Gene name - orb
Synonyms - oo18 RNA-binding protein
Cytological map position - 94E11-13
Function - RNA binding protein
Symbol - orb
Genetic map position -
Classification - RRM family
Cellular location - cytoplasmic
The contents of the Drosophila egg form a highly organized ensemble of protein systems, ready to abandon the quietude of the oocyte for the actively developing zygote once fertilization has occurred. Four maternal systems exist in the pre-fertilized egg. They will eventually determine the anterior-posterior axis and the dorsoventral axis in the developing embryo. The gene orb is involved in two of these four systems and the processes they govern.
orb is considered a gene of both the posterior group and the dorsal group. It functions in the transport of mRNAs coding for two essential proteins that govern polarity in the egg: Oskar (anterior/posterior) and Gurken (dorsal/ventral). ORB protein contains two RNA recognition motifs characteristic of proteins that bind the 3' messenger RNA. The correct distribution of both Oskar and Gurken mRNAs is dependent on ORB during the middle phase of oogenesis, stages 8-10. Oskar and Gurken are crucial to appropriate anterior-posterior and dorso-ventral patterning. Osk protein recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, Nanos. During oogenesis ORB protein is enriched in the cortical region that underlies the oocyte plasma membrane, especially in the anterior dorsal region and at the posterior pole of the oocyte. Gurken mRNA is usually localized to the anterior dorsal region of the egg during this period, while Oskar mRNA is localized to the posterior pole. orb mutation interfers with both localization processes. Gurken acts both early and late in the oocyte assmblage process. Consequently, since orb is involved in both phases of Gurken mRNA localization, orb also acts both early and late in oocyte assembly (Christerson, 1994).
Two other genes, cappuccino and spire, are also required for the proper determination of both axes. There is little doubt that proper RNA localization requires a large protein complex to shuttle transcripts to specific sites. In the egg, RNA localization represents a macrocosm of similar phenomena that must occur in normal cells in the process of determining cell polarity and alternative fates for cells during mitosis. The fly egg provides developmental biology a peek at these phenomena. It remains to be seen if the same processes function during normal cell division to generate intracellular mRNA asymmetries, comparable to the localizations occurring in the egg.
The critical step in the targeting of posterior determinants during the establishment oocyte polarity axes is the localization of Oskar (OSK) mRNA to the pole and its on-site translation. The role of the Drosophila CPEB homolog, the orb gene, in the OSK mRNA localization pathway has been analyzed. The expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orbmel, little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation in orb mutant ovaries are correlated with a reduction in the length of the OSK poly(A) tails. OSK mRNA is found in immunoprecipitable complexes with Orb protein in ovaries. The OSK 3' UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of OSK mRNA and that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of 'masked' mRNAs (Chang, 1999).
What role does Orb protein play in the translation of OSK mRNA localized at the posterior pole in vitellogenic oocytes? One model that could potentially account for the current findings posits that translationally masked OSK mRNA would be deposited from the nurse cells into the anterior of the oocyte and then transported to the posterior pole by the Stauffen protein. Good candidates for ensuring that OSK mRNA is not translated in the nurse cells or at the anterior of the oocyte would be the Bruno protein and p50 protein. When the masked OSK mRNA arrives at the posterior pole, it would be bound by an Orb protein complex that is already anchored in the posterior cytoskeleton. Orb protein would then activate OSK mRNA translation, most probably by promoting polyadenylation (Chang, 1999 and references therein).
Orb plays an early role during oogenesis in controlling entry into meiosis. The oocyte is the only cell in Drosophila that goes through meiosis with meiotic recombination, but several germ cells in a 16-cell cyst enter meiosis and form synaptonemal complexes (SC) before one cell is selected to become the oocyte. Using an antibody that recognises a component of the SC or the synapsed chromosomes, an analysis was carried out of how meiosis becomes restricted to one cell, in relation to the other events in oocyte determination. Although Bicaudal-D and egalitarian mutants both cause the development of cysts with no oocyte, they have opposite effects on the behavior of the SC: none of the cells in the cyst form SC in BicD null mutants, whereas all of the cells do in egl and orb mutants. Furthermore, unlike all cytoplasmic markers for the oocyte, the SC still becomes restricted to one cell when the microtubules are depolymerised, even though the BicD/Egl complex is not localised. These results have lead to the proposal of a model in which BicD, Egl and Orb control entry into meiosis by regulating translation (Huynh, 2000).
