oskar


DEVELOPMENTAL BIOLOGY

Oocyte

See the embryonic expression pattern of osk at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Oskar mRNA is present in the germarium before 16 cell clusters bud to form individual egg chambers. The Oskar mRNA is confined to the oocyte, the one cell among the 16 within the cluster that is not a nurse cell. By late stage 8, OSK mRNA is still present throughout the oocyte, but has become more concentrated at the anterior margin and the posterior pole. By stage 9, (middle egg chamber), OSK mRNA is no longer concentrated at the anterior margin, and soon becomes completely localized to the posterior pole. Localization takes place prior to the onset of cytoplasmic streaming and prior to bulk flow from the nurse cells into the oocyte. During stage 10A, OSK mRNA begins to accumulate at high levels in the nurse cells.

Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos

Drosophila segmentation is governed by a well-defined gene regulation network. The evolution of this network was investigated by examining the expression profiles of a complete set of segmentation genes in the early embryos of the mosquito, Anopheles gambiae. There are numerous differences in the expression profiles as compared with Drosophila. The germline determinant Oskar is expressed in both the anterior and posterior poles of Anopheles embryos but is strictly localized within the posterior plasm of Drosophila. The gap genes hunchback and giant display inverted patterns of expression in posterior regions of Anopheles embryos, while tailless exhibits an expanded pattern as compared with Drosophila. These observations suggest that the segmentation network has undergone considerable evolutionary change in the dipterans and that similar patterns of pair-rule gene expression can be obtained with different combinations of gap repressors. The evolution of separate stripe enhancers in the eve loci of different dipterans is discussed (Goltsev, 2004).

Anopheles lacks bicoid and contains a lone Hox3 gene that is more closely related to zen and specifically expressed in the serosa. How is hunchback activated in the presumptive head and thorax in Anopheles? The homeobox gene orthodenticle can substitute for bicoid in Tribolium. However, orthodenticle does not appear to be maternally expressed in Anopheles, but instead, staining is strictly zygotic and restricted to anterior regions, similar to the pattern seen in Drosophila. Sequential patterns of orthodenticle, giant, and hunchback expression are established by differential threshold readouts of the Bicoid gradient in Drosophila. It is possible that an unknown maternal regulatory gradient emanating from the anterior pole is responsible for producing similar patterns of expression in Anopheles. It is proposed that this unknown regulatory factor may be localized to the anterior pole by Oskar. Oskar coordinates the assembly of polar granules and is essential for the localization of Nanos in the posterior plasm. It might also localize one or more unknown determinants in anterior regions of Anopheles embryos (Goltsev, 2004).

Stimulation of endocytosis and actin dynamics by Oskar polarizes the Drosophila oocyte

In Drosophila, localized activity of oskar at the posterior pole of the oocyte induces germline and abdomen formation in the embryo. Oskar has two isoforms, a short isoform encoding the patterning determinant and a long isoform of unknown function. This study shows by immuno-electron microscopy that the two Oskar isoforms have different subcellular localizations in the oocyte: Short Oskar mainly localizes to polar granules, and Long Oskar is specifically associated with endocytic membranes along the posterior cortex. Cell biological and genetic analyses reveal that Oskar stimulates endocytosis, and its two isoforms are required to regulate this process. Furthermore, long F-actin projections at the oocyte posterior pole are described that are induced by and intermingled with Oskar protein. It is proposed that Oskar maintains its localization at the posterior pole through dual functions in regulating endocytosis and F-actin dynamics (Vanzo, 2007).

This investigation of the subcellular localization of Oskar in the Drosophila oocyte has uncovered an unanticipated function of Oskar in endocytosis. Using immuno-electron microscopy, it was found that Short and Long Oskar are mostly concentrated on distinct cellular structures in the oocyte—the polar granules and the endocytic compartment, respectively. Using stereological methods and functional assays, it has been shown that endocytosis levels are asymmetric around the cortex of wild-type oocytes, and that high levels of endocytosis at the posterior pole require Oskar expression. In addition, oskar mutant oocytes exhibit both a reduced endocytosis of yolk proteins and a reduction in endocytic structures at the posterior plasma membrane. Finally, a function of Oskar was identified in the asymmetric organization of the F-actin cytoskeleton in the oocyte. These data strongly suggest that both isoforms are potent regulators of endocytosis and F-actin dynamics (Vanzo, 2007).

