel ovaries, its possible that K(10) repression may be somehow linked to this spatial transition in orb regulation (Chang, 2001).

Although the K(10) mutation has quite dramatic effects on Orb expression in orb^mel ovaries, there are no obvious changes in Orb expression in K(10) mutant ovaries that are wild type for orb. It seems possible that there may be some special features of the orb^mel mutation that make it especially sensitive to K(10) repression. However, genetic interaction experiments suggest that K(10) also negatively regulates expression of the wild-type orb gene. An important unanswered question is the mechanism of regulation. Here, there is a problem of compartmentalization. For example, since ORB mRNA is thought to be synthesized in nurse cells, K(10) protein is unlikely to influence transcription. Even effects on the localization/translation of orb mRNA must be indirect. Further studies will clearly be required to understand how K(10) regulates orb expression (Chang, 2001).

The hiiragi (hrg) gene encodes a poly(A) polymerase (PAP), an enzyme that attaches adenylyl residues to the 3' untranslated region of mRNAs. The single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo (Juge, 2002). Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP during embryogenesis does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. Thus regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development. hrg is also probably essential to cell viability since strong hrg mutant germline clones do not survive. The PAP encoded by this gene is involved in polyadenylation of oskar mRNA in oocytes. This indicates that although the reactions of nuclear and cytoplasmic polyadenylation are not identical, a single PAP is responsible for both in Drosophila (Juge, 2002).

The molecular mechanism of cytoplasmic polyadenylation has been analysed extensively in Xenopus oocytes, and some aspects of the reaction are similar to that of nuclear polyadenylation. Nuclear polyadenylation consists of endonucleolytic cleavage of pre-mRNAs followed by the synthesis of a poly(A) tail onto the upstream cleavage product. Poly(A) addition can be reconstituted in vitro from three purified mammalian factors: poly(A) polymerase (PAP), cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein II [PABP2, the nuclear poly(A)-binding protein]. CPSF is a complex of four proteins that binds the polyadenylation signal AAUAAA located upstream of the cleavage site. Recognition of the poly(A) site also requires cleavage stimulation factor (CstF) that binds to a GU/U-rich element downstream of the cleavage site and interacts with CPSF. PAP catalyses the polyadenylation reaction, but is also required for efficient cleavage of pre-mRNAs in vitro. PAP by itself does not recognize pre-mRNAs specifically. Specificity requires the AAUAAA element and CPSF that binds PAP through its 160 kDa subunit. Even in the presence of CPSF, PAP activity remains weak; it is again stimulated by binding of PABP2 to the poly(A) tail. Together, CPSF and PABP2 stimulate PAP activity by holding PAP on the RNA such that a full-length poly(A) tail is synthesized in a single processive event. When the poly(A) tail has reached its complete length, elongation is no longer processive and becomes slow and distributive. PABP2 is required for this poly(A) tail length control (Juge, 2002 and references therein).

In Drosophila, the role of CPEs has not been addressed, and the polyadenylation signal is dispensable in some cases, since embryonic cytoplasmic polyadenylation occurs on a bicoid engineered mRNA deleted for this element. Although genes encoding the four subunits of CPSF are present in the Drosophila genome, their role in cytoplasmic polyadenylation has not been determined. The Drosophila homolog of CPEB is the Orb protein. orb encodes germline-specific proteins different in male and female, and its function has been determined in the female germline. Strong orb mutants arrest oogenesis early, before the formation of the 16-cell cyst that would normally differentiate into nurse cells and one oocyte. Using a weaker allele, orb^mel, Orb was shown to be required for anchoring of oskar mRNA at the posterior pole of the oocyte. However, this could result from a failure in oskar mRNA translation since Oskar protein is required for anchoring its own mRNA at the posterior pole. A recent study suggests that Orb could have a function analogous to that of CPEB in cytoplasmic polyadenylation. In orb mutant egg chambers, the level of Oskar protein is decreased and poly(A) tails of oskar mRNAs are shortened (Juge, 2002 and references therein).

To address a possible role for hrg in cytoplasmic polyadenylation during early development, germline clones homozygous for hrg^PAP45, hrg^PAP21 or hrg^PAP12 were induced. No germline clones were obtained for any of these mutants, possibly as a result of a requirement of PAP for cell viability. Therefore genetic interactions were studied between hrg and orb, which is known to be involved in cytoplasmic polyadenylation. Females homozygous for the weak orb^mel allele produce egg chambers at all stages and lay eggs, 30% of which show a ventralized phenotype. hrg lethal mutants act as dominant enhancers of the orb^mel phenotype, since hrg^PAP45/+; orb^mel and hrg^PAP21/+; orb^mel females lay almost no eggs. In these females, oogenesis stops most frequently at stage 7/8, after which egg chambers degenerate, even though one or two stage 14 oocytes per ovary can be observed. Poly(A) tails of oskar mRNA are shortened in orb mutant ovaries. The defect of these poly(A) tails were analyzed in hrg^- /+; orb^mel mutants by PAT assays. oskar mRNA poly(A) tails were measured to be up to 135 residues in wild-type ovaries. These poly(A) tails are weakly reduced in orb^mel, but severely reduced in hrg^- /+;orb^mel double mutant ovaries, their maximal length reaching 40- 50 residues. These short poly(A) tails do not result from the oogenesis defect in hrg^- /+; orb^mel females, since unrelated mutants that stop oogenesis early show wild-type poly(A) tails of oskar mRNA. These poly(A) tails were also found to be of wild-type length in hrg^PAP21/+ and hrg^PAP45/+ ovaries. This shows that the strong shortening of oskar poly(A) tails in hrg^- /+; orb^mel mutants does not result from an additive effect of two phenotypes, but from a synergistic effect of the two mutants due to a simultaneous decrease in PAP and Orb protein levels. This strongly suggests that hrg and orb are involved together in cytoplasmic polyadenylation. This was confirmed by measurements of poly(A) tails of a control mRNA, sop, which is thought not to be regulated by cytoplasmic polyadenylation. Poly(A) tails of sop mRNAs are unaffected in orb^mel as well as in hrg^- /+; orb^mel mutant ovaries. It was verified that shortening of oskar mRNA poly(A) tails in hrg^- /+; orb^mel mutants leads to a reduction of Oskar protein level, by immunostaining of ovaries with anti-Oskar. Oskar accumulates at the posterior of the oocyte from stage 9 onwards. The amount of Oskar decreases in orb^mel oocytes. This amount decreases again in hrg^- /+; orb^mel oocytes to a barely detectable level (Juge, 2002).

Taken together, these results show that hrg and orb cooperate in poly(A) tail lengthening during cytoplasmic polyadenylation and that alteration of this process affects protein accumulation and oogenesis (Juge, 2002).

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date revised: 1 September 2020

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