A Bicoid self-inhibitory domain

A small domain of Bcd located immediately N-terminal to the homeodomain (residues 52-91) represses Bcd activity in Drosophila cells. This domain, referred to as a self-inhibitory domain, works as an independent module that does not rely on any other sequences of Bcd and can repress the activity of heterologous activators. This domain of Bcd does not affect its properties of DNA binding or subcellular distribution. A Bcd derivative with point mutations in the self-inhibitory domain severely affects pattern formation and target gene expression in Drosophila embryos. Evidences suggest that the action of the self-inhibitory domain requires a Drosophila co-factor(s), other than CtBP or dSAP18. These results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen during embryonic development is dependent on the downregulation of its own activity through an interaction with a novel co-repressor(s) or complex(es) (Corado, 2002).

Analysis of embryos from bcd(A52-56) transgenic females reveals a dominant, gain-of-function effect causing developmental defects in both head and abdominal regions. These phenotypes share resemblance to those caused by a Bcd-VP16 fusion protein which contains the strong activation domain VP16. Interestingly, an excessive amount of wild-type Bcd produced from six copies of bcd can also cause head and abdominal defects in a fraction of the embryos. It is relevant to note that a bcd cDNA transgene in the P-element vector pCaSpeRBcdBglII has been estimated to produce, on average, approx. half the amount of Bcd protein as an endogenous bcd gene. Compared with wild-type Bcd expressed from six copies of bcd, Bcd(A52-56) can cause embryonic defects at a much higher penetrance (100% in line 18A) and at a much lower concentration (~1/8). Two copies of wild-type bcd cDNA transgene caused only moderate abdominal defects at a low frequency in most of the lines examined. This observation is consistent with the estimate that two copies of the transgene are equivalent to only one copy of genomic bcd (Corado, 2002).

The head defects caused by Bcd(A52-56), like those caused by Bcd-VP16 and excessive amounts of wild-type Bcd, are presumably due to a failed or incomplete head involution resulting from the posterior shift of the fate map. It is possible that both Bcd-VP16 and Bcd(A52-56) may have additional molecular consequences associated with their strong activation functions. It remains to be determined whether, for example, Bcd(A52-56) causes the developmental defects, in part, by activating zygotic genes that are normally not targets of Bcd in embryos (Corado, 2002).

The expression domains of hb and otd in embryos containing Bcd(A52-56) are expanded dramatically towards the posterior. Interestingly, no obvious increase in the intensity of their expression is observed in these embryos. It is possible that hb and otd are expressed, in response to the Bcd gradient, at levels that are already maximal in wild-type embryos. According to this idea, the consequence of the stronger activator Bcd(A52-56) is not an elevated level of hb and otd expression, but rather, a posterior shift of their expression domains. It has been shown that the activating strength of an activator can actually influence its in vivo DNA-binding ability. In particular, activators with stronger activation domains can bind DNA at lower concentrations in vivo, presumably because a stronger interaction with the basal transcription machinery can facilitate their DNA binding function at low concentrations. Although these experiments demonstrate that both wild type Bcd and Bcd(A52-56) have a similar affinity to a single Bcd binding site in vitro, a dramatic posterior shift of the hb and otd expression domains in embryos containing Bcd(A52-56) suggests that Bcd(A52-56) may have a significantly higher in vivo affinity to both enhancers. Furthermore, since Bcd(A52-56) is a much stronger activator, it is possible that hb and otd can be activated by fewer Bcd(A52-56) molecules (than wild-type molecules) in the more posterior part of the embryo (Corado, 2002).

Another domain of Bcd (residues 300-340, alanine-rich) also exhibits an inhibitory function. Besides their different physical locations and amino acid compositions, there are several other important differences between the self-inhibitory domain delineated in this report and that newly described domain. (1) The self-inhibitory domain described in this study represses transcription over 20-fold in deletion assays, whereas a single alanine-rich domain only represses transcription threefold (its effect is significantly enhanced when multimerized). A Bcd derivative lacking the alanine-rich region also causes a posterior shift of the hb expression domain in embryo, though less dramatically than Bcd(A52-56). (2) The self-inhibitory domain can work on heterologous activation domains, in addition to those from Bcd, suggesting an active repression mechanism. (3) This domain has been systematically dissected by deletion and point mutations. (4) Finally, and most importantly, while point mutations in the self-inhibitory domain cause severe developmental defects, the entire C-terminal half of Bcd, including the alanine-rich domain, can be deleted (Corado, 2002 and references therein).

Although transgenic studies demonstrate that the self-inhibitory function of Bcd is important for proper embryonic pattern formation in Drosophila, it is not completely clear how this function is regulated by other developmental cues. The action of the self-inhibitory domain is not responsible for Tor-dependent repression upon cellularization, although the possibility cannot be ruled out that the self-inhibitory domain may play a contributory role in this process. In addition, it has been shown that self-inhibitory domains of other proteins are involved in synergistic activation with co-factors. The self-inhibitory domain of Bcd may similarly participate in synergistically activating transcription with other Drosophila factors, such as Hb. Furthermore, since the N-terminal region of Bcd is also engaged in self-association and cooperative DNA binding, enhancer architecture (i.e. the arrangement of DNA sites for Bcd) may influence how Bcd molecules are positioned on different enhancers and, thus the availability of the self-inhibitory domain for interacting with the proposed co-repressor(s). Given its intricate morphogenetic role in instructing embryonic patterning, an intriguing possibility exits that Bcd itself may function as an active repressor in a context-dependent manner during embryonic development (Corado, 2002).

Bicoid mRNA localization and regulation

Bicoid localization in the oocyte can be divided into two phases: previtellogenic, in which Bicoid mRNA is localized to the apex of nurse cells, and vitellogenic, in which Bicoid is transported along the microtubule network to the anterior portion of the oocyte. The latter localization event requires genes of the anterior group.