The localization of the two Oskar isoforms to distinct subcellular structures during oogenesis may account for the previous unexplained observation of their distinct segregation during early embryogenesis, when the primordial germ cells form. Indeed, during these stages, Short Oskar accumulates in the pole cells, the future germ cells. During oogenesis, Short Oskar mainly concentrates in the polar granules. This localization is consistent with the previously reported function of this isoform in the establishment of the germ cell lineage in the embryo, a process thought to be instructed by polar granules. In contrast, Long Oskar is selectively excluded from pole cells in the early embryo and shows a specific affinity for membranous structures during oogenesis. Thus, the differential localization of the two Oskar isoforms that originates in oogenesis could persist over the lifetime of the two proteins and specify distinct destinies—Short Oskar being incorporated in the germ cells, and Long Oskar not (Kempkens, 2006).

Distinct localizations of the two Oskar isoforms could be due to the amino-terminal extension of Long Oskar (M1M2). However, this extension is unlikely sufficient to explain the specific association of Long Oskar with endocytic membranes, since Short Oskar can also associate with these, at a very low level. This extension might either increase the membrane affinity of Long Oskar and/or provide additional elements for efficient targeting/recruitment. It is noteworthy that Long Oskar is not an integral membrane protein. Neither a signal peptide, required for ER targeting, nor a significant hydrophobic stretch, required for membrane insertion, are apparent in its primary sequence. It is therefore presumably a peripheral protein recruited from the ooplasm to the cytosolic face of the endocytic membrane (Vanzo, 2007).

The absence of Long Oskar from polar granules is surprising for two reasons. First, Long Oskar is required for the maintenance of Short Oskar at the posterior pole of the oocyte. This implies that the maintenance function of Long Oskar must operate in an indirect way. Second, in addition to its specific amino-terminal extension, Long Oskar contains the entire Short Oskar peptide. This suggests that the N-terminal extension inhibits polar granule association of Long Oskar, in addition to promoting its association with membranes. As the polar granule component Vasa is not found on Long Oskar-positive membranes, this extension might also prevent Long Oskar from recruiting/assembling polar granule components, explaining its failure to specify the germline and the abdomen (Vanzo, 2007).

In oskar null oocytes, clathrin-mediated endocytosis is affected, both in its efficiency and its asymmetry. The aberrant prominence of flat, clathrin-coated areas of plasma membrane, instead of coated pits and vesicles, in those oocytes suggests impairment of endocytosis at an early stage, possibly in the initial invagination of the plasma membrane. This reveals that Oskar is a novel regulator of endocytosis (Vanzo, 2007).

The finding that sole expression of Long Oskar in oskar null oocytes triggers the formation of long and dense membrane sheet invaginations whose formation is abrogated upon coexpression of Short Oskar from a second transgene provides plausible explanations for the function of Oskar isoforms. Long Oskar could trigger an early step in the formation of clathrin-mediated membrane invaginations, which, in the absence of Short Oskar, fail to pinch off as coated vesicles and, instead, become protrusions, as observed in the dynamin mutant shibire (shits) at the restrictive temperature. Very reminiscent of abortive coated extensions also described in shibire oocytes, numerous clathrin-coated buds forming at the tip and sides of the large plasma membrane invaginations were identified in oocytes expressing only Long Oskar. In these oocytes, the invaginations may eventually detach and mature into yolk granules. The fact that these invaginations are no longer observed when Short Oskar is present suggests that Short Oskar could have a specific function in the subcellular localization and/or activation of dynamin at the posterior pole. The second, not exclusive, possibility is that Long Oskar activates membrane invagination through a clathrin-independent mechanism of endocytosis that Short Oskar antagonizes/overcomes to promote classical clathrin-mediated endocytosis. Clathrin-independent endocytosis has long been proposed and has recently been shown to involve tubular pleiomorphic structures very similar to those observed in the ooplasm of oocytes expressing Long Oskar only (Vanzo, 2007).

