374
*
Recent advances shed light on the cellular processes that cooperate during oogenesis to produce a fully patterned egg, containing all the maternal information required for embryonic development. Progress has been made in defining the early steps in oocyte specification and it has been shown that progression of oogenesis is controlled by a meiotic checkpoint and requires active maintenance of the oocyte cell fate. The function of Gurken signalling in patterning the dorsal–ventral axis later in oogenesis is better understood.
Anteroior–posterior patterning of the embryo requires activities of bicoid and oskar mRNAs, localised within the oocyte. A microtubule motor, Kinesin, is directly implicated in localisation of oskar mRNA to the posterior pole of the oocyte.
incomplete cytokinesis, to generate a cyst of 16 cells interconnected by ring canals (for reviews about oogenesis and cyst development see [2,3]). How one of these 16 cells is selected to become the oocyte is one of the central questions in axis formation. Oocyte selection is a gradual process in which several cells initially accumulate oocyte features before one cell is singled out as the definitive oocyte and the 15 others develop as nurse cells. The failure to select an oocyte, as in Bicaudal D ( BicD ) and egalitarian
( egl ) mutants, typically results in the formation of a 16 nurse-cell cyst [4,5]. Although the exact mechanism by which the oocyte is selected is still unclear, recent detailed descriptions of cyst development have helped subdivide oocyte selection into different steps and distinguish three different processes that define this event (see Figure 1 and
Table 1). Oocyte selection requires localisation of cytoplasmic markers, restriction of meiosis, and migration of centrioles into the oocyte.
Addresses
European Molecular Biology Laboratory, Meyerhofstra
β e 1,
Postfach 10.2209, D-69012 Heidelberg, Germany
* e-mail: riechman@embl-heidelberg.de
† e-mail: ephrussi@embl-heidelberg.de
Current Opinion in Genetics & Development 2001, 11 :374–383
0959-437X/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
A/P
BicD egl
Egfr anterior–posterior
Bicaudal D egalitarian
Epidermal growth factor receptor
DSB double-strand DNA breaks dsRBD double-stranded RNA-binding domain
D/V
Grk dorsal–ventral
Gurken
MT microtubule
MTOC microtubule organising center
TCE translational control element
Embryonic axis formation in Drosophila is the direct consequence of symmetry-breaking events that take place throughout oogenesis. Oogenesis starts with the first asymmetric division of a germline stem cell to generate a new stem cell and a cystoblast. Four divisions of the cystoblast will generate a cyst of 16 cells, one of which will become the oocyte. The events leading to oocyte specification and patterning of the oocyte and surrounding follicular epithelium provide an attractive model for the study of basic cellular processes such as cytoskeletal dynamics, cell to cell signalling, RNA localisation and translational control. Here we describe developments over the past two years, since the last publication of a review in this journal on the topic [1].
The functional unit of oogenesis, the egg chamber, is produced in the germarium. Stem cell divisions occur at the anterior tip of the germarium, giving rise to a new stem cell and a cystoblast. Cystoblasts in turn divide four times, with
What initial cue determines which of the 16 cells in a cyst is to become the oocyte? There is growing evidence that the fusome, a continuous vesicular organelle linking the 16 cells via the ring canals, gives this initial cue. The fusome is polarised as of the first cystoblast division, when one daughter cell inherits more fusome material than does the other. This unequal distribution of fusome is maintained until the 16-cell cyst has formed. As the unequal distribution of fusome is the earliest visible asymmetry within a cyst, it has been proposed that a first and crucial element in oocyte specification is provided by the asymmetric fusome [6].
Two genes, which encode Dynein heavy chain and Lis-1, are required for fusome integrity and for selection of the oocyte [7,8 • ,9 • ]. The fact that these genes interact in many different processes in Drosophila and in other organisms
(see [10] and references therein) suggests that they also interact in fusome formation. Formation of a correctly branched fusome requires its stable association with microtubules (MTs) during the cystocyte divisions, and dynein is essential for this association. It is therefore possible that the failure of Dynein heavy chain 64C and Lis-1 mutants to select an oocyte is the consequence of an earlier function of the encoded proteins in regulating the MT-dependent formation of a correctly branched fusome.
The fusome and the MT cytoskeleton are interdependent for their function. During the cystocyte divisions, MTs are required for formation of a proper fusome and, once a
16-cell cyst has formed, the fusome is required to organise a polarised MT cytoskeleton within the cyst [11 • ]. A polarised MT network is required for one of the three features of oocyte selection: transport of specific cytoplasmic markers. These mRNAs and proteins are transported along MTs, towards the microtubule organising centre (MTOC) of the cyst, located within the future

Axis formation during Drosophila oogenesis Riechmann and Ephrussi 375
Figure 1
Bic D
Fusome
Centrioles
MT minus ends
MT
Synaptonemal complex
Oblique
Aligning
Sretching
Straightening
Lens-shaped
+
+
+
+
+
+
+
+
+
Region
1
Region
2a
Region
2b
Region
3
Patterning in the germarium. The figure summarises results described in references [11 according to [11
•
,17
•
,25
••
,26,27]. Shapes of the cysts are drawn
•
]. At the anterior tip of the germarium (far left) germline stem cells divide, giving rise to a new stem cell and a cystoblast. In region 1, cystoblasts divide four times with incomplete cytokinesis to form a cyst of 16 cells connected by ring canals
(not shown). The fusome, a continuous vesicular organelle, connects the cystoblasts via the ring canals. The cyst contains two cells with four ring canals, two cells with three, four cells with two and eight cells with one. In region 2, one of the cells with four ring canals is selected to become the oocyte. MT minus ends, centrioles, BicD protein and
Current Opinion in Genetics & Development other cytoplasmic markers accumulate within the oocyte. In addition, the formation of the synaptonemal complex, which is initially detected in four cells, becomes restricted to the oocyte. MT are not depicted at stages younger than budding cysts. In these, MT are focused in the oocyte and project into the nurse cells. At this stage, the MT minus ends, centrioles, and BicD are localised at the anterior of the oocyte.
When the cyst moves from region 2b to region 3, these structures move from the anterior to the posterior end of the oocyte. As oocyte selection is a continuous process, not all of the depicted stages are simultaneously present in one germarium in vivo . See Table 1 for further description.
oocyte. In addition to the fusome, two genes, BicD and egl , are required for the establishment of this polarised MT cytoskeleton, and BicD and Egl proteins themselves are amongst the first proteins transported into the oocyte
[12–15]. As this transport is MT-dependent, the role of
Dynein as a MT-based motor provides another explanation for the oocyte selection defects in Lis-1 and Dynein heavy chain 64C mutants, in which impaired transport of BicD,
Egl and other factors into the oocyte might prevent its specification.
While the transport of cytoplasmic factors into the oocyte is MT-dependent, the two other features of oocyte selection — restriction of meiosis and migration of the centrioles — seem to be independent of the MT network.