RNA regulatory element BLE1 directs the early steps of bicoid mRNA localization. Development 118: 1233-43>

Deployment of the Bicoid morphogen gradient in early Drosophila embryos requires the prelocalization of BCD mRNA to the anterior pole of the egg. This anterior localization is mediated by a cis-acting localization signal contained within the 3' untranslated region of the BCD mRNA. One essential element, BLE1, specifically directs the early steps of localization in nurse cells. Many deletions within the BCD mRNA 3' untranslated region impair but do not prevent localization. One deletion specifically interferes with a later step in localization. Thus the BCD mRNA localization signal appears to consist of multiple different elements, each responsible for different steps in the localization process (Macdonald, 1993).

exl protein specifically binds BLE1, a bicoid mRNA localization element, and is required for one phase of its activity

Localization of mRNAs, a crucial step in the early development of some animals, has been shown to be directed by cis-acting elements that presumably interact with localization factors. EXU binds to BLE1, an RNA localization element from the Drosophila Bicoid mRNA. Using mutations in BLE1, a correlation has been found between in vitro Exuperantia-like (EXL) binding and an early previtellogenic phase of in vivo localization directed by BLE1, implicating EXL in that localization event. The same phase of localization is disrupted in exuperantia mutants, suggesting that exl and exuperantia proteins interact (Macdonald, 1995).

Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis

The exuperantia (exu) gene is necessary for this localization of BCD mRNA. A chimaeric gene encoding a fusion between the Acquorea victoria green fluorescent protein (GFP) and the EXU protein is expressed in female germ cells. The fusion protein rescues an exu null allele, restoring full fertility to females, and is expressed and localized in a temporal and spatial pattern similar to native EXU. The fusion protein is found in particles concentrated at ring canals, where transport occurs between nurse cells and the oocyte. Drugs such as colchicine and taxol that affect microtubule stability alter localization of the particles. The particles may be ribonucleoprotein complexes or vesicles which transport BCD mRNA along microtubules and target it to the anterior oocyte cortex (Wang, 1994).

Components acting in localization of bicoid mRNA are conserved among Drosophila species

How highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning? Two components are involved in that process: the exuperantia gene, required for an early step in localization, and the cis-acting signal that directs BCD mRNA localization. The cloned D. melanogaster exu gene has been used to identify the exu genes from D. virilis and D. pseudoobscura. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of BCD mRNA, demonstrating that function is conserved (Luk, 1994).

Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes

Localization of Bicoid (BCD) mRNA to the anterior and oskar (OSK) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. exuperantia (exu) is implicated in BCD mRNA localization, but its role in this process is not understood. Various studies have shown that localized messages are organized into particles, suggesting that a large protein complex may be involved in recognizing, transporting, and anchoring localized messages. Exu is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins is the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. The Exu-Yps complex also contains OSK mRNA. exu-null mutants are defective in OSK mRNA localization in both nurse cells and the oocyte. It is proposed that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte (Wilhelm, 2000 and references therein).

Genetic screens have identified several mutants that have patterning defects due to the mislocalization of BCD and/or OSK mRNAs. Mutations in some genes, such as swallow and staufen, cause only partial disruption of BCD mRNA localization late in oogenesis. However, in exuperantia mutants, defects in BCD mRNA localization occur early in oogenesis and result in BCD mRNA being uniformly distributed in the mature oocyte. Time-lapse confocal microscopy has shown that green fluorescent protein (GFP)-Exu forms particles that move in a microtubule-dependent manner and accumulate at the anterior and posterior of the oocyte. Immunoelectron microscopy has also revealed that Exu is a component of large electron-dense structures called sponge bodies (Wilhelm, 2000 and references therein).

The pathways by which anterior- and posterior-localized mRNAs arrive at their destinations are poorly understood, although it is generally believed that these RNAs are recognized by different proteins and utilize distinct transport machineries. However, it is proposed that anterior- and posterior-localized mRNAs begin their localization process in the nurse cells using a similar complex, with Exu serving as a common core component. In this model, one of Exu's functions is as a component of an mRNA transport complex, since GFP-Exu particles have been observed to move in a microtubule-dependent manner. Consistent with this idea, both OSK and BCD mRNA accumulate in apical patches within nurse cells, and exu mutants disrupt this localization pattern for both mRNAs. It is also proposed that the Exu complex transports mRNAs from the nurse cells to the oocyte as well as within the oocyte, although these transport steps also can be achieved through other redundant mechanisms, such as nurse cell dumping and cytoplasmic streaming. Although the above model places Exu as part of a transport complex, it should be noted that Exu might contribute to the establishment of anchoring once mRNAs reach their final destination (Wilhelm, 2000 and references therein).

After arriving in the oocyte, BCD- and OSK-containing RNPs must be sorted so that BCD becomes anchored at the anterior, whereas OSK is transported to the posterior pole. Since Yps, Exu, BCD mRNA, and OSK mRNA all first colocalize at the anterior, it is proposed that this sorting decision occurs at the anterior of the oocyte. Evidence for this anterior sorting model comes from genetic studies of staufen (stau) and tropomyosin II (TmII) that show that these proteins do not interfere with anterior localization but rather block the release and transport of OSK transcripts to the posterior. The molecular basis for this sorting decision is unclear, but may involve modifications to the transport machinery or the recruitment of additional factors (Wilhelm, 2000 and references therein).

Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis

Whereas Exu and Yps associate with one another independently of mRNA, another component of the ribonucleoprotein complex, the DEAD-box protein, Me31B, associates with Exu and Yps in a RNase-sensitive manner. Me31B is dispensable for the transport of the associated mRNA and proteins molecules to oocytes. Exu, OSK and BicaudalD mRNAs can be transported to the oocyte even in the absence of Me31B. Nevertheless, Me31B is essential for the translational silencing of OSK and BicaudalD mRNAs during their transport to the oocyte. This suggests that Me31B and the Exu-Yps complex bind different regions of the same RNA molecule. These data lead to the speculation that the assembly of a cytoplasmic RNP complex is achieved by binding of functionally different proteins to discrete regions of an oocyte-localizing RNA (Nakamura, 2001).

Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner

In the later vitellogenic stage in BCD RNA localization, after transfer of the RNA from nurse cells to the oocyte, Staufen protein is required in order to anchor the Bicoid mRNA at the anterior pole of the Drosophila egg. Staufen protein colocalizes with BCD mRNA at the anterior. This localization depends upon its association with the mRNA. Upon injection into the embryo, BCD transcripts specifically interact with Staufen. The required sequences have been mapped to three regions of the 3'UTR, each of which is predicted to form a long stem-loop. The resulting Staufen-BCD 3'UTR complexes form particles that show a microtubule-dependent localization. Since Staufen is also transported with Oskar (OSK) mRNA during oogenesis, Staufen associates specifically with both OSK and BCD mRNAs to mediate their localizations, but at two distinctly different stages of development (Ferrandon, 1994).