In any event, it is speculated that maintenance of a physiological ratio of the two isoforms (estimated to 1/4, Long/Short Oskar) is crucial to stimulate the clathrin-mediated endocytosis. Consistent with this, overexpressing Long Oskar in wild-type oocytes also induces an alternative, non-clathrin-dependent endocytic pathway, confirming that the balance of expression of the two Oskar isoforms is critical (Vanzo, 2007).

In contrast to Long Oskar, whose sublocalization agrees with a function in endocytosis, it is not obvious how Short Oskar could directly regulate endocytosis and/or Long Oskar function. Only residual membrane association of Short Oskar is detected in transgenic oocytes expressing this isoform alone. However, it is possible that Long Oskar enhances this association in wild-type oocytes. In this hypothesis, Short Oskar might interact functionally with the endocytic membrane to directly regulate either endocytosis or Long Oskar activity. Alternatively, Short Oskar might act indirectly by promoting local concentration of critical regulators of clathrin-mediated endocytosis at the posterior pole, possibly dependent on polar granule assembly. Further investigation of the specific function of Short Oskar in endocytosis will require the development of molecular tools circumventing the requirement for Long Oskar in Short Oskar expression and localization (Vanzo, 2007).

Long Oskar is required for efficient maintenance of Short Oskar at the posterior pole of the oocyte. The finding that Short Oskar largely accumulates in polar granules during oogenesis therefore implies that Long Oskar is essential for polar granule maintenance or integrity. Polar granules are only transiently found in close proximity to endocytic membranes on which Long Oskar localizes, when they form at stage 9. Then, as they mature from stage 9 to stage 10, they become larger, denser, and move away from the area where endocytosis occurs, to accumulate more internally at the edge of the endocytic zone. This observation suggests that polar granules are not maintained at the posterior pole by direct anchoring to the endocytic membranes (Vanzo, 2007).

The cortical F-actin cytoskeleton has been implicated in posterior anchoring of Oskar in oocytes, but the mechanism by which it acts was not addressed. In light of the present results, an attractive hypothesis is that polar granule anchoring involves the F-actin projections that are observed at the posterior pole of wild-type oocytes. These projections would be of sufficient length to span the first micrometer internally from the plasma membrane and to contact the underlying polar granules. In addition, they become detectable from stage 10 onward, when anchoring is required. These projections are largely reduced in oskar null oocytes. As Long Oskar expression per se does not restore their formation, this model suggests the existence of a positive feedback loop mechanism of maintenance, in which polar granules, possibly in concert with Long Oskar, could enhance their own anchoring at the posterior pole (Vanzo, 2007).

In light of the subcellular localization of Long Oskar, its unique competence to anchor at the posterior of the oocyte is quite notable. Interestingly, in addition to its well-documented role in nutrient uptake, endocytosis has emerged as a mechanism restricting the localization of proteins to plasma membrane subdomains in different polarized cells. In yeast, polarized exocytosis coupled with local endocytic recycling localizes membrane proteins at growing shmoo tips. More recently, endocytic trafficking has been shown to localize epithelial polarity proteins and restrict membrane receptor-dependent signaling during cell migration. By analogy, Long Oskar might be maintained and concentrated by continuous endocytic cycles at the posterior pole. By upregulating endocytosis, Oskar might promote its own accumulation, in addition to that of other membrane-associated factors, at the posterior pole. Consistent with this, Oskar has been shown to enhance the posterior accumulation of Rab11, a small GTPase recruited to endocytic membranes and required for endocytic recycling in the oocyte (Vanzo, 2007).