Analysis of the first visible sign of meiosis, formation of the synaptonemal complex, reveals that BicD and Egl control entry into and progressive restriction of meiosis to one cell within the cyst [16,17 • ]. However, depolymerisation of the
MT network, and the consequent delocalisation of BicD and Egl, has no effect on restriction of meiosis to the oocyte. This indicates that this aspect of oocyte specification is MT-independent and that BicD and Egl control meiosis before they are localized to the oocyte [17 • ]. In contrast to restriction of meiosis to the oocyte, the third feature of oocyte selection, migration of the nurse cell centrioles into the oocyte, is not affected in BicD and egl mutants. The fact that the centrioles translocate normally in these mutants, which lack a polarised MT network, suggests that another component, such as the fusome, might guide centrosome migration [18 • ].
Further steps in oocyte specification and other aspects of axis formation are inhibited by a meiotic checkpoint that senses the presence of unrepaired double-strand DNA

376 Pattern formation and developmental mechanisms
Table 1
Steps in oocyte selection.
Region in the germarium
Shape of the cyst
Stages in restriction of oocyte fate
Description
2a
2a
2a
2a
2a
2b
2b
2b
3
Oblique
Oblique
Aligning
Stretching
Stretching
Straightening
Straight/ lens-shaped and bulging
Budding
Egg chamber
(stage 1 of oogenesis)
1
2
3
4
5
6
7
No sign of SC formation
Two cells with SC : two pro-oocytes reach zygotene stage of meiosis; meiotic checkpoint is active
MT : equally distributed and closely associated with the fusome; minus ends are not focused
Centrioles : localise to the tips of the fusome
Four cells with SC : two pro-oocytes are in pachytene and two cells with three ring canals reach zygotene; DSBs are repaired and meiotic checkpoint is passed
MT : closely associated with the fusome; minus ends focus into the center of the cyst
Centrioles : localise to the tips of the fusome
Two cells with SC : two pro-oocytes still have complete SC while cells with the three ring canals lose their SC
BicD/Orb : start accumulating in the two pro-oocytes
MT : reduced density of the network; minus ends reflect the localisation of BicD/Orb
Centrioles : migrate along the fusome towards the pro-oocytes
Two cells with SC : both pro-oocytes with identical SC
BicD/Orb : concentrate in one of the two pro-oocytes
MT : reduced density of the network; minus ends reflect the localisation of BicD/Orb
One cell with SC : one pro-oocyte loses SC and reverts to the nurse cell pathway
BicD/Orb : restricted to one pro-oocyte
MT : reduced density of the network; minus ends reflect the localisation of BicD/Orb
Oocyte is selected
BicD/Orb : restricted to one pro-oocyte
MT : high density in the oocyte; minus ends focus into the oocyte
Centrioles : predominantly in the oocyte and still associated with the fusome
Fusome : regresses but still associates with MTs
MT : focus into the oocyte; some still associate with fusome remnants; minus ends are no longer fusome associated and predominantly localise to the anterior part of the oocyte
BicD/Orb are localised anterior of the oocyte nucleus
Centrioles : dissociate from fusome remnants and predominantly localise to the anterior to the oocyte nucleus
Oocyte at the posterior of the newly formed egg chamber
BicD/Orb , MT minus ends and Centrioles move from the anterior towards the posterior of the oocyte
SC becomes more compact and disappears
The table subdivides oocyte selection into distinct stages. This subdivision is based on the results of references [11
•
,17
•
,25
••
]. As oocyte selection is a continuous process, not all of the stages are necessarily present simultaneously in one germarium in vivo . See also Figure 1.
breaks (DSB). Meiosis requires the induction of DSB to allow recombination, and spindle-B , spindle-C and okra are involved in their subsequent repair [19]. The phenotype of spindle-B , spindle-C and okra mutants includes a delay in oocyte specification and dorsal–ventral (D/V) defects in late oogenesis, the latter due to reduced levels of the transforming growth factor
α homologue Gurken (Grk), a signalling protein essential for axis formation [19,20,21 •• ].
The patterning defects are suppressed by mutations in mei-W68 , required for the generation of DSB, and in mei-41 , which encodes the Drosophila homologue of a component of the yeast DNA-damage checkpoint. This indicates that the axial patterning defects — and most likely also the delay in oocyte selection in spindle-B , spindle-C and okra mutants, are caused by the presence of unrepaired DSB and that normal progression of oogenesis is controlled by a meiotic checkpoint that detects unrepaired DSB [21 •• ].
The molecular targets of the checkpoint pathway are not entirely clear. The similar patterning defects of spindle-B , spindle-C , and okra mutants and those of vasa mutants, make Vasa, which is required for accumulation of Grk, a candidate for such a target [21 •• ,22,23]. However, the checkpoint seems to have additional targets, as a delay in oocyte selection is only detected in the spindle mutants, but not in vasa mutants [17 • ].
Progression of oogenesis requires not only the inactivation of the meiotic checkpoint but also active maintenance of oocyte fate, as revealed by null alleles of par-1 [24,25 •• ]. In such alleles, the oocyte is initially specified but loses its character as the egg chamber leaves the germarium and ultimately adopts a nurse cell fate. Determination of oocyte fate occurs when the cyst reaches the end of the germarium, and is accompanied by the translocation of BicD, the MTOC and the centrioles from the anterior to the posterior of the

Axis formation during Drosophila oogenesis Riechmann and Ephrussi 377
Figure 2
Patterning of the oocyte. (a) Repolarisation of the oocyte MT cytoskeleton by Grk signalling at stage 6–7 of oogenesis. Left panel: in the germline, mRNAs and proteins (red arrows) are produced in the nurse cells and transported along MT into the ooctyte.
Somatic follicle cells surrounding the germline cells are subdivided into two competence domains, the mainbody follicle cells and the terminal follicle cells. Only the terminal follicle cells are competent to adopt posterior fate after receiving the Grk signal. Middle panel: induction of posterior follicle cell fate leads to the production of an unidentified back signal by the posterior follicle cells. Right panel: the back signal results in the reorganisation of the oocyte MT cytoskeleton. The MTOC at the posterior pole disassembles and MT nucleate at the anterior cortex. After this repolarisation has occurred, the oocyte nucleus moves in a
MT-dependent manner to an anterior corner of the oocyte. The position of the oocyte nucleus defines the dorsal side of the egg chamber, as it determines the region where the second
Grk signal will induce dorsal cell fates [see (c)].
(b) Localisation of bicoid and oskar mRNA.
A stage 9 egg chamber is shown. bicoid mRNA and oskar mRNA are produced in the nurse cells and transported through the ring canals into the oocyte. After passing the ring canals, bicoid mRNA accumulates at the anterior cortex of the oocyte, while oskar mRNA and Staufen protein are transported by Kinesin to the plus ends of the MT, towards the posterior pole. (c) Patterning of the egg by the second Grk signal. A stage 10 egg chamber is shown. Grk protein, associated with the oocyte nucleus in the antero-dorsal corner of the oocyte, induces the formation of dorsal chorion structures.