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization in order to anchor the BCD mRNA in the anterior cytoplasm. STAU protein contains five double-stranded RNA-binding motifs common to a family of dsRNA-binding proteins. Endogenous STAU protein associates specifically with injected BCD mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. The STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. Two loops contact each other through base pairing. One possibility is that this pairing occurs within the molecule, thus forming an element of tertiary structure called a pseudoknot. Attempts to model the putative pseudoknot in BCD mRNA were unsuccessful because it was impossible to bend the structure sufficiently to allow these two single-stranded loops to base-pair with one another. This suggests that the two loops base-pair with each other, but do so between two different mRNA molecules. Base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intra-molecular; thus dimers or multimers of the RNA localization signal must be associated with STAU protein in these particles. The BCD mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization (Ferrandon, 1997).

RNA recognition by a Staufen double-stranded RNA-binding domain

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, the third dsRBD from Drosophila Staufen has been studied. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and the amino acids required for this interaction have been identified. By mutating these residues in a staufen transgene, it has been shown that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of Bicoid and Oskar mRNAs. Using high-resolution NMR, the structure of the complex between dsRBD3 and an RNA stem-loop was determined. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins (Ramos, 2000).

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation

Drosophila Staufen protein is required for the localization of Oskar mRNA to the posterior of the oocyte, the anterior anchoring of Bicoid mRNA and the basal localization of Prospero mRNA in dividing neuroblasts. Analysis of an alignment of the Stau homologs reveals that the only regions of the protein to have been conserved during evolution are the five dsRBDs and a short region within an insertion that splits dsRBD2 into two halves. The M. domestica and D. melanogaster proteins show an average of 67% amino acid identity within the dsRBDs, but less than 15% in the rest of the protein. dsRBD2 and dsRBD5 were originally described as 'half domains' showing similarity to the dsRBD consensus only over the C-terminal portion of the domain. However, the conservation extends over a region corresponding to the length of a whole domain, and these domains should therefore be considered as complete, albeit divergent. The only other obvious homology between these proteins is a short region, one which is rich in proline and aromatic amino acids, within the insertion that splits dsRBD2. Since the regions of the protein essential for its activity are expected to be conserved during evolution, the dsRBDs and this proline-rich region are likely to mediate all of the functions of Stau, including its ability to bind both mRNA and the factors that localize Stau-mRNA complexes. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with Oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes Oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of OSK mRNA, and dsRBD5 is required for the derepression of Oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of Prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport (Micklem, 2000).

Since Stau is also required for the anterior anchoring of BCD mRNA, it was of interest to determine whether the Stau lacking dsRBD5 and the RBD2 loop could rescue the BCD mRNA localization defect of a stau null mutation. Although both mutant proteins are expressed at similar levels to endogenous Stau, neither anchors BCD mRNA at the anterior. Surprisingly, both constructs almost completely rescue the stau head phenotype, even though they do not restore the wild-type localization of the BCD mRNA. Whereas 100% of the embryos laid by stau homozygous mutant females at 18°C lack all or part of the head skeleton, over two-thirds of the embryos laid by the transgenic stau minus females have wild-type heads, and the rest have much milder head defects than are found in stau mutants alone. This suggests that Stau plays a second role in the regulation of BCD mRNA expression that is independent of its function in localization. In contrast to its role in anchoring, this activity does not require dsRBD5 or the insertion in dsRBD2 (Micklem 2000).

Both StauDeltaloop2 and StauDeltadsRBD5 partially rescue the stau head phenotype, even though they do not restore the wild-type localization of BCD mRNA. Thus, more Bcd activity must be produced from the mislocalized mRNA in the presence of these mutant proteins than in stauD3 alone, indicating that they provide a function for Stau that is independent of its role in anchoring. A comparison of the phenotypes produced by vasa;exu and stau;exu double mutants also indicates that Stau has a second function in the regulation of BCD mRNA. exu mutants block the localization of BCD mRNA early in oogenesis, and result in a uniform distribution of the RNA along the anterior-posterior axis of the embryo, while both vasa and stau mutants prevent the formation of the pole plasm, and therefore lack Nanos activity, which represses BCD mRNA translation. Despite the identical distributions of BCD RNA in these genotypes, vasa;exu embryos develop anterior head structures everywhere, indicating that they contain high levels of Bcd activity, whereas stau;exu form only thoracic structures. Thus, the removal of Stau reduces the level of Bcd expression, in the absence of any effect on mRNA localization. Two explanations for this localization-independent function of Stau can be envisioned. Stau binding could protect BCD RNA from degradation, and therefore increase the total amount of RNA. Alternatively, Stau could enhance the efficiency of BCD translation, in much the same way as it does for OSK mRNA (Micklem, 2000 and references therein).

Since Stau has been conserved throughout animal evolution, it seems likely that the homologs will fulfil similar functions in mRNA localization and translational control in other organisms. In support of this view, recent evidence indicates that mammalian Stau mediates mRNA transport along microtubules in neurons. The mouse and human Stau genes share an extra region of homology (not found in the insect homologs) that resembles the microtubule-binding domain of MAP1B, and this region of HsStau binds to microtubules in vitro. It will therefore be interesting to see whether this domain or the insertion in dsRBD2 is required for the microtubule-dependent movement of Stau in neurons (Micklem, 2000).

A small predicted stem-loop structure mediates oocyte localization of Drosophila K10 mRNA

fs(1)K10 mRNA transport and anterior localization is mediated by a 44 nucleotide stem-loop structure. A similar putative stem-loop structure is found in the 3' untranslated region of the Drosophila ORB mRNA, suggesting that the same factors mediate the transport and anterior localization of both K10 and ORB mRNAs. Apart from ORB, the K10 TLS (transport/localization sequence) is not found in any other localized mRNA, raising the possibility that the transport and localization of other mRNAs, e.g., Bicoid, Oskar and Gurken, are mediated by independent sets of cis- and trans-acting factors. K10 TLS overrides the activity of Oskar cis-regulatory elements that mediate the late stage movement of the mRNA to the posterior pole (Serano, 1995).

cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila

In Drosophila, the dorsal-ventral polarity of the egg chamber depends on the localization of the oocyte nucleus and the Gurken RNA to the dorsal-anterior corner of the oocyte. Gurken protein presumably acts as a ligand for the Drosophila EGF receptor (torpedo/DER) expressed in the somatic follicle cells surrounding the oocyte. cornichon is a gene required in the germline for dorsal-ventral signaling. cornichon, gurken, and torpedo also function in an earlier signaling event that establishes posterior follicle cell fates and specifies the anterior-posterior polarity of the egg chamber. Mutations in all three genes prevent the formation of a correctly polarized microtubule cytoskeleton required for proper localization of the anterior and posterior determinants bicoid and oskar and for the asymmetric positioning of the oocyte nucleus (Roth, 1995).