In addition to being actin dependent, Oskar anchoring also relies on an uncharacterized actin-independent mechanism. This mechanism was inferred from the observation that drug-induced F-actin depolymeriation caused only mild Oskar-anchoring defects in wild-type, in vitro-cultured oocytes. In light of the cureent findings, it is proposed that endocytosis might be this alternative mechanism. Consistent with this, the posterior localization of a Long Oskar reporter construct is essentially unaffected by actin depolymerization. Significant Oskar-anchoring defects are provoked by F-actin depolymerization in homer mutant oocytes. Although the molecular function of Homer in anchoring is unknown, it has been proposed that the protein is a key player in the actin-independent anchoring mechanism. Strikingly, using immuno-electron microscopy, it has been observed that a fraction of Homer associates with endocytic membranes and partially colocalizes with Long Oskar-containing endocytic structures. This observation supports a role of Homer and endocytosis in the mechanism of Long Oskar maintenance (Vanzo, 2007).

In conclusion, this work has revealed unexpected cellular functions of Oskar. Beyond its known functions in posterior patterning and germline induction, Oskar regulates asymmetry in clathrin-mediated endocytosis and F-actin organization in the Drosophila oocyte. It is proposed that, by regulating these two cellular processes, in positive feedback loops, Oskar isoforms promote their own maintenance at the posterior pole, thus reinforcing oocyte polarity (Vanzo, 2007).

Effects of Mutation or Deletion

Embryos derived from oskar females lack the specialized pole plasm including polar granules. They also lack pole cells, the zygotic stem cells for the gonads. In addition, the abdominal region remains unsegmented and eventually dies. Transplantation of cytoplasm from normal embryos into mutant embryos reveals that osk-dependent activity is strictly localized at the posterior pole and has two distinct functions. In mutant embryos the activity will normalize pole cell formation when transplanted into the posterior pole. Furthermore, osk activity can provoke the formation of a second "posterior center" at the anterior (Lehmann, 1986).

osk mutants fall into two classes: one in which OSK mRNA localization is normal, and one in which initial localization to the posterior pole is normal, but then becomes delocalized.

Oskar is involved in Drosophila long-term memory

Memory formation after olfactory learning in Drosophila displays behavioral and molecular properties similar to those of other species. Particularly, long-term memory requires CREB-dependent transcription, suggesting the regulation of 'downstream' genes. At the cellular level, long-lasting synaptic plasticity in many species also appears to depend on CREB-mediated gene transcription and subsequent structural and functional modification of relevant synapses. To date, little is known about the molecular-genetic mechanisms that contribute to this process during memory formation. Two complementary strategies were used to identify these genes. From DNA microarrays, 42 candidate memory genes were identified that appear to be transcriptionally regulated in normal flies during memory formation. Via mutagenesis, 60 mutants with defective long-term memory have been independently identified and molecular lesions have been identified for 58 of these. The pumilio translational repressor was found from both approaches, along with six additional genes with established roles in local control of mRNA translation. In vivo disruptions of four genes, staufen, pumilio, oskar, and eIF-5C, yield defective memory. It is concluded that convergent findings from the behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio/staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification (Daubnau, 2003).

The 60 memory mutants were generated with enhancer-trap transposons, which often drive reporter genes (lacZ or GFP) in patterns of expression similar to those of the endogenous genes they disrupt. Thus, reporter gene expression patterns were examined for milord-1 and -2 (pum), norka (oskar), and krasavietz (eIF-5C) in the adult CNS. Each of these enhancer-trap memory mutants shows common reporter gene expression in the mushroom body, an anatomical focus with a demonstrated role for olfactory memory. The norka and krasavietz strains carry a PGAL4 transposon that can drive expression of GFP in neuronal somata and processes. These data clearly reveal a common site of expression in a subset of intrinsic mushroom body neurons (Kenyon cells) that comprise the α and β lobes. The milord-1 and milord-2 strains, in contrast, carry a PlacZ transposon that expresses β galactosidase only in somata and, thus, only around the calyx region of mushroom bodies (Daubnau, 2003).