The formation of the dorsal appendages requires the combined activities of Grk, emanating from the oocyte, and Dpp, which is expressed in the neighbouring anterior follicle cells (light blue). Grk signalling also guides the dorsal migration of the border cells, which are required for the formation of a functional micropyle. Grk also represses the expression of pipe , in the dorsal follicle cells, and thereby restricts the region in which a ventralising signal is produced. (d) Localisation of anterior and posterior determinants in the egg. A stage 14 egg with dorsal appendages and micropyle is shown. Before egg activation, bicoid mRNA (blue) is anchored at the anterior pole and remains translationally dormant. oskar and nanos mRNAs as well as
Oskar and Nanos proteins are anchored at the posterior pole. Upon egg activation, bicoid mRNA is translated and diffuses from
(a)
(b)
(d)
Micropyte
Mainbody follicle cells
Dorsal appendages bcd
MT
(c) mRNA
Grk the anterior pole forming a morphogen gradient, while a Nanos protein gradient forms from the posterior pole. The two
Terminal follicle cells
Back signal
Repolarisation of the MT network and nuclear migration
Dpp
Induction of dorsal cell fates
Border cells osk nos
Posterior follicle cells
Grk
Guidance of border cells
Repression of pipe mRNA and protein mRNA and protein bcd mRNA osk mRNA protein gradients pattern the embryo along the A/P axis by regulating zygotic gap gene expression.
Pipe
Current Opinion in Genetics & Development oocyte, constituting the first sign of polarity within the oocyte itself (see Figure 1; [25 •• ,26,27]). The fact the posterior shift of oocyte markers fails in par-1 mutants that eventually lose oocyte identity suggests that there might be a causal relationship between the translocation and maintenance of oocyte fate. However, maelstrom , spindle-A and spindle-B mutants, in which translocation of the oocyte markers is disrupted, maintain an oocyte nonetheless [26,27].
This suggests that the posterior translocation of the oocyte markers is not strictly required for maintenance of oocyte fate.

378 Pattern formation and developmental mechanisms
A complete egg chamber is formed at the posterior end of the germarium by encapsulation of the 16-cell cyst in a layer of somatic epithelial cells. After the egg chamber leaves the germarium, the oocyte is polarised by two signalling events, both of which are induced by the epidermal growth factor receptor (Egfr) ligand, Grk, associated with the oocyte nucleus. The first Grk signal induces the follicle cells overlaying the oocyte to take on posterior fate (Figure 2a), and the second Grk signal induces the follicle cells closest to the oocyte nucleus to adopt dorsal fate (Figure 2c; [28,29]). The different response of the dorsal and posterior follicle cells to the same signal is the consequence of an earlier subdivision of the follicle cells into two competence domains. The terminal follicle cells at the anterior and posterior ends of the egg chamber are competent to differentiate as posterior follicle cells if they receive the Grk signal, while the remaining follicle cells, the mainbody follicle cells, can only adopt dorsal fates in response to Grk [30].
The first Grk signalling event, which occurs by stage 7 of oogenesis [31 •• ], induces the posterior follicle cells to respond by sending an unidentified signal back to the oocyte, resulting in the repolarisation of its MT cytoskeleton (Figure 2a; [28,29]). The nature of the reverse signal is still unknown but two genes have been identified that are required in the follicle cells for transduction of this signal. One of these genes, Merlin , encodes a member of the ERM family of proteins, which are thought to function as linkers between the cytoskeleton and the apical membrane. Hence, Merlin may act in intracellular targeting of the signal to the apical membrane of the posterior follicle cells [32 • ]. Successful transduction of the signal seems to require the extracellular matrix, as follicle cells with mutant Laminin A, an extracellular matrix component, are unable to signal to the oocyte to induce its repolarisation [33 • ].
In response to the reverse signal, the MTOC at the posterior of the oocyte disassembles and MTs nucleate from the anterior and lateral cortices of the oocyte (Figure 2a).
This reorganisation of the MT network is necessary for the migration of the oocyte nucleus to an antero-lateral position where, at stage 9, the second Grk signal induces dorsal follicle cell fate. Formation of a proper D/V axis requires the controlled export of grk mRNA from the oocyte nucleus and regulated distribution of the RNA and protein in the antero-dorsal corner of the oocyte, ensuring precisely localised signalling. Squid seems to have a central role in these processes as in squid mutants, grk RNA is mislocalised along the entire anterior cortex, resulting in dorsalised eggs [34]. squid encodes a heterogeneous nuclear RNAbinding protein and the finding that Squid protein binds grk mRNA, the nuclear import protein Transportin and the translational repressor Bruno, suggests that Squid couples nuclear export of grk mRNA with its localisation and translation [35 • ].
Grk signalling in the antero-dorsal corner of the oocyte controls D/V patterning in at least three ways. It restricts the generation of a ventralising signal to the ventral follicle cells, specifies the fate of the dorsal follicle cells, and guides a group of migrating follicle cells, the border cells
(Figure 2c and see [36,37] for reviews about Grk signalling).
Grk signalling controls D/V axis formation by restricting the expression of pipe to the ventral follicle cells. This restriction is crucial, as pipe expression links the dorsal Grk signal and the ventral signal that polarises the D/V axis [38]. It is not entirely clear whether Grk acts as a long-range morphogen that represses pipe expression in the follicle cells directly, or whether Grk represses pipe indirectly, by inducing production of an inhibitory signal in the dorso-lateral follicle cells [39 • ]. The finding that an inhibitor of Egfr activity is required in the ventral follicle cells to allow pipe expression provides evidence that Egfr activity is present along the whole D/V axis. The Cbl family of proteins is thought to negatively regulate Egfr activity by targeting the activated receptor tyrosine kinase complex for degradation. In ventral follicle cells mutant for the Drosophila homologue of cbl ,
Egfr target genes are ectopically activated, and pipe expression is abolished. This indicates that cbl acts in the ventral follicle cells to ensure the absence of Egfr activity, which is a prerequisite for pipe expression [40 • ].
In addition to this long-range effect of Grk signalling in
D/V patterning, Grk also acts in a locally restricted manner, in cell fate specification on the dorsal side. Here, Grk signalling induces production of dorsal chorion structures, such as the dorsal appendages, formation of which is confined to the anterior third of the dorsal mainbody follicle cells. The positioning of the dorsal appendages to this region of the mainbody follicle cells is achieved by the combined activities of Grk, originating from the oocyte nucleus, and Dpp, emanating from the neighbouring anterior follicle cells (Figure 2c). The intersection of Grk and Dpp signalling in this region determines the specification and positioning of the dorsal appendages [31 •• ].
The third function of Grk signalling is in the guidance of the border cells, a group of anterior terminal follicle cells that delaminate from the follicular epithelium and migrate posteriorly between the nurse cells to the oocyte. When they reach the oocyte, the border cells migrate dorsally, towards the oocyte nucleus. This dorsal migration is essential for the formation of a functional micropyle in the eggshell, which allows sperm entry. Egfr activation in the border cells directs their migration towards the source of
Grk signalling in the antero-dorsal corner of the egg chamber. Interestingly, while Grk activates the Raf-MAP kinase pathway in the dorsal follicle cells, the same signal triggers a different, yet unidentified, pathway in the border cells. Thus, the different responses of the two follicle cell types to Grk signalling — dorsal cell fate specification

Axis formation during Drosophila oogenesis Riechmann and Ephrussi 379 versus migration — are elicited through the activation of different pathways [41 • ].