In the course of a study on the role of inscuteable (insc) during oogenesis, it has been found that an anti Insc antibody recognizes a nuclear structure that is present in some of the germ cells in regions 2a to 3 of the germarium. However, this staining does not disappear in germline clones of protein null allele insc 22, indicating that it is due to a cross-reaction of the antibody. Nevertheless, the staining pattern is very reminiscent of that expected for a component of the synaptonemal complex (SC), and therefore the staining was analyzed further, since this would be the first marker identified for the SC structure in Drosophila. Several lines of evidence indicate that the antiserum does indeed label the SC or a component associated with its formation. (1) The nuclear staining colocalizes with DNA, and has a morphology that corresponds exactly with the observed behaviour of the SC in electron micrographs. The staining is dotty in very early region 2a when the SC starts to form, becomes more thin and thread-like when the chromosomes are fully synapsed, and then becomes more compact in region 3, when the meiotic chromosomes condense to form the karyosome. (2) This structure first appears at the stage when the cysts enter into meiosis. The mitotic cysts in region 1 of the germarium express Bam protein, but this disappears after the final division when the cysts move from region 1 to region 2a of the germarium. The nuclear staining is only detectable in cysts that no longer show any Bam expression, indicating that it labels a postmitotic structure. (3) The spatial distribution of the signal within the cyst precisely follows that described for the SC at the EM level. The signal first appears in two cells in early region 2a and spreads to four cells per cyst in the middle of 2a, before it is restricted to two cells, and finally to one cell in region 2b. Ovaries from females that are mutant for C(3)G were examined, since these are the only characterized mutants that completely abolish the formation of the SC at the electron microscope level. C(3)G encodes the fly homolog of yeast Zip1 and mammalian SCP1, components of the transverse filament of the SC, and the effects of the C(3)G mutation on the SC are therefore likely to be direct (Szauter and Hawley, personal communication to Huynh, 2000). The nuclear structure stained by this antibody is absent in C(3)G mutant cysts, even though the localization of Orb protein to the oocyte occurs normally. Thus, the antibody acts as a marker for the formation of the SC, although the molecular nature of the epitope recognized is not known (Huynh, 2000).
A detailed analysis of the behavior of the SC in comparison to that of cytoplasmic markers for oocyte determination, such as Orb and Bic-D proteins, reveals a number of distinct steps in the restriction of oocyte fate to one cell. The SC first appears in early region 2a cysts in the nuclei of two cells, which are presumably the pro-oocytes. The punctate appearance of the SC suggests that they are at the zygotene stage of meiotic prophase 1. The next one or two cysts per germarium have four cells in synapsis. Two of these cells have four ring canals (the pro-oocytes) and contain an almost continuous SC, typical of the pachytene stage, while the two cells on either side, presumably the cells with three ring canals, contain a zygotene-like SC. In the middle of region 2a, the SC disappears from the two cells with three ring canals, but the two pro-oocytes still have complete SCs, and accumulate Orb and Bic-D proteins. Soon afterwards, Orb and Bic-D become concentrated in only one of these cells, providing the first sign that this pro-oocyte has been selected to become the oocyte. However, the SC still appears identical in both pro-oocytes at this stage. The SC disappears from one pro-oocyte as the cyst enters region 2b, and the cell that remains in meiosis is always the one that has already accumulated Orb or Bic-D. Finally, SC becomes more compact in region 3 and a hole forms in its middle, before it disappears soon after the cyst leaves the germarium. This comparison of the behavior of nuclear and cytoplasmic markers for the oocyte reveals two important features about how oocyte fate becomes restricted to one cell. (1) The two pro-oocytes are already different from the other 14 cells in the cyst in early region 2a, as they both start to form SC at this stage. BicD and Orb only accumulate in these cells in mid 2a, about two cysts further down the germarium. (2) Orb and Bic-D become restricted to the oocyte before any sign of oocyte identity can be deduced from the behavior of the SC (Huynh, 2000).
A cyst can progress through the normal pattern of SC localization to one cell in the presence of high concentrations of colcemid, suggesting restriction of SC to one cell is not mediated by microtubules. Unlike the microtubules, BicD, orb and egl mutations disrupt all steps in the restriction of the SC to one cell, and this leads to two important conclusions: (1) BicD and Egl must have a function that is independent of microtubules, even though they are required for the establishment or maintenance of the MTOC in the oocyte; (2) this function of BicD, Egl and Orb does not depend on their own localisation to the oocyte, since all three proteins are completely delocalized after colcemid treatments, yet the SC still becomes restricted to one cell. Although both BicD and egl mutations give rise to cysts in which all 16 cells appear identical, they have different effects on the behavior of the SC itself. In BicD null germline clones, none of the cells form a detectable SC, whereas all cells reach the full pachytene stage in egl mutants (Huynh, 2000).