An existing mouse polyclonal antibody was to determine the expression pattern of Pum protein in the adult CNS. Consistent with the pattern of enhancer-trap expression for milord-1 and -2, Pum immunoreactivity is detected broadly in the CNS but appears to be expressed at high levels in mushroom body neurons. Strong immunoreactivity appears to be perinuclear in Kenyon cells, whereas weaker, punctate expression is detected in mushroom body neuropil (calyx). This antibody shows appreciable specificity on Western blots of embryonic extracts. It was not possible to use pumilio null mutants to establish antibody specificity for adult brain tissue, however, because the null mutants are not viable as adults. Coexpression in mushroom bodies of the reporter genes for oskar, pum, and eIF-5C and anti-Pum immunostaining are consistent with the notion that these genes function together in the CNS during long-term memory formation (Daubnau, 2003).

STAU already has been implicated in mRNA localization in mammalian CNS. In cultured hippocampal neurons, for instance, STAU has a punctate, somato-dendritic distribution and is a component of large RNP-containing neural granules, which themselves are associated with microtubules. These neural granules seem to play an analogous role in targeting mRNA translation to subcellular (synaptic) compartments in neurons, as do STAU-containing RNP particles (polar granules) in Drosophila oocytes. In cultured hippocampal neurons, neural granules are located near dendritic spines, appear to dissociate in response to local synaptic activity, and thereby release translationally repressed mRNAs. This process has been proposed as a mechanism for synapse-specific modification via local protein synthesis in response to neural activity in vitro. The data indicate that this staufen-dependent pathway underlies memory formation per se. Moreover, the further identification of oskar as a memory mutant and of moesin and orb as confirmed candidate memory genes suggests that additional genetic components of the machinery used for mRNA translocation and translation in oocytes also may function in neurons (Daubnau, 2003).

Combined with these observations from the literature, the data suggest a molecular mechanism for synapse-specific delivery of gene products during long-term memory formation. (1) Behavioral training results in the activation of CREB-mediated transcription, and nascent mRNAs are packaged into an RNP complex, a neural granule. In addition to staufen, oskar, and moesin, these granules may well include other components of polar granules such as mago and faf. (2) These neural granules then are transported into dendritic shafts along an organized microtubule network, as proposed above for vertebrate neurons. These activity-induced transcripts may be delivered nonselectively throughout the neuron or selectively to sites of recent synaptic activity. In either case, packaged mRNAs probably are translationally quiescent while in transport, thereby preventing ubiquitous expression of protein products. The data implicate pumilio as part of this translational repression complex. Finally, synapse-specific modification may result from the depolarization-dependent release of neural granule-associated mRNAs and a concomittant translational derepression (Daubnau, 2003 and references therein).

Local derepression of translation, in part, may involve phosphorylation of CPEB (orb) by aurora kinase, resulting in cytoplasmic polyadenylation and the dissociation of MASKIN from eIF-4E, which then allows interaction between eIF-4E and eIF-4G. Release of eIF-4E via phosphorylation of other 4E binding proteins also may promote assembly of the rest of the translation initiation complex. The presence during synaptic or behavioral plasticity of several other persistently active kinases also might contribute to such phosphorylation. CPEB-mediated translational activation in Xenopus oocytes, for instance, is associated with phosphorylation of ORB by CDC2 kinase (which is a dimer of CycB and CDC2) and ubiquitin-mediated degradation of Orb, perhaps modulated by faf or another ubiquitin hydrolase. Here again, DNA chip and memory mutant experiments have identified several of these additional components (Daubnau, 2003 and references therein).

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is a particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. A subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. Evidence is provided that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation (Werz, 2005).