The organisation of the cytoskeleton is crucial in the establishment of polarity within the oocyte, as the cytoskeleton mediates the localisation and the anchoring of mRNAs that determine the poles of the oocyte and the embryo.
Mutations in cap , a gene encoding a putative actin-binding protein, cause accumulation of ectopic F-actin in the oocyte, indicating a function of cap in regulating cortical actin polymerisation. Interestingly, the defects in the actin cytoskeleton are accompanied by an aberrant polarisation of the MT network. In wildtype oocytes at stages 8 and 9, the
MT cytoskeleton is polarised such that the MT plus ends are enriched at the posterior pole, however, in cap mutants the MT plus ends frequently focus at ectopic sites around the cortex [42 • ]. This indicates that the organisation of the oocyte actin cytoskeleton influences the polarisation of the
MT network. In contrast to cap mutations, weak par-1 alleles and mutations in rab-11 , a gene encoding a small
GTPase required for targeting of vesicles, cause no visible actin defects but only an aberrant organisation of MTs. In these mutants, the plus ends of the MT do not focus at the posterior pole, as in the wild type, nor to ectopic cortical sites, as in cap mutants, but to the center of the oocyte
[43 • ,44,45 • ]. Par-1 is a serine/threonine kinase and the ability of its mammalian homologue MARK to regulate MT dynamics by phosphorylating MT-associated proteins suggests a direct influence of Par-1 on the MT network.
The MT-dependent localisation of bicoid and oskar mRNA to the anterior and posterior pole of the oocyte defines the A/P axis of the embryo. Both mRNAs are produced in the nurse cells, transferred into the oocyte, and become localised at the poles of the oocyte, where they are anchored (Figure 2b).
bicoid mRNA, at the anterior pole, is translated after fertilisation to produce a morphogen gradient that patterns the anterior region of the embryo [46]. oskar mRNA, at the posterior pole, directs assembly of the pole plasm, containing determinants of the abdomen and germline [47]. Recent work sheds light on the mechanisms by which these mRNAs and the proteins they encode are localised within the oocyte.
The fact that bicoid mRNA localisation within the oocyte is
MT-dependent and that the RNA colocalises with the minus ends of MT has led to the proposal that, in the oocyte, bicoid mRNA is transported along MT, to their minus ends. Recent experiments show that fluorescently labelled bicoid RNA injected into nurse cells at stage 9 becomes localised at the anterior of the oocyte, but that
RNA injected directly into the oocyte is localised in a nonpolar fashion to all regions of the oocyte cortex, with the exception of the posterior pole. While MT appear enriched at the anterior of the oocyte, they are also detected all around the cortex, with the exception of the posterior pole, consistent with the cortical localisation of bicoid RNA injected into the oocyte. Hence, the MT cytoskeleton does not appear as highly polarised along the A/P axis as was previously thought. How then is the polarised transport of bicoid mRNA within the oocyte achieved? In a technically impressive series of experiments it was shown that bicoid RNA injected into nurse cells, withdrawn and then injected into the oocyte of another egg chamber localises specifically to the anterior cortex of the oocyte. Hence, it appears that bicoid RNA associates with essential anterior-targeting factors in the nurse cells, prior to its transport to the oocyte [48 •• ].
The finding that Swallow, which is required for bicoid mRNA localisation within the oocyte [49], binds Dynein light chain suggests that Swallow couples bicoid mRNA in the oocyte to the plus end-directed motor Dynein [50 • ].
This association with Dynein could indicate a function of
Swallow in active transport of bicoid mRNA to the anterior pole. However, the fact that bicoid RNA injected into swallow mutant oocytes is transported to the cortex, supports a model in which binding of Swallow to Dynein is involved in bicoid mRNA anchoring at the anterior, rather than in its transport [48 •• ]. Localised bicoid mRNA remains translationally dormant at the anterior of the oocyte until egg activation, when its short poly(A) tail is elongated by cytoplasmic polya-denylation, resulting in bicoid translational activation [51]. Staufen, a double-stranded RNA-binding
(dsRBD) protein, anchors bicoid RNA at the anterior during the late stages of oogenesis, and enhances the level of Bicoid expression in the embryo [52 •• ]. Bicoid protein, translated from the concentrated source of localised RNA, diffuses towards the posterior of the embryo, resulting in the Bicoid gradient required for zygotic gap gene regulation and formation of anterior structures [46].
New insight into the mechanism of oskar mRNA localisation has come from two recent studies, one of Staufen and the other of Kinesin I. Staufen protein presumably forms a complex with oskar mRNA, as both colocalise at all stages of oogenesis and as Staufen is required for localisation and translation of oskar mRNA [53,54]. Staufen contains five dsRBD, two of which have now been studied in detail.
dsRBD2 is only able to bind RNA if a protein loop splitting the domain is removed. Staufen protein from which the loop splitting dsRBD2 is deleted associates with oskar mRNA and activates its translation in vivo but is not able to mediate its MT-dependent localisation to the posterior pole. Thus, Staufen dsRBD2 may mediate the interaction of oskar mRNA with the localisation machinery. In contrast, Staufen dsRBD5 is dispensable for oskar mRNA localisation and does not bind RNA in vitro , but is required for oskar translational activation, presumably due to interaction with other proteins [52 •• ]. A long awaited result, after years of assumption that oskar mRNA is localised by active transport on MT, is the demonstration that localisation of the Staufen– oskar mRNA complex to the posterior pole is abolished in Kinesin heavy chain mutants. This

380 Pattern formation and developmental mechanisms strongly suggests that the plus-end directed motor,
Kinesin, transports the Staufen– oskar mRNA complex to the MT plus ends, near the posterior pole [55 •• ].
Localisation of oskar mRNA involves more proteins than
Staufen and Kinesin, as suggested by the purification of a ribonucleoprotein complex containing oskar mRNA. In addition to oskar mRNA, this complex contains
Exuperantia, the RNA-binding protein Yps and five unidentified proteins, suggesting a role for these proteins in oskar mRNA localisation or anchoring at the posterior pole [56].
Localised expression of the posterior determinant Oskar is effected by enrichment of oskar mRNA to the posterior pole, translational repression of unlocalised RNA, and localised translational activation of the mRNA at the posterior pole ([54,57] and references therein). The RNA-binding protein Bruno, binds to oskar mRNA in vitro and has been implicated in repression of oskar translation in vivo [54,58].
Translational repression of oskar mRNA by Bruno has now been demonstrated in vitro , using recombinant Bruno in conjunction with translation extracts produced from
Drosophila ovaries and embryos [59,60]. Although oskar mRNA does not require a poly(A) tail for either its translation or repression in vitro , the observation that the oskar poly(A) tail is unusually short in orb mutants, in which no
Oskar protein is detected, suggests an involvement of the poly(A) tail in efficient translation of oskar mRNA in vivo . A possible role of Orb in the polyadenylation of oskar mRNA is supported by its homology to the cytoplasmic polyadenylation element binding protein CPEB [61].