Microtubules are a general component of mRNA localization systems in Drosophila oocytes

Some of the spatial cues which direct early patterning events in Drosophila embryogenesis are maternal mRNAs localized in the oocyte during oogenesis. Microtubules are required for localization of these mRNAs during oogenesis. However, the RNAs show a differential sensitivity to microtubule inhibitors. Anterior localization of Bicaudal-D, Fs (1) K10, and ORB RNAs is completely disrupted following even mild drug treatments. Bicoid RNA localization is intermediate in its response to microtubule drugs, while Oskar RNA localization is much more resistant. In addition, the localized RNAs respond differently to taxol, a microtubule stabilizing agent. The differences among these RNAs suggest that factors other than microtubules are required to maintain the positions of localized RNAs in the oocyte. Microtubules are also required for the preferential accumulation of these transcripts in the previtellogenic oocyte, consistent with the idea that these mRNAs are transported by a microtubule-dependent mechanism to the oocyte (Pokrywka, 1995).

RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization

Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anteroposterior axis in Drosophila oocytes. Protein Kinase A (PKA) mutations act in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of Bicoid and Oskar RNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition; Oskar RNA fails to accumulate at the posterior, and Bicoid RNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns for Notch and Delta mutants suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells (Lane, 1994).

Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases

The female sterile (3) homeless gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. Transport and localization of Bicoid and Oskar messages during vitellogenic stages are strongly disrupted in homeless mutants. Examination of the microtubule structure with anti-alpha-Tubulin antibodies reveals aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork (Gillespie, 1995).

Egg ligation alters the Bcd protein gradient and segmentation gene expression in embryos of Drosophila

A concentration gradient of the anterior morphogen Bicoid plays a key role in the specification of cell fates in the early Drosophila embryo. Introduction of a membrane barrier across the embryo results in increased levels of BCD protein on the anterior side of the barrier and decreased levels on the posterior side, consistent with a blockage in the postulated anterior-to-posterior translocation of BCD protein. The expression patterns of downstream segmentation genes are in large part consistent with their regulation by the BCD morphogen. However, some aspects of the patterns do not correlate with the altered BCD distribution, suggesting that other morphogens also regulate the anteroposterior pattern (Boring, 1993).

maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA

A mutant, maelstrom (mael), is described that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, Bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell (Clegg, 1997).

Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development

Translational recruitment of maternal mRNAs is an essential process in early metazoan development. To identify genes required for this regulatory pathway, a collection of Drosophila female-sterile mutants was examined for defects in translation of maternal mRNAs. This strategy has revealed that maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of Bicoid and Toll mRNAs. Cortex embryos contain a Bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the Bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine [poly(A)] tail. Injection of polyadenylated Bicoid mRNA into cortex embryos allows translation, demonstrating that insufficient polyadenylation prevents endogenous Bicoid mRNA translation. In contrast, Nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. cortex embryos are fertilized, but arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development (Lieberfarb, 1996).

A system of repressor gradients spatially organizes the boundaries of Bicoid-dependent target genes

The C-terminal half of the Grau protein consists of eight conserved C2H2-type zinc finger motifs, identifying it as a new member of the C2H2-type zinc finger protein family. Other than the zinc finger motifs, there are at least two notable features in the Grau protein. (1) There is a cluster of acidic residues extending from Asp146 to Asp172 (17 of 27 residues are Asp or Glu). Similar acidic domains have been found in many transcription factors, for example Gal4, and function as transcription activating regions. (2) There are two predicted bipartite nuclear localization signals within the Grau protein. The first is located from residue Arg382 to Lys399, and the second is from Lys522 to Thr539. Grau-GFP is localized to nuclei in germarium cells and in early stage egg chambers, in stage 7, stage 8 and stage 9 egg chambers. Expression is observed in both follicle cells (fc) and nurse cells (nc). Very little or no Grau-GFP signal is detected in the oocyte nucleus (Chen, 2000)

Embryos from female flies homozygous for grau or cort mutations show very similar defects, suggesting that the two gene products participate in the same developmental pathway. A more careful investigation allowed for the identification of a difference between grau and cort mutants. While no embryos laid by cort homozygous females hatch into larvae, a small percentage of grau embryos develop into adult flies. This limited fertility allows for a test of interaction between the two genes. The small percentage of developing embryos derived from grau homozygous females (~2%-5%) is abolished when the females are also heterozygous for a cort mutation (Chen, 2000).

To examine whether Grau mRNA expression is affected by cort, grau expression was examined in ovaries from cort mutant females. The expression of Grau RNA is not affected. On the contrary, grau homozygous ovaries have severely reduced levels of Cort transcript. Analysis was carried out of RNAs isolated from ovaries containing different dosages of grau function: wild-type, heterozygous and null. With decreasing dosage of grau function, decreasing amounts of cort transcript are detected. These results indicate that grau acts upstream of cort, probably by regulating cort transcription. The presence of small amounts of cort transcript in grau null ovaries may account for the leaky phenotype of the grau mutants. To determine if Grau protein can regulate the transcription of cort directly, the ability of Grau to bind to a region of the cort promoter was examined in vitro. Recombinant GST-Grau protein binds to a 32 bp region of the cort promoter (Chen, 2000).