Although this study largely focus on the maternal effect mutants bicoid and oskar, it is likely that the principles uncovered are of broader significance. Segmentation mutants acting downstream of bicoid and oskar, including mutants of gap genes (Krüppel, knirps), pair-rule genes (odd, fushi-tarazu) and segment polarity genes (wg, hedgehog, engrailed) induce expression of hid. These mutants are characterized by loss of larval tissue. As in the case of bicoid and oskar, hid expression is upregulated during stage 9 of embryogenesis in the regions of the mutant embryos that are later deleted in the larvae. In addition, hid mutants rescue the cuticle phenotype of armadillo mutants. Finally, hid expression accompanied by TUNEL-positive cell death was found in dorsal and Toll10b mutants, which cause dorsalizing and ventralizing phenotypes, respectively, along the dorsoventral axis of Drosophila embryos. Thus, these data support the notion that upregulation of hid appears to be a common trigger for a caspase-dependent cell death program in mis-specified cells of patterning mutants (Werz, 2005).

Furthermore, mutations affecting imaginal disc development result in loss of the adult appendage due to inappropriate cell death. It is currently being determined whether these mutants also require hid expression to develop the final phenotypes. Moreover, many gene disruptions in mice result in inappropriate cell death in the tissue that requires the function of the disrupted gene, suggesting that similar mechanisms might exist in mammalian development. Finally, cell death may be an important contributing factor to human congenital birth defects. Thus, an understanding of the underlying mechanisms is of general interest (Werz, 2005).

Interestingly, not all segment polarity mutants analyzed induce hid expression and cell death. Embryos mutant for patched, which encodes the hedgehog receptor, were not found to express hid and do not exhibit increased cell death, although hedgehog mutants both upregulate hid and contain increased amounts of cell death. The reasons for these differences are not known, but partial redundancy might account for lack of hid expression in patched mutants. The Drosophila genome encodes another patched homolog, patched-related, which might provide the survival requirement for mis-specified cells in patched mutants (Werz, 2005).

Mis-specified cells in bicoid and oskar mutants induce expression of hid. No increased reaper or grim expression was observed in these mutants. However, expression of reaper has been reported in crumbs mutants, which affect epithelial integrity. X-ray-treated embryos also preferentially respond by upregulation of reaper, rather than hid. Although crumbs mutants were not analyzed for hid expression, it appears that cells contain several developmental checkpoints, which activate different cell death-inducing regulators depending on the type of abnormal cellular development (Werz, 2005 and references therein).

Mis-specified cells can survive if an alternative survival pathway is provided. The example presented here is the acron into telson transformation in bicoid mutants, which is mediated by the torso signaling pathway. Although the cells giving rise to telson structures at the anterior tip are mis-specified based on Abd-B-labeling experiments, they survive because they receive a survival signal from the torso signaling system. In this case, transformation rather than cell death is favored. It has been shown that activation of the Ras/Mapk pathway protects cells from hid-induced apoptosis, both by transcriptional repression of hid and by phosphorylation of Hid protein by Mapk. Because Torso, which encodes a receptor tyrosine kinase (RTK), is known to activate Ras and Mapk, tests were performed to see whether manipulation of active Mapk levels using a gain-of-function allele, MapkSem, can suppress hid expression and cell death in bicoid mutants. However, this was found not to be the case. Thus, torso appears to protect mis-specified cells independently of Mapk activation (Werz, 2005).

The hid bicoid double mutant analysis reveals that the transformation of anterior into posterior identity expands beyond the telson, and that this expansion undergoes hid-induced cell death in bicoid single mutants. The rescued cells secrete larval cuticle elements, suggesting that mis-specified cells have the developmental capacity to terminally differentiate. However, in hid+ background, they instead die, presumably because equivalent survival signals are lacking. It is proposed that mis-specified cells undergo cell death if no alternative survival pathway is provided to protect them (Werz, 2005).