Oskar recruits the other posterior pole plasm components, including Vasa protein and nanos mRNA, which encodes the abdominal determinant of the embryo [47,62]. Vasa, a
DEAD-box RNA helicase, is essential for posterior patterning and germline formation in the embryo and has been proposed to play a role in translation of several germline mRNAs, such as oskar and nanos [63–65]. The demonstration that Vasa interacts directly with the Drosophila homologue of yeast translational initiation factor 2 (dYF2), and that vasa and dIF2 interact genetically in abdominal patterning and germ cell formation, provides further evidence that Vasa is directly involved in translational control in the germline [66 • ].
A gradient of Nanos protein emanating from the posterior of the embryo negatively regulates translation of hunchback mRNA, allowing posterior zygotic gap gene activation and abdominal patterning to proceed. nanos mRNA is concentrated at the posterior of the oocyte during the last stages of oogenesis by RNA localisation coupled with translational control. nanos mRNA is translationally repressed throughout the oocyte and early embryo, and is selectively derepressed in the posterior pole plasm [67,68]. nanos mRNA localisation to the posterior pole is mediated by multiple redundant elements, some of which overlap with the translational control element (TCE) that mediates repression of nanos mRNA. This overlap of regulatory elements with mutually exclusive functions suggests that translational activation of nanos mRNA is achieved by localisation factors at the posterior pole that interfere with the binding of translational repressors [69 •• ]. Several proteins bind the TCE directly, including Smaug [69 •• ,70], which represses nanos mRNA translation in the embryo [71 •• ] and in vitro [72]. The finding that Smaug also interacts biochemically with the pole plasm protein Oskar, which is required for activation of nanos translation, suggests that Smaug is a central component of nanos translational regulation [71 •• ]. Analysis of the mechanism negatively regulating Nanos synthesis outside of the posterior pole suggests that accumulation of ectopic
Nanos is prevented, not by inhibition of translation of nanos mRNA during initiation or elongation, but by a novel mechanism possibly involving cotranslational degradation of nascent peptides [73].
While key molecules that pattern the early Drosophila embryo have been known for over a decade, understanding the mechanisms that control their localisation and restrict their expression has required a detailed genetic and cell biological inspection of oogenesis, reaching back to its earliest stages, in search of the earliest polarity cues. The asymmetric distribution of fusome within the cyst, from the first division onwards, may define the later oocyte.
Three cellular features, the enrichment of cytoplasmic markers, centriole migration and the restriction of meiosis, together indicate oocyte fate. Progression of oocyte development is controlled at various steps, as revealed by the existence of a meiotic checkpoint and the necessity to actively maintain oocyte fate. During oocyte development, activation of the Egfr receptor by Grk is crucial for polarisation of the egg chamber. Signalling by Grk, which is repeated and diverse, most probably controls patterning along the whole dorso-ventral axis, and in addition provides a critical cue in cell migration. The involvement of
Kinesin in localisation of oskar mRNA to the posterior pole of the oocyte provides strong evidence for active transport of the posterior determinant along microtubules.
We thank many colleagues for generously sharing with us their unpublished results. We apologise for not mentioning many interesting publications, which we were unable to cite due to space limitations. We thank Francesca
Peri and Shoko Yoshida for comments on the manuscript and Nicole C
Grieder and Jean-Rene Huynh for comments on Table 1. We are grateful to
Nicola Berns for drawings. Veit Riechmann was supported by EMBO fellowship ALTF 508-1998.
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
1.
van Eeden F, St Johnston D: The polarisation of the anteriorposterior and dorsal-ventral axes during Drosophila oogenesis.
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3.
de Cuevas M, Lilly MA, Spradling AC: Germline cyst formation in
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Genetics 1991 ,
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6.
de Cuevas M, Spradling AC: Morphogenesis of the Drosophila fusome and its implications for oocyte specification.
Development
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7.
McGrail M, Hays TS: The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila .
Development 1997 ,
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8.
Liu Z, Xie T, Steward R: Lis1, the Drosophila homolog of a human
• lissencephaly disease gene, is required for germline cell division and oocyte differentiation.
Development 1999 , 126 :4477-4488.
Lis-1 null alleles are lethal. In germline clones of null alleles, cysts are produced that fail to divide normally and in which no oocyte is specified. This phenotype is accompanied by an aberrantly formed fusome.
9.
Swan A, Nguyen T, Suter B: Drosophila Lissencephaly-1 functions
• with Bic-D and dynein in oocyte determination and nuclear positioning.
Nat Cell Biol 1999 , 1 :444-449.
Lis-1 null mutants are defective in cystoblast divisions and oocyte selection
(see annotation [8 • ]). Weak Lis-1 mutants provide residual function to determine an oocyte but fail to localise mRNAs and proteins within the oocyte. In addition, positioning of the oocyte nucleus after stage 8 is affected, indicating a function of Lis-1 in nuclear migration or anchoring. Lis-1 protein is localised to the oocyte cortex as of stage 5.
10. Morris R: A rough guide to a smooth brain.
Nat Cell Biol 2000 ,
2 :E201-E202.
11. Grieder NC, de Cuevas M, Spradling AC: The fusome organizes the
• microtubule network during oocyte differentiation in Drosophila .
Development 2000 , 127 :4253-4264.
A careful and detailed description of the behaviour of the MT cytoskeleton, the fusome and the centrioles during cyst development showing that the fusome is required for organisation of the MT cytoskeleton. This is revealed by a disrupted polarisation of the MT in hts mutants, which lack the fusome.
12. Suter B, Romberg LM, Steward R: Bicaudal-D , a Drosophila gene involved in developmental asymmetry: localized transcript accumulation in ovaries and sequence similarity to myosin heavy chain tail domains.
Genes Dev 1989 , 3 :1957-1968.
13. Wharton RP, Struhl G: Structure of the Drosophila BicaudalD protein and its role in localizing the the posterior determinant nanos .
Cell 1989 , 59 :881-892.
14. Theurkauf WE, Alberts M, Jan YN, Jongens TA: A central role for microtubules in the differentiation of Drosophila oocytes.
Development 1993 , 118 :1169-1180.
15. Mach JM, Lehmann R: An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in
Drosophila .
Genes Dev 1997 , 11 :423-435.
16. Carpenter AT: Egalitarian and the choice of cell fates in Drosophila melanogaster oogenesis.
Ciba Found Symp 1994 , 182 :223-246.
17.
Huynh JR, St Johnston D: The role of BicD , egl , orb and the
• microtubules in the restriction of meiosis to the Drosophila oocyte.
Development 2000 , 127 :2785-2794.