The grau and cort genes are the only two mutations identified so far that cause female meiotic arrest (Lieberfarb, 1996; Page, 1996). The chromosome configuration in the mutant eggs suggests a block at meiotic metaphase-anaphase transition. Very little is known about the mechanisms that regulate this transition in meiosis. Much progress has been made, however, in elucidating the similar process during mitosis. Such studies have shown that the metaphase-anaphase transition in mitosis requires the destruction of cell cycle regulators, such as cyclin B and the anaphase inhibitor Pds1, through ubiquitin-mediated proteolysis. Since the degradation of anaphase inhibitors and mitotic cyclins allows progression through mitosis, similar inhibitors may also be present during meiosis. One possible role for grau and cort may be in the regulated degradation of these meiotic anaphase inhibitors and cyclins. The fact that Grau protein regulates cort transcription provides a possible explanation for the grau mutant phenotype. In the absence of Grau protein function, cort transcription is severely reduced. This may result in the production of much less Cort protein than is required for the metaphase-anaphase transition. Since no mutant phenotype has been observed in mitosis or male meiosis (Page, 1996), grau appears to be required specifically during female meiosis. The hypothesis that grau and cort may be necessary for the degradation of certain cell cycle regulators may also account for the persistent cortical microtubules in the mutant eggs. The inappropriate presence of meiotic Cyclin B results in persistent maturation promoting factour (MPF) activity. Since MPF activity has been shown to affect microtubules, the cortical microtubule phenotype could be a result of MPF persistence at metaphase II (Chen, 2000).

How does Grau, a transcription factor, and its downstream target Cort, affect cytoplasmic polyadenylation and translational activation of Bicoid mRNA? Translation of stored maternal mRNAs has to be coordinated with the act of egg activation or fertilization. Many maternal mRNAs that remain silent in arrested oocytes do not become cytoplasmic polyadenylated and translationally activated until egg activation or fertilization. It is conceivable that an inhibitor(s) of cytoplasmic polyadenylation and translation may be present or the cytoplasmic polyadenylation machinery in the arrested egg needs to be activated. In these cases, fertilization or egg activation would generate a signal(s) to remove the inhibitor or to activate the cytoplasmic polyadenylation machinery. With Cort being a potential meiotic cell cycle regulator, it is possible that the pathway to which grau and cort belong is required to remove the inhibitory factor of Bicoid mRNA polyadenylation or to activate the cytoplasmic polyadenylation machinery upon egg activation. The final answer to this question awaits further investigation of translational control during early development as well as the identification of its relationship to the meiotic cell cycle (Chen, 2000).

BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA

BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA

The early stages of Drosophila development rely extensively on posttranscriptional forms of gene regulation. Deployment of the anterior body patterning morphogen, the Bicoid protein, requires both localization and translational regulation of the maternal Bicoid mRNA. Evidence is provided that the Bicoid mRNA is also selectively stabilized during oogenesis. A protein, Bicoid stability factor (BSF: CG10302), has been identified and isolated that binds specifically to IV/V RNA, a minimal form of the Bicoid mRNA 3' untranslated region that supports a normal program of mRNA localization during oogenesis. Mutations that disrupt the BSF binding site in IV/V RNA or substantially reduce the level of BSF protein lead to reduction in IV/V RNA levels, indicating a role for BSF in RNA stabilization. The BSF protein is novel and lacks all of the characterized RNA binding motifs. However, BSF does include multiple copies of the PPR motif, whose function is unknown but appears in other proteins with roles in RNA metabolism (Mancebo, 2001).

To search for proteins that may regulate the activity or distribution of BCD mRNA, a focus was placed on the IV/V region, a 271-nt portion of the BCD 3' UTR that supports a normal pattern of mRNA localization during oogenesis. Unlike the complete 3' UTR, the IV/V RNA lacks redundant information for the initial step of BCD mRNA localization. Specifically, the localization activity of IV/V can be greatly reduced by a point mutation (G4496U), while the same mutation has only a subtle and transient effect on the activity of the complete 3' UTR. The absence of functional redundancy is a prerequisite for experiments in which an attempt is made to correlate a protein binding site with a biological activity (Mancebo, 2001).

Extracts prepared from Drosophila ovaries were tested for the presence of proteins that bind to IV/V RNA using a UV cross-linking assay. A number of proteins bind under these conditions. The assays were also performed in the presence of increasing amounts of competitor RNA as an initial test for binding specificity. Most of the bands detected in the assay are unaffected by addition of the competitor, but the binding of four proteins, p55, p70, p80, and BSF, is clearly reduced. To explore a possible role for any of the proteins in BCD mRNA localization, RNA probes corresponding to the isolated parts of IV/V (predicted stem-loops IV and V) were used in separate binding assays; RNAs IV and V have no localization activity in vivo and thus may fail to bind one or more localization factors. Many proteins bind equally well to all probes. Two proteins, p55 and p70, bind to V RNA but not IV RNA, suggesting that they recognize sites contained entirely within V. Finally, p80 and BSF bind much better to IV/V RNA than to either of the isolated parts (which do not support mRNA localization) and are thus the best candidates to act in mRNA localization. To explore further a possible role for the cross-linking proteins in BCD mRNA localization, a binding assay was done with a point-mutated IV/V RNA (G4496U) that interferes with mRNA localization in vivo. The G4496U mutation has no effect on the binding of any of the proteins detected in this assay. Although this result does not rule out involvement of any of the binding proteins in BCD mRNA localization, it does suggest that other roles may be more likely (Mancebo, 2001).

The strategy for testing the role of BSF was to first identify mutations in IV/V RNA that interfere with BSF binding in vitro and then to determine the consequences of these same mutations in vivo. LS mutagenesis was used to create a series of 27 mutants that collectively alter most of the 271 nt of IV/V. Each mutant replaces a 10-nt segment of IV/V with a synthetic sequence. Wild-type and mutant IV/V RNAs were used as probes in binding assays. One mutant RNA, LS15, is most severely impaired in BSF binding. Several others (e.g., LS19 and LS20) are also impaired but to a lesser extent. Almost all of the same LS mutants were also tested in vivo. Each mutant was introduced into a reporter construct, transgenic fly strains were established, and patterns of mRNA localization in transgenic ovaries were monitored by in situ hybridization. For the LS15 mutant, no localized reporter mRNA was detected in any of four independent transgenic fly stocks, and the underlying cause was unique among all mutants tested: the LS15 mutant fails to accumulate any mRNA. In comparison, the LS11 mutant, which is completely defective in mRNA localization, retains normal levels of transgene mRNA. The simplest interpretation of these results is that the LS15 mutant destabilizes the IV/V RNA. The data point to the likelihood that LS15 mRNA is unstable and that instability is the consequence of the defect in BSF binding (Mancebo, 2001).