An alternative survival mechanism might also operate in other developmental mutants where transformation rather than cell death occurs. Mutations in the sev RTK and its ligand boss result in transformation of the R7 photoreceptor cell into a non-neuronal cone cell. Survival of this cell could be mediated by the Drosophila Egf receptor (Egfr), another RTK, which is required to maintain cell survival in the developing eye disc. Accordingly, activation of the Ras/Mapk pathway by Egfr would inhibit hid expression and support survival of the presumptive R7 photoreceptor cell. This interpretation is also consistent with observations that egfr- clones are small and undergo cell death, and that this death can be suppressed in hid mutants. Thus, transformation of the R7 photoreceptor to a cone cell rather than R7 cell death in sev and boss mutants could occur because of survival signaling by the Egfr (Werz, 2005).

The hid bicoid double mutant analysis suggests that mis-specified cells can continue to develop and differentiate. Yet, they die. Presumably, this cell death protects the organism from potentially dangerous cells. For example, it is conceivable that in mammals, surviving mis-specified cells might lie dormant in the host organism for years. During this time, they might acquire additional genetic alterations that could drive the progressive transformation of these cells into malignant cancer. In wild-type embryos, mis-specification probably occurs in cells in isolation, and elimination of these cells does not interfere with development and survival of the organism. Only in extreme situations, such as the patterning mutants analyzed in this study, is the mis-specification caused by aberrant development so severe that the affected organism dies (Werz, 2005).

The cause of mis-specification in each segmentation mutant is different. Usually, the expression of other segmentation genes is shifted and expanded, resulting in flattened gradients. Yet, irrespective of the cause of mis-specification, most of these mutants have in common that they induce hid expression. It is currently unknown how the mis-specified fate of cells is recognized, and how hid expression is induced. One possibility might be that the protein gradients established by bicoid+ and oskar+, as well as other segmentation genes are used as readout for proper cellular specification. The steepness of protein gradients as a means to determine life or death decisions has recently been proposed. Such a model would imply that cells are able to determine their position in a graded field and compare this readout with their neighbors. Because in bicoid and oskar mutants these gradients do not form, the concentration difference between neighboring cells would be zero. If the concentration difference between two neighboring cells is below a crucial threshold, they induce the expression of hid and undergo cell death. This model could also explain embryonic pattern repair, which was described in embryos that express six copies of the bicoid gene. In these embryos, the head and thorax primordia are expanded because of the presence of six copies of bicoid. However, this expansion is corrected for by induction of cell death, and relatively normal larvae develop. In this case, the Bicoid protein gradient does form, but would be flatter compared with wild type. Thus, the concentration difference between neighboring cells would be below a critical threshold, sufficient to induce hid-dependent cell death. However, it is largely unknown how cells compare their position in a graded field with those of their neighbors. It has been proposed that short-range cell interactions mediated via the cell-surface proteins Capricious and Tartan provide cues that support cell survival during wing development. Cells unable to participate in these interactions are eliminated by cell death. It is unclear, however, whether short-range interactions are sufficient to explain the cell death phenotype in bicoid and oskar mutants (Werz, 2005).

Irrespective of the underlying mechanism for sensing mis-specification, the current results highlight the role of an active gene-directed process that removes mis-specified cells during development. However, if a survival mechanism is provided, mis-specified cells can survive and adopt a different fate. In wild-type embryos, mis-specification probably occurs in cells in isolation, and hence is difficult to study. However, in bicoid and oskar mutants, large regions of neighboring cells are mis-specified and undergo cell death simultaneously, providing a unique opportunity to clarify the signals that initiate cell death in situations where cells are developmentally mis-specified (Werz, 2005).

A translation-independent role of oskar RNA in early Drosophila oogenesis

The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring that lack both abdominal segments and a germline, thus defining the 'posterior group phenotype'. Common to these classical oskar alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented by oskar nonsense mutant alleles, as well as by oskar transgenes, the protein-coding capacities of which have been annulled. Moreover, expression of the oskar 3' untranslated region (3'UTR) is sufficient to rescue the egg-less defect of the RNA null mutant. This analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein. These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte (Jenny, 2006).