BicD and Egl are part of the same protein complex, and it is therefore surprising that they have opposite phenotypes. It is suggested that BicD and Egl may have different functions. BicD is required to enhance SC formation in the cells that normally enter meiosis, whereas Egl functions to repress SC formation in the other cells of the cyst. The strongest mutations in orb have a very similar effect on SC formation as do egl mutants, suggesting that Orb protein is also involved in this repression. Given the colocalization of Orb with Egl and BicD, it will be interesting to determine whether it is part of the same protein complex (Huynh, 2000).
The discovery that the restriction of SC to one cell requires neither microtubules nor the localization of BicD, Egl and Orb raises the question of how this asymmetry arises. It has previously been suggested that BicD and Egl function in the transport of meiosis promoting factors and oocyte determinants from the future nurse cells into the oocyte. Although this could still be the case if this transport occurs either very early in region 2a or along some non-microtubule cytoskeletal network, such as actin, this model cannot easily explain why BicD and egl mutations have opposite effects on SC formation. An alternative model is preferred in which BicD, Egl and Orb are required to interpret a pre-existing asymmetry that is set up in region 1 (Huynh, 2000 and references therein).
The divisions that give rise to the cyst are asymmetric with respect to the fusome, and recent data strongly suggest that this structure, or some factor associated with it, somehow marks the future oocyte. If this is correct, this unidentified mark could regulate the BicD/Egl complex, so that it performs different functions in the different cells of the cyst. For example, the Egl-dependent activity of the complex could repress SC in the cells that do not inherit the factor, and the BicD-dependent activity could enhance its formation in the cells that do, thereby explaining the different phenotypes of the null mutations in the two genes. It is interesting to note that BicD protein is phosphorylated, and that mutations that disrupt this phosphorylation give rise to egg chambers with 16 nurse cells. Thus, this post-translational modification could be responsible for the spatial regulation of the activity of the BicD/Egl complex (Huynh, 2000).
Although these results suggest that BicD and Egl have functions that are independent of the microtubules, the nature of this activity is unclear. However, a number of lines of evidence suggest that these proteins may be involved in translational control. (1) BicD was originally identified because two single amino acid changes in the gene produce a dominant bicaudal phenotype in which Oskar mRNA is mis-expressed at the anterior of the oocyte. Since Oskar translation is normally repressed unless the RNA is localized to the posterior pole, these mutant BicD proteins must not only trap Oskar RNA at the anterior, but also relieve translational repression. Mutations in egl suppress the BicD gain-of-function phenotype, while extra copies of egl enhance it, indicating that the ectopic translation of Oskar mRNA requires the formation of the BicD/Egl complex. The second argument for a role of BicD and Egl in translational control comes from the discovery that orb null mutations give a very similar phenotype to egl mutants. Orb protein, which contains two RNA-binding motifs, has recently been shown to associate with the 3'UTR of Oskar mRNA, and is required for its efficient translation. Similarly, the Xenopus Orb homolog, CPEB, binds to elements in the 3'UTRs of a number of mRNAs, and induces the polyadenylation and translational activation of these mRNAs during oocyte maturation. Furthermore, the Spisula solidissima (clam) homolog plays a role not only in translational activation, but also in repression, since it binds to masking elements in the 3'UTRs of cyclin mRNAs to prevent their translation before fertilization. Thus, Orb functions as a regulator of translation, and can act as both a repressor and an activator in other species. This raises the possibility that the BicD/Egl complex exerts different effects in the cells of the cyst by controlling the inhibitory and activating functions of Orb. For example, Orb could repress the translation of factors required for SC formation in the future nurse cells, and activate their translation in the pro-oocytes and oocyte. If this model is correct, the selection of the oocyte would occur by a similar mechanism to the other asymmetries that are generated later in oogenesis, which are also all based on the translational regulation of asymmetrically localized mRNAs, such as Bicoid, Gurken and Oskar (Huynh, 2000 and references therein).
The behavior of the SC indicates that the determination of the oocyte occurs in two steps. The two pro-oocytes must have been selected by early region 2a, because they already behave differently from the other 14 cells of the cyst at this stage, but the development of the cyst remains symmetric until the end of 2a, when BicD and Orb disappear from the losing pro-oocyte. It has been proposed that the choice between the two pro-oocytes could depend on competition between these cells as they progress through meiosis, with the cell that is more advanced becoming the oocyte and then inhibiting its neighbor. However, the results presented here argue against this model: (1) cytoplasmic factors, such as BicD and Orb, are concentrated in one cell before there is any visible difference between the SCs in the two pro-oocytes; (2) the cytoplasmic aspects of oocyte determination occur normally in C(3)G mutants, which completely lack the SC, and in meiW68 mutants, which fail to initiate meiotic recombination. Thus, any competition between these two cells must be independent of SC formation and recombination (Huynh, 2000 and references therein).