BSF is a protein of 1,412 amino acids. The most notable feature of the BSF sequence is the presence of seven copies of the PPR motif, with four copies adjacent to one another near the amino terminus and three copies dispersed over the carboxyl-terminal half of the protein. The PPR motif, which is usually about 35 amino acids long, has no assigned function but has already been identified in over 200 proteins that are widely represented in plant organelles. Two proteins that contain the motif have been characterized genetically, and each plays a role in RNA metabolism. Notably, the PPR-containing PET309 protein has been shown to act in either processing or stabilization of certain RNAs. Based on these observations, BSF is a member of a new protein family that may have a common function in RNA metabolism (Mancebo, 2001 and references therein).

Sequence comparisons of BSF with those of the GenBank database identify two proteins that are most closely related, a human leucine-rich protein of unknown function (expectation [E] value of <10-136) and a predicted Drosophila protein (E <10-68). Both proteins also contain multiple copies of the PPR motif, although the extensive homology between BSF and the leucine-rich protein is not limited to these repeated structural elements (Mancebo, 2001).

Flies transheterozygous for bsf1 mutation and the deficiency Df(2L)M36F-S5 are viable and fertile, with no obvious morphological defects in oogenesis. When eggs from such females are fertilized by wild-type or bsf1 sperm, they progress normally through embryogenesis and display no cuticular pattern defects (development is arrested later for bsf1/bsf1 individuals because of the 25E lethal mutation on the chromosome). As a first test for a molecular defect in the ovaries of bsf1/Df(2L)M36F-S5 females, the level of endogenous BCD mRNA was examined but no substantial difference was found relative to the wild type. However, the apparent mRNA instability phenotype associated with LS15 (the LS mutant defective in BSF binding) is detected for transgenes containing only the IV/V portion of the BCD mRNA 3' UTR, while deletion from the complete BCD 3' UTR of the region corresponding to LS15 has no substantial effect on mRNA levels. Thus, LS15 can block the action of only a single component of a redundant stabilizing system, and reduction of BSF activity would only be detected when redundancy is eliminated. Accordingly, the level of the reporter mRNA bearing the wild-type IV/V RNA in flies transheterozygous for bsf1 and Df(2L)M36F-S5 was examined. Compared to control flies, the bsf mutant flies display a consistent three- to five-fold reduction in the level of wild-type IV/V RNA. There is no direct evidence that proves a mechanism by which a reduction in BSF levels reduces the level of IV/V RNA. Nevertheless, the fact that BSF binds to sequences within IV/V RNA argues for a posttranscriptional role, an interpretation consistent with the mRNA stabilization role suggested for BSF by the LS mutant analysis (Mancebo, 2001).

The subcellular distribution of BSF protein was determined by immunofluorescent detection in whole-mount ovaries. At all stages of oogenesis the protein is cytoplasmic. During the previtellogenic stages of oogenesis BSF is present in both the nurse cells and the oocyte at similar levels. Within the nurse cells BSF appears primarily in regions surrounding the nuclei, and within these regions the protein is often concentrated in a punctate pattern. As oogenesis proceeds, the tight association of BSF with nurse cell nuclei is lost. The particulate appearance of BSF is enhanced, but the particles are more evenly dispersed throughout the cytoplasm of the nurse cells. In the oocyte the BSF levels are reduced relative to the nurse cells. At no time does BSF appear to be concentrated at sites of BCD mRNA accumulation, at either the apical regions of the nurse cells or the anterior margin of the oocyte (Mancebo, 2001).

The consequence of mutating either the IV/V RNA stability element or bsf is the elimination or reduction, respectively, of the reporter mRNA bearing the IV/V 3' UTR. In contrast, mutation of bsf has no discernable effect on endogenous BCD mRNA. Similarly, deletion of the LS15 region from the full BCD 3' UTR is tolerated, and the deletion mutant RNAs are readily detectable. The striking context dependence of mutating either the cis or trans components of this RNA stabilization system suggests that there is redundancy in the stabilization process: sequences outside IV/V are sufficient for stabilization, and another factor(s) can perform the same function as BSF. This redundancy is not surprising given the redundancy already demonstrated for localization of BCD mRNA. Indeed, the IV/V subdomain of BCD RNA was used in this work because it lacks the mRNA localization redundancy of the full 3' UTR (Mancebo, 2001).

An alternative explanation for the observation that a mutation in the RNA stabilization element leads to a reduction in the level of IV/V RNA while a deletion of the stabilization element from the full BCD 3' UTR appears to have no affect is suggested by known mechanisms of mRNA stabilization. Specifically, binding of the iron response element protein to sequences in the transferrin receptor mRNA 3' UTR blocks endonucleolytic attack and thus stabilizes the mRNA. It is possible that the LS15 mutant disrupts the BSF binding site but not a nearby nucleolytic cleavage site and thus confers instability. In contrast, the larger deletion mutants that do not affect stability of the BCD 3' UTR might eliminate both the protection and cleavage elements, making them resistant to targeted degradation. Distinguishing among these and other possible explanations will require more detailed analysis of the cis-acting elements (Mancebo, 2001).

In Drosophila, dorsoventral polarity is established by the asymmetric positioning of the oocyte nucleus. In egg chambers mutant for cap 'n' collar, the oocyte nucleus migrates correctly from a posterior to an anterior-dorsal position, where it remains during stage 9 of oogenesis. However, at the end of stage 9, the nucleus leaves its anterior position and migrates towards the posterior pole. The mislocalization of the nucleus is accompanied by changes in the microtubule network and a failure to maintain Bicoid and Oskar mRNAs at the anterior and posterior poles, respectively. Gurken mRNA associates with the oocyte nucleus in cap 'n' collar mutants and initially the local secretion of Gurken protein activates the Drosophila EGF receptor in the overlying dorsal follicle cells. However, despite the presence of spatially correct Grk signaling during stage 9, eggs laid by cap 'n' collar females lack dorsoventral polarity. cap 'n' collar mutants, therefore, allow for the study of the influence of Grk signal duration on DV patterning in the follicular epithelium (Guichet, 2001).