The new oskar alleles oskA87 and osk187 reveal that oskar plays a crucial role during early oogenesis. This early function of oskar is unusual, since it is mediated by oskar RNA, and not by Oskar protein. The low but detectable amount of oskar RNA observed in osk187 mutants indicates that a threshold exists, below which egg chambers fail to develop. These findings raise the possibility that many protein encoding genes may fulfill additional non-coding functions and thus increase the spectrum of gene function (Jenny, 2006).

What might the function of oskar RNA be during early oogenesis? Oskar protein serves as a scaffold for the assembly of cytoplasmic structures essential for germline development, the polar granules, at the posterior pole of the oocyte and embryo. During early oogenesis, oskar RNA might provide a similar scaffold function for assembly of cytoplasmic complexes essential for the progression of oocyte development. RNAs are associated with proteins in ribonucleoprotein complexes during most of their existence, from their emergence as nascent transcripts in the nucleus, during nucleo-cytoplamic transport, to their final cytoplasmic localization, translation or degradation. Staufen protein requires oskar RNA for its transport from the nurse cells into the oocyte. In addition, it is the oskar 3'UTR that mediates accumulation of Staufen protein in the oocyte. This reveals that the well-known mutual interdependence of Staufen and oskar mRNA in their localization during oogenesis is mediated by interaction of Staufen with the oskar 3'UTR. Of the candidate proteins examined, only Staufen showed a clearly altered distribution in the oskar RNA null mutant, yet Staufen itself does not appear to play a role during early oogenesis. It is therefore reasonable to assume that oskar RNA acts as a structural partner for the transport into the oocyte of additional, so far unidentified proteins or RNAs essential for its development. In this regard it is interesting to note that both VgT RNA and the non-coding Xlsirts RNA have been shown to mediate anchoring of several RNAs at the vegetal pole of the Xenopus oocyte (Jenny, 2006).

Alternative functions of the oskar 3'UTR are also plausible. In particular, the oskar 3'UTR might bind and sequester a negative regulator that, in its free form (i.e. in an oskar RNA null background), inhibits early oogenesis. One candidate that has been shown to bind to the oskar 3'UTR is the translational regulator Bruno. However, overexpression of Bruno - at least in the presence of wild-type levels of oskar mRNA - does not cause a phenotype similar to that of the oskar RNA null mutant. Thus, to fully understand the mechanism of oogenesis arrest resulting from absence of oskar mRNA, it will be important to identify other proteins and RNAs binding to the oskar 3'UTR that are required for egg chamber development (Jenny, 2006).

Oskar controls morphology of polar granules and nuclear bodies in Drosophila

Germ cell formation in Drosophila relies on polar granules, which are large ribonucleoprotein complexes found at the posterior end of the embryo. The granules undergo characteristic changes in morphology during development, including the assembly of multiple spherical bodies from smaller precursors. Several polar granule components, both protein and RNA, have been identified. One of these, the protein Oskar, acts to initiate granule formation during oogenesis and to recruit other granule components. To investigate whether Oskar has a continuing role in organization of the granules and control of their morphology, advantage was taken of species-specific differences in polar granule structure. The polar granules of D. immigrans fuse into a single large oblong aggregate, as opposed to the multiple, distinct, spherical granules of D. melanogaster embryos. D. immigrans oskar rescues the body patterning and pole cell defects of embryos from D. melanogaster oskar- mothers, and converts the morphology of the polar granules to that of D. immigrans. The nuclear bodies, which are structures that appear to be closely related to polar granules, are also converted to the D. immigrans type morphology. It is concluded that oskar plays a persistent and central role in the polar granules, not only initiating their formation but also controlling their organization and morphology (Jones, 2007).


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oskar: Biological Overview | Evolutionary Homologs | Regulation | Factors affecting Oskar translation | Factors affecting Oskar localization | Developmental Biology | Effects of Mutation

date revised: 17 March 2008

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