Although meiosis is not required for oocyte determination, it can clearly influence this process, as demonstrated by the results on the spn genes. Several lines of evidence indicate that mutations in spnB, C and D disrupt the repair of dsDNA breaks during meiotic recombination, activating a checkpoint pathway that inhibits Gurken mRNA translation and the formation of the karyosome. The results presented here strongly suggest that this checkpoint also inhibits the determination of the oocyte, since the SC becomes restricted to one cell much later than in wild type in these mutants. This phenotype also allows the time when recombination occurs to be narrowed down. This process cannot begin until the SC forms in early region 2a, but the double-strand DNA breaks have to be repaired before the two cells with three ring canals exit meiosis, since this stage is delayed in spnC mutants, indicating that the checkpoint pathway has already been activated (Huynh, 2000).
Activation of the meiotic checkpoint causes a change in the mobility of Vasa protein, leading to the suggestion that the patterning defects seen in spn mutants result from the inhibition of Vasa by this pathway. The results presented here show that the SC becomes restricted to one cell at the normal time in most vasa mutant cysts. Thus, the delay in oocyte determination in spn mutants cannot be a consequence of the inhibition of Vasa, suggesting that the checkpoint pathway has additional targets that control oocyte selection (Huynh, 2000).
One problem in the study of cyst development in region 2 has been the difficulty in ordering the various developmental events that occur in this region. Using this marker for the SC, the behavior of this structure relative to the localization of cytoplasmic factors like Orb and BicD could be followed, and these could be correlated with the data from EM studies on the behavior of the SC, and the centrioles. On the basis of this comparison, a number of distinct stages in the restriction of oocyte fate to one cell can be distinguished: (1) The first cyst in region 2a shows no sign of SC, but Bam protein has already disappeared. (2) The two pro-oocytes reach the zygotene stage of meiosis in early region 2a, and start to form SC. (3) Soon afterwards, the two cells with three ring canals also form SC. The SC in the pro-oocytes has reached its maximum length, indicating that they have reached the pachytene stage. The dsDNA breaks generated during recombination must have already been repaired, since the meiotic checkpoint can arrest the pattern of SC staining at this stage. EM data also suggest that intracellular transport begins at this point, since the first signs of the migration of the centrioles towards the pro-oocytes can be seen when the two cells with three ring canals are in meiosis, and this may correlate with the first appearance of a focus of microtubules in the cyst in the middle of region 2a. (4) The SC disappears from the two cells with three ring canals in the middle of region 2a, but the two pro-oocytes still have complete SCs. Orb and Bic-D start to accumulate in the pro-oocytes at this stage. The centrioles have migrated to either side of the largest ring canal, which connects the two pro-oocytes, and the first signs of 'nutrient streaming' appear, since elongated mitochondria can be seen inside the ring canals in electron micrographs. (5) All of the steps in cyst development so far are symmetric, relative the largest ring canal, and the first asymmetry becomes evident in cysts numbers 5 and 6, when Orb and Bic-D become concentrated in one cell. The centrioles also start to move into the oocyte, and the largest ring canal is presumably open, because mitochondria can now be seen inside it. However, both pro-oocytes still contain an identical intact SC at this stage. (6) As the cyst enters region 2b, one pro-oocyte loses its SC and reverts to the nurse cell pathway of development. The pro-oocyte that remains in meiosis and becomes the oocyte is always the cell that has already accumulated Orb and Bic-D. The cytoplasm of the oocyte now contains all of the centrioles, BicD and Orb proteins, and an obvious MTOC, which nucleates microtubules that extend into the other 15 cells of the cyst. Thus, both the nucleus and cytoplasm of the oocyte are clearly different from the other cells of the cyst by this stage. Immediately afterwards, the oocyte starts to behave differently from the other cells in the cyst, as it moves to the posterior during the transition between region 2b and region 3. At the same time, the karyosome forms, and the SC becomes more compact, before disappearing soon after the cyst leaves the germarium (Huynh, 2000).
orb has two transcripts, each initiating from a different promoter. The ovarian and early embryonic transcript is 4.5 kb in length and contains four exons. The male specific transcript is 3.2 kb in length and consists of three exons. The second and third exon of the male specific transcript correspond to the third and fourth exons of the ovarian transcript, except that the transcription of the male third exon terminates early (Lantz, 1992).
cDNA clone length - 4737 for ovarian
Bases in 5' UTR -782 for ovarian; 318 for testicular
Exons (ovarian) - four
Exons (testicular) - three
Bases in 3' UTR - 1104 for ovarian; 216 for testicular
The predicted proteins contain two regions with similarity to the RRM family of RNA-binding proteins. In the N-terminal portion of the protein there is a glycine/ulanine rich region of about 100 amino acids located between two opa repeats (Lantz, 1992).
date revised: 15 December 99
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.