In cnc mutant egg chambers, nuclear movement occurs normally. The nucleus remains cortically localized even after its posterior displacement. Since interference with components of the dynactin complex leads to the dissociation of the nucleus from the cortex, it is believed that the dynactin complex is not affected by the loss of cnc function. However, the polarization of the microtubule network is aberrant in stage 10A cnc oocytes. Higher numbers of microtubules accumulate in the posterior region of the oocyte at the expense of the anterior cortical ring, which dominates the microtubule network of wild-type stage-9 to -10A egg chambers. This second microtubule reorganization could either be the cause of or result from the late displacement of the nucleus. In the first case, cnc would be required for a process that stabilizes and maintains the microtubule polarity after stage 8. Prolonged signaling from posterior follicle cells might be necessary to suppress the reestablishment of microtubule organizing centers (MTOCs) at the posterior pole. The reception of such a signal or its transmission to the cytoplasm might be impaired in the absence of cnc function. In this model, the reassembly of MTOCs in posterior regions would lead to the redistribution of free tubulin and consequently weaken anterior MTOCs. The nucleus would subsequently migrate towards these ectopic posterior MTOCs. BCD mRNA also would become mislocalized since it is known to move, like the nucleus, towards the minus ends of microtubules, i.e., towards the MTOCs (Guichet, 2001).

In the other scenario, cnc would be required specifically for oocyte nucleus anchoring at the anterior cortex. Anterior anchoring might be necessary since there is a massive influx of cytoplasm from the nurse cells to the anterior pole of the oocyte during egg chamber growth. If the nucleus is not properly anchored, these transport processes might dislodge the nucleus from the anterior pole. Why would this mispositioning of the nucleus lead to the reorganization of the microtubule network? Such microtubule reorganizations have not been described in other mutant backgrounds where the nucleus does not reach the anterior cortex, such as grk, cni, mago, and DLis-1. It has been shown that the nucleus gets encaged by microtubules when it arrives at the anterior pole in wild-type oocytes, indicating that the anteriorly localized nucleus acquires a microtubule-nucleating activity. This activity might remain associated with the mispositioned nucleus in cnc egg chambers and might subsequently cause the increased microtubule density in the posterior half of the cnc oocytes (Guichet, 2001).

In both scenarios, the mislocalization of OSK mRNA remains somehow enigmatic. OSK should not localize to the same region to which BCD is transported. However, normal OSK transport from the anterior to the posterior might just be blocked by the mispositioned nucleus and its associated microtubules. Thus OSK might be trapped in the vicinity of the ectopic nucleus on its way to the posterior pole (Guichet, 2001).

The process of mRNA localization, often used for regulation of gene expression in polarized cells, requires recognition of cis-acting signals by components of the localization machinery. Many known RNA signals are active in the contexts of both the Drosophila ovary and the blastoderm embryo, suggesting a conserved recognition mechanism. Variants of the bicoid mRNA localization signal were used to explore recognition requirements in the embryo. bicoid stem-loop IV/V, which is sufficient for ovarian localization, is necessary but not sufficient for full embryonic localization. RNAs containing bicoid stem-loops III/IV/V localize within the embryo, demonstrating a requirement for dimerization and other activities supplied by stem-loop III. Protein complexes that bind specifically to III/IV/V and to fushi tarazu localization signals copurified through multiple fractionation steps; this suggests that these complexes are related. Binding to these two signals is competitive but not equivalent. Thus, the binding complexes are not identical but appear to have some components in common. A model is proposed for a conserved mechanism of localization signal recognition in multiple contexts (Snee, 2005).

Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins

The swallow (sww) gene encodes a novel protein whose function in oogenesis is not well understood, and the observation that it is required for the localization of two anteriorly positioned RNAs, Bicoid and Hu-li tai shao (hts), provides an opportunity for a comparative study of the role sww plays in RNA localization. Further, the reported differences between HTS and BCD RNA localization raise several questions: To what extent are the sww-mediated localizations of the two RNAs similar or different? Do the localizations of HTS and BCD RNAs share other molecular and biochemical requirements? Are there other RNAs that exhibit a dependence on sww for proper localization in the oocyte (Pokrywka, 2000)?

A detailed characterization of the phenotypes associated with each of eight sww alleles was initiated as a means of investigating the role of sww in oogenic patterning. Several previously unreported RNA localization defects have been observed. Although BCD RNA localization is often lost completely in sww oocytes, in a high proportion of cases, BCD RNA is localized inappropriately along the periphery of the mature oocyte. In several sww mutants, a portion of the BCD mRNA population becomes concentrated at the posterior pole of the oocyte during late oogenesis. Several sww mutations also result in oskar RNA localization defects, consistent with a global role for sww in cytoskeletal regulation or organization. A detailed temporal and spatial analysis of HTS RNA localization in sww mutants and in drug-treated ovaries reveals many similarities to BCD RNA localization, and implies the two independent localization events are accomplished by the same mechanism (Pokrywka, 2000).

Localization of Bicoid messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia, swallow and staufen genes. swa encodes a protein of 548 amino acids that contains a coiled-coil domain and a region with remote similarity to an RNA-recognition motif. SWA mRNA shows an even distribution during oogenesis in the nurse cells as well as the oocyte, with higher levels being found in the nurse cells. Swa protein transiently co-localizes with BCD mRNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of BCD mRNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and BCD mRNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. Dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, has been identified as a Swa-binding protein. It is proposed that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport BCD mRNA along microtubules to their minus ends at the anterior pole of the oocyte (Schnorrer, 2000).