A novel marker allows easy visualisation of the restriction of meiosis to one cell in the cyst. All cells of the cyst enter meiosis in egl mutants, whereas none of the cells does so in BicD null mutants. Cysts in which MTs have been depolymerised by drugs restrict meiosis to one cell, although oocyte localisation of
BicD and Egl is inhibited. These observations suggest that BicD and Egl act before they are localised to the oocyte to control meiosis in the cyst. A model is proposed in which translational control regulates entry into meiosis.
18. Bolivar J, Huynh J, Lopez-Schier H, Gonzalez C, St Johnston D,
•
Gonzalez-Reyes A: Centrosome migration into the Drosophila oocyte is independent of BicD , egl and the organisation of the microtubule cytoskeleton.
Development 2001 , 128 :1889-1909.
Centrosome migration takes place in BicD and egl mutants, which lack a polarized MT cytoskeleton, suggesting that centrosomes do not migrate along MT. In contrast, centrosome migration in blocked in Dhc64C null mutant germline clones, in which the fusome is initially normally polarised but disrupts soon after the 16-cell cyst has formed. This indicates a role of the fusome in centrosome migration.
19. Ghabrial A, Ray RP, Schüpbach T: okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis.
Genes Dev 1998 , 12 :2711-2723.
20. Gonzalez-Reyes A, Elliott H, St Johnston D: Oocyte determination and the origin of polarity in Drosophila : the role of the spindle genes.
Development 1997 , 124 :4927-4937.
21. Ghabrial A, Schüpbach T: Activation of a meiotic checkpoint
•• regulates translation of Gurken during Drosophila oogenesis.
Nat
Cell Biol 1999 , 1 :354-357.
Spindle-B, Spindle-C, and Okra are involved in the repair of DSB during meiosis, and mutants in the corresponding genes have defects in karyosome formation and in accumulation of Grk protein, resulting in ventralisation of the embryo. These defects are suppressed by mutations in mei-W68 , encoding a topoisomerase-II-like protein thought to induce DSB, and mei-41 , a homologue of the yeast mec1 gene, which is required to delay the cell cycle in the presence of repair intermediates in yeast. The suppression by mei-W68 and mei-41 suggests that a meiotic checkpoint, which senses the presence of unrepaired DSB, must be active to provoke the phenotype. Patterning defects similar to those of spindle-B , spindle-C , and okra are seen in vasa mutants, but they are not suppressed by mei-41 , placing vasa downstream of the meiotic checkpoint. Vasa, which is required for translation of mRNAs crucial for oocyte patterning could be one target of the meiotic checkpoint.
22.
Tomancak P, Guichet A, Zavorszky P, Ephrussi A: Oocyte polarity depends on regulation of gurken by Vasa.
Development 1998 , 125 :1723-1732.
23. Styhler S, Nakamura A, Swan A, Suter B, Lasko P: Vasa is required for Gurken accumulation in the oocyte, and is involved in oocyte differentiation and germline cyst development.
Development
1998 , 125 :1569-1578.
24. Cox DN, Lu B, Sun TS, Williams LT, Jan YN: Drosophila par-1 is required for oocyte differentiation and microtubule organization.
Curr Biol 2001 , 11 :75-87.
25. Huynh JR, Shulman J, Benton R, St Johnston D: PAR-1 is required for
•• the maintenance of oocyte fate in Drosophila .
Development 2001 ,
128 :1201-1209.
A null allele of par-1 is lethal. In germline clones of this allele, an oocyte is specified, as shown by restriction of meiosis, migration of the centrosomes and accumulation of Orb in one cell of the cyst. However, when the cyst reaches the posterior end of the germarium and the first signs of polarity within the oocyte become apparent, mutant oocytes lose their identity: the oocyte exits meiosis, loses Orb protein and adopts a nurse-cell fate.
26. Pare C, Suter B: Subcellular localization of Bic-D: GFP is linked to an asymmetric oocyte nucleus.
J Cell Sci 2000 , 113 :2119-2127.
27.
Clegg NJ, Findley SD, Mahowald AP, Ruohola-Baker H: maelstrom is required to position the MTOC in stage 2-6 Drosophila oocytes.
Dev Genes Evol 2001 , 211 :44-48.
28. Gonzalez-Reyes A, Elliott H, St Johnston D: Polarization of both major body axes in Drosophila by gurken-torpedo signalling.
Nature 1995 , 375 :654-658.
29. Roth S, Neuman-Silberberg FS, Barcelo G, Schüpbach T: cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in
Drosophila .
Cell 1995 , 81 :967-978.
30. Gonzalez-Reyes A, St Johnston D: Patterning of the follicle cell epithelium along the anterior-posterior axis during Drosophila oogenesis.
Development 1998 , 125 :2837-2846.
31. Peri F, Roth S: Combined activities of Gurken and
••
Decapentaplegic specify dorsal chorion structures of the
Drosophila egg.
Development 2000 , 127 :841-850.
Grk can induce posterior follicle cell fate as late as stage 6, close to the time when the oocyte nucleus starts its anterior migration. Further, Grk can induce posterior and dorsal cell fates simultaneously in neighbouring cells, indicating that timing does not play a role in the different response of the mainbody and terminal follicle cells to Grk signal. Dpp signalling is involved in the specification of the dorsal appendages. These structures require combined signalling from both Grk and Dpp for their specification and positioning.
32. MacDougall N, Lad Y, Wilkie GS, Francis-Lang H, Sullivan W, Davis I:
•
Merlin , the Drosophila homologue of Neurofibromatosis-2 , is specifically required in posterior follicle cells for axis formation in the oocyte.
Development 2001 , 128 :665-673.
Posterior follicle cells do not require Merlin function to receive the Grk signal. However, mutant follicle cells are unable to transduce the signal that is required for the reorganisation of the oocyte MT cytoskeleton, resulting in oocytes with a grk -like phenotype. Merlin belongs to the family of ERM proteins — which are thought to be involved in the apical intracellular targeting of signals — suggesting that Merlin is involved in directing the signal towards the posterior follicle cell apical membrane, in close proximity to the oocyte.

382 Pattern formation and developmental mechanisms
33. Deng WM, Ruohola-Baker H: Laminin A is required for follicle cell
• oocyte signaling that leads to establishment of the anteriorposterior axis in Drosophila .
Curr Biol 2000 , 10 :683-686.
laminin A encodes a component of the extracellular matrix and its function is required in the posterior follicle cells for the transduction of the signal that leads to the reorganisation of the oocyte MT cytoskeleton. Posterior follicle cell clones with laminin A mutations cause a grk -like phenotype, with bicoid mRNA localisation at the anterior and posterior pole, centrally localised oskar mRNA, and failure of the oocyte nucleus to migrate anteriorly.
34. Kelley RL: Initial organization of the Drosophila dorsoventral axis depends on an RNA-binding protein encoded by the squid gene.
Genes Dev 1993 , 7 :948-960.
35. Norvell A, Kelley RL, Wehr K, Schüpbach T: Specific isoforms of
•
Squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis.
Genes Dev 1999 , 13 :864-876.
Squid, a heterogeneous nuclear RNA-binding protein, is required for localisation of grk mRNA and Grk accumulation in the antero-dorsal corner of the oocyte. Squid binds grk mRNA, the nuclear import protein Transportin, and the translational repressor Bruno, suggesting that Squid links grk mRNA localisation and translation. Different Squid isoforms appear to perform different functions in this process. One of these localises to the oocyte nucleus, binds
Transportin, and localises grk mRNA indicating a role in nuclear export of the
RNA. Another isoform, which is predominantly localised in the cytoplasm, is less effective at localising grk mRNA but supports localised accumulation of
Grk protein, indicating its role in translation.
36. Nilson LA, Schüpbach T: EGF receptor signaling in Drosophila oogenesis.
Curr Top Dev Biol 1999 , 44 :203-243.
37.
van Buskirk C, Schüpbach T: Versatility in signalling: multiple responses to EGF receptor activation during Drosophila oogenesis.
Trends Cell Biol 1999 , 9 :1-4.
38. Sen J, Goltz JS, Stevens L, Stein D: Spatially restricted expression of pipe in the Drosophila egg chamber defines embryonic dorsal-ventral polarity.
Cell 1998 , 95 :471-481.
39. Jordan KC, Clegg NJ, Blasi JA, Morimoto AM, Sen J, Stein D,
•
McNeill H, Deng WM, Tworoger M, Ruohola-Baker H: The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through Notch activation.
Nat Genet 2000 , 24 :429-433.
Mirror (Mirr) is a homeodomain-containing transcription factor, which is expressed in the antero-dorsal follicle cells in a grk-dependent manner. mirr mutants show a dorsal expansion of the pipe expression domain, and produce ventralised eggshells. Overexpression of Mirr represses pipe expression at a distance and results in dorsalisation of the eggshell. In addition, mirr restricts the expression domain of fringe . A model is proposed in which
Notch activation at the mirr / fringe expression boundary causes secretion of a molecule that represses pipe expression.
40. Pai LM, Barcelo G, Schüpbach T: D-cbl , a negative regulator of the
•
Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis.
Cell 2000 , 103 :51-61.
Members of the Cbl family of proteins are known to negatively regulate activated receptor tyrosine kinases such as Egfr. In cbl mutant ventral follicle cells, pipe expression is abolished and Egfr target genes are ectopically activated, cell-autonomously and in a grk -dependent manner causing production of dorsalised embryos. These results suggest that Egfr activity is present along the whole D/V axis and that cbl downregulates Egfr signalling in the ventralmost cells to allow pipe expression.
41. Duchek P, Rørth P: Guidance of cell migration by EGF receptor
• signaling during Drosophila oogenesis.
Science 2001 ,
291 :131-133.
Border cell migration occurs in two phases: in the first, the cells migrate posteriorly, towards the oocyte and, in the second, they migrate dorsally, towards the oocyte nucleus. The guidance signal in the first phase of their migration is unknown; in the second they are guided by Grk signal emanating from the oocyte nucleus.
42. Baum B, Li W, Perrimon N: A cyclase-associated protein regulates
• actin and cell polarity during Drosophila oogenesis and in yeast.
Curr Biol 2000 , 10 :964-973.
Cap proteins have been shown to inhibit actin polymerisation by sequestering monomeric actin. Drosophila Cap protein preferentially localises to the oocyte.
In cap mutants ectopic F-actin accumulates, suggesting that Cap regulates actin polymerisation in the oocyte. F-actin accumulation is accompanied by aberrant MT polarity and mislocalisation of oskar and bicoid mRNAs, indicating an influence of the actin cytoskeleton on the polarisation of the MT network.
43. Shulman JM, Benton R, St Johnston D: The Drosophila homolog of
•
C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localization to the posterior pole.
Cell 2000 ,
101 :377-388.
Par-1 function is required for maintenance of oocyte fate in the germarium, as revealed by a par-1 null allele (see annotation [25
••
]). Oocytes mutant for weak par-1 alleles proceed normally through early oogenesis but show an aberrantly polarised MT cytoskeleton during later stages. Although the MTs seem to nucleate normally in these alleles, the MT plus ends do not focus at the posterior pole but at the center of the oocyte. As a result, oskar mRNA is mislocalised to the middle, while bicoid mRNA localisation is not affected.
Interestingly, mutants for the C. elegans homologue of par-1 also show defects in axis formation raising the possibility of an evolutionarily conserved mechanism in establishment of polarity (see also reference [44]).
44. Tomancak P, Piano F, Riechmann V, Gunsalus KC, Kemphues K ,
Ephrussi A: A Drosophila melanogaster homologue of
Caenorhabditis elegans par-1 acts at an early step in embryonicaxis formation.
Nat Cell Biol 2000 , 2 :458-460.
45. Jankovics F, Sinka R, Erdelyi M: An interaction type of genetic
• screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte.
Genetics 2001 , in press.
Rab11 is a small monomeric GTPase involved in various vesicle transport processes. Drosophila Rab11 is required for normal polarisation of the MT network. In Rab11 mutant oocytes, the MT plus ends do not focus at the posterior of the oocyte but instead focus at its center, where oskar mRNA is ectopically localised.
46. Driever W: Maternal control of anterior development in the
Drosophila embryo.
In The development of Drosophila melanogaster, volume 1. Edited by Bate M, Martinez-Arias A. New York: Cold Spring
Harbor Laboratory Press; 1993:301-324.
47.
Ephrussi A, Lehmann R: Induction of germ cell formation by oskar .
Nature 1992 , 358 :387-392.
48. Cha B-J, Koppetsch BS, Theurkauf WE: In vivo analysis of bicoid
• mRNA localization: implications for microtubule function in embryonic axis formation.
Cell 2001, in press.
The authors analyse bicoid RNA localisation by injecting fluorescently labelled RNA into nurse cells and oocytes. Injection into the nurse cells results in bicoid RNA transport to the anterior of the oocyte. However, injection into stage 9 oocytes results in localisation to all cortical surfaces, with the exception of the posterior pole. bicoid mRNA typically colocalises with the MT minus ends and consistent with this, re-examination of the MT network shows that MTs nucleate all around the oocyte cortex with the exception of the posterior pole. Thus, the MT cytoskeleton of stage 9 oocytes appears to be polarised in cortical-central gradient, rather than in a clear A/P gradient.
These initial experiments suggested that either the localisation of bicoid
RNA to the oocyte anterior is achieved by trapping of the RNA upon its entry at the anterior of the oocyte, or that nurse cells factors are essential for specific targeting of the RNA to the anterior. To determine which of the two hypotheses is correct, the authors performed experiments, in which particles of bicoid RNA, which assemble after injection of the RNA into the nurse cells, were withdrawn and injected into the oocyte of a different egg chamber.
Such pre-assembled particles of bicoid RNA injected into the oocyte localise specifically to anterior of the oocyte in an exuperantia and MT-dependent fashion, indicating that anterior targeting factors associate with bicoid RNA in the nurse cells.
49. Berleth T, Burri M, Thoma G, Bopp D, Richstein S, Frigerio G, Noll M,
Nüsslein-Volhard C: The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo.
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50. Schnorrer F, Bohmann K, Nüsslein-Volhard C: The molecular motor
•
Dynein is involved in targeting Swallow and bicoid RNA to the anterior pole of Drosophila oocytes.
Nat Cell Biol 2000 ,
2 :185-190.
In swallow mutants, bicoid mRNA localises normally to the anterior pole, but by stage 10b the RNA spreads towards the posterior pole indicating a function of Swallow in bicoid mRNA localisation within the oocyte. Swallow protein is enriched at the anterior pole of the oocyte as of stage 10 and this localisation is MT-dependent. Swallow binds dynein light chain providing a link between bicoid mRNA and a MT-dependent motor protein.
51. Lieberfarb ME, Chu T, Wreden C, Theurkauf W, Gergen JP,
Strickland S: Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development.
Development
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Axis formation during Drosophila oogenesis Riechmann and Ephrussi 383
52. Micklem DR, Adams J, Grünert S, St Johnston D: Distinct roles of
•• two conserved Staufen domains in oskar mRNA localization and translation.
EMBO J 2000 , 19 :1366-1377.
Staufen is required for the localisation of oskar mRNA to the posterior pole and is involved in its translation. Staufen contains five double stranded RNAbinding domains (dsRBD) which are conserved during evolution. dsRBD5 is unable to bind RNA but is required for derepression of oskar mRNA translation at the posterior pole. Three other dsRBDs, dsRDB1,3 and 4, bind
RNA in vitro , while the RNA binding of dsRDB2 is prevented by an insertion that splits this domain into two halves. Removal of the insertion results in
RNA binding activity of the domain but interferes with the ability of Staufen to localise oskar mRNA to the posterior. The authors propose a model in which RNA binding of three dsRBDs leads to a high local concentration of
RNA that in turn induces dsRBD2 to loop out the insertion and adopt an
RNA-binding configuration. This conformational switch would then allow the coupling of the Staufen–RNA complex to the localisation machinery.
53. St Johnston D, Beuchle D, Nüsslein-Volhard C: staufen , a gene required to localize maternal RNAs in the Drosophila egg.
Cell
1991 , 66 :51-63.
54. Kim-Ha J, Kerr K, Macdonald PM: Translational regulation of oskar mRNA by Bruno, an ovarian RNA-binding protein, is essential.
Cell
1995 , 81 :403-412.
55. Brendza RP, Serbus LR, Duffy JB, Saxton WM: A function for
••
Kinesin I in the posterior transport of oskar mRNA and Staufen protein.
Science 2000 , 289 :2120-2122.
Germline clones mutant in kinesin heavy chain produce oocytes with a normally polarised MT network and normally localised bicoid mRNA. However,
Staufen and oskar mRNA are not transported to the posterior pole, but accumulate at the anterior. This strongly suggests that the Staufen– oskar mRNA complex is transported by Kinesin to the MT plus ends at the posterior pole.
56. Wilhelm JE, Mansfield J, Hom-Booher N, Wang S, Turck CW,
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57.
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′′ and 3
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58. Webster PJ, Liang L, Berg CA, Lasko P, Macdonald PM: Translational repressor Bruno plays multiple roles in development and is widely conserved.
Genes Dev 1997 , 11 :2510-2521.
59. Lie YS, Macdonald PM: Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts.
Development 1999 , 126 :4989-4996.
60. Castagnetti S, Hentze MW, Ephrussi A, Gebauer F: Control of oskar mRNA translation by Bruno in a novel cell-free system from
Drosophila ovaries.
Development 2000 , 127 :1063-1068.
61. Chang JS, Tan L, Schedl P: The Drosophila CPEB homolog, Orb, is required for Oskar protein expression in oocytes.
Dev Biol 1999 ,
215 :91-106.
62. Breitwieser W, Markussen FH, Horstmann H, Ephrussi A: Oskar protein interaction with Vasa represents an essential step in polar granule assembly.
Genes Dev 1996 , 10 :2179-2188.
63. Markussen FH, Michon AM, Breitwieser W, Ephrussi A: Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly.
Development 1995 , 121 :3723-3732.
64. Rongo C, Gavis, ER, Lehmann R: Localization of oskar RNA regulates oskar translation and requires Oskar protein.
Development 1995 , 121 :2737-2746.
65. Gavis ER, Lunsford L, Bergsten SE, Lehmann R: A conserved
90 nucleotide element mediates translational repression of nanos
RNA.
Development 1996 , 122 :2791-2800.
66. Carrera P, Johnstone O, Nakamura A, Casanova J, Jäckle H, Lasko P:
•
Vasa mediates translation through interaction with a Drosophila yIF2 homolog.
Mol Cell 2000 , 5 :181-187.
Stable accumulation of Oskar, Grk and Nanos has been shown to require
Vasa, a DEAD-box RNA helicase. Vasa interacts directly with the Drosophila homolog of yeast translation initiation factor 2 (dIF2), and females doubly heterozygous for vasa and dIF2 mutations show defects in abdomen and germline formation. This provides strong evidence that Vasa regulates translation of several germline RNAs.
67.
Gavis ER, Lehmann R: Translational regulation of nanos by RNA localization.
Nature 1994 , 369 :315-318.
68. Bergsten SE, Gavis ER: Role for mRNA localization in translational activation but not spatial restriction of nanos RNA.
Development
1999 , 126 :659-669.
69. Crucs S, Chatterjee S, Gavis ER: Overlapping but distinct RNA
•• elements control repression and activation of nanos translation.
Mol Cell 2000 , 5 :457-467.
Synthesis of Nanos protein is restricted to the posterior pole of the oocyte and embryo, where nanos mRNA is enriched. A 90-nucleotide bipartite RNA element (TCE), whose secondary structure is important for binding of
Smaug repressor, mediates translational repression of unlocalized nanos mRNA in the bulk cytoplasm. nanos mRNA localisation elements overlap the
TCE, suggesting a mechanism leading to the switch between repression and localisation-dependent activation of nanos mRNA translation.
70. Smibert CA, Wilson JE, Kerr K, Macdonald PM: Smaug protein represses translation of unlocalized nanos mRNA in the
Drosophila embryo.
Genes Dev 1996 , 10 :2600-2609.
71. Dahanukar A, Walker JA, Wharton RP: Smaug, a novel RNA-binding
•• protein that operates a translational switch in Drosophila .
Mol Cell
1999 , 4 :209-218.
Smaug protein was previously identified as a biochemical species that binds to a region involved in nanos mRNA translational control. Smaug is a novel
RNA-binding protein that represses nanos translation in the bulk cytoplasm of the embryo. Smaug also interacts directly with Oskar, the key component of the pole plasm that recruits, and is required for, nanos mRNA translational activation. Hence, Smaug may mediate the switch between repression and activation of translation of nanos RNA.
72. Smibert CA, Lie YS, Shillinglaw W, Henzel WJ, Macdonald PM:
Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro .
RNA 1999 , 5 :1535-1547.
73. Clark IE, Wyckoff D, Gavis ER: Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism.
Curr Biol 2000 , 10 :1311-1314.