Electron-microscopic studies have revealed a concentration of Exu in discrete subcellular particles, called sponge bodies. These structures consist of endoplasmic-reticulum-like cisterna, embedded in an amorphous electron-dense mass. Swa protein alone is not sufficient to concentrate BCD mRNA anteriorly, since in exu mutants BCD mRNA reaches the oocyte but is not localized correctly, even though the localization of Swa appears normal. Thus, Exu may be involved in preparing or modifying BCD mRNA, thereby allowing the later anterior localization of this RNA while Exu itself is not concentrated anteriorly but is dispersed throughout the oocyte. Although it has not yet been shown directly that BCD mRNA is indeed concentrated in the sponge bodies, the strikingly similar pattern of localization of Exu protein and BCD mRNA in patches in the nurse cells strongly suggests that Exu targets BCD mRNA to the sponge bodies. In the current model, during this Exu-dependent phase of BCD mRNA localization, the RNA assembles, together with unknown components, into ribonucleoprotein particles (RNPs) in the sponge bodies. These RNPs are released into the oocyte. The BCD mRNA localization step in the oocyte crucially depends on Swa. Swa interacts with the BCD mRNA , which is complexed with trans-acting factors, and thus allows the transport of both Swa protein and BCD mRNA by the dynein motor along microtubules to the anterior pole of the oocyte. The Swa-dependent transport does not occur until BCD mRNA has arrived in the oocyte, since Swa cannot be detected in the nurse cells and the transport of BCD mRNA from nurse cells to the oocyte is intact in swa mutants. Binding of Swa to Ddlc-1 appears to be required for the localization of Swa, since a mutant Swa construct that lacks the dynein-binding domain is not transported. However, the binding of Swa to the dynein light chain alone seems not to be sufficient for the localization of Swa, as mutations in the C-terminal part of Swa abolish correct Swa localization but not Ddlc-1 binding. The results of pulldown experiments indicate that the Swa C terminus may contain a regulatory region or recruit a binding partner that is involved in regulating the dynein-dependent transport. One possible additional factor involved in the transport process is the dynactin complex, which has been suggested to be an essential co-factor for dynein (Schnorrer, 2000 and references therein).

Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. A large multiprotein complex has been isolated that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth (Sm), the known localization factor Swallow, and the kinesin family member Nod. Localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization (Arn, 2003).

Promise for understanding RNA recognition in bcd mRNA localization has come from identification of a minimal localization signal directing normal patterns of localization during oogenesis (but not embryogenesis) and lacking at least one form of redundancy. This minimal signal, the IV/V RNA (comprising stem-loops IV and V of the complete signal), can be inactivated by subtle mutations. The availability of such mutants facilitates a biochemical approach to identify recognition factors, since bona fide recognition factors should have reduced affinity for these RNAs. A gel mobility shift assay was used to detect the specific association of two large ovarian protein complexes with IV/V RNA. Notably, both complexes display reduced affinity for inactive IV/V mutant RNAs, making them strong candidates to act in localization. This study characterizes one of these complexes and demonstrates that a complex component not previously implicated in mRNA localization is required in vivo for localization of IV/V RNA, the full bcd localization signal, and an additional anterior-directed message (Arn, 2003).

Three of the identified components of the recognition complex are known RNA binding proteins. Notably, none of these proteins alone displays the observed binding specificity of the complex. PABP binds to the polyA tails of mRNAs, as well as to lower affinity non-A-rich sites. The IV/V RNA used in binding assays is not polyadenylated, and direct binding of PABP to IV/V RNA by crosslinking is not detected. Sm is orthologous to hnRNPL, a member of the hnRNP family of proteins involved in many posttranscriptional gene regulation events through binding to diverse RNA sequences. A highly related protein, Xenopus VgRBP60, has been implicated in mRNA localization in Xenopus oocytes through binding to the Vg1 mRNA localization signal. Sm also fails to crosslink to IV/V RNA. Mod is a multifunctional protein that associates with DNA in chromatin and RNA in the nucleolus (Perrin, 1999). Preferred RNA substrates for binding by Mod have been identified by in vitro selection experiments (Perrin, 1999), but these sequences do not appear within the IV/V RNA. Recombinant Mod binds to IV/V RNA, but this binding lacks the specificity observed in the intact recognition complex. It is possible that the binding specificity of the complex is conferred by a single protein yet to be identified, if this protein interacts with the RNA in a manner not detected by UV crosslinking (Arn, 2003).

An alternative explanation, which the authors favor, is that binding of the recognition complex to IV/V RNA is the sum of multiple interactions, none of which by themselves has the binding affinity and specificity displayed in the gel shift assay. The notion of multiple components in a protein complex making individual contributions to nucleic acid binding affinity is well established. A spectacular example is the ribosome, and recent descriptions of its structure reveal contacts of many proteins with each rRNA (Arn, 2003).

Several types of evidence support or are consistent with this model for recognition of the bcd mRNA localization signal. (1) There is direct evidence suggesting that multiple proteins within the complex bind the IV/V RNA. The competition binding assays reveal a gradual alteration of IV/V complex mobility as increasing amounts of competitor RNA are added, consistent with incremental loss of proteins from the complex. Moreover, at least three known RNA binding proteins are present in the complex (Arn, 2003).

(2) The large size of the bcd mRNA localization signal suggests that it would be bound by multiple proteins. The intact signal is over 600 nt, and the minimal IV/V version used here is still 273 nt. Isolated structural components of IV/V RNA lack localization activity, yet a linker-scanning mutagenesis reveals that most IV/V sequences are nonessential. These seemingly contradictory results can be explained by the following model: multiple binding sites are required, but the consequences of losing only one binding site will be limited to a modest reduction in overall affinity and a partial or variable loss of localization. How can the fact be explained that point mutations in one region of IV/V do abolish localization be explained? One option is that these mutations affect the folding of IV/V, and thus indirectly alter the binding sites for multiple proteins rather than altering the sequence of a single binding site (Arn, 2003).

(3) Although mutations in exu, swa, and stau, all implicated in regulating bic localization, in fact disrupt localization of bcd mRNA, none has the phenotype expected for a failure of recognition: a complete absence of localization. All of these mutants retain early localization to the oocyte, as well as some degree of anterior localization. There are other reasonable explanations for the absence of mutants of this class: genetic screens may not have been saturating, or mutants may exist that have defects in early development masking later roles in the ovary (as the early lethality of a mod mutant obscures its role in oogenesis). However, it is quite possible that no single gene is absolutely essential for recognition of the bcd localization signal, as would be the case if recognition is the result of summing many interactions. Mutants which only reduce the efficiency or consistency of bcd mRNA localization will produce some viable progeny, and are unlikely to be detected in screens for maternal-effect lethal or female sterile mutants. The mRNA localization defects of mod mutant clones fit well with this model. Significant disruptions of localization are observed, but these are variable and not every oocyte is equally affected. The mod minus localization phenotype may be among the most severe possible, since it is the sole complex component detected to bind IV/V by crosslinking, and it is the most abundant identified complex component (Arn, 2003).

A model of recognition such as is described for the bcd localization signal is consistent with data regarding cis-acting signals from a variety of localized transcripts. A common paradigm for localized mRNAs, such as bcd, nos, and osk in flies, and Vg1 in Xenopus, is a large localization signal that cannot be significantly reduced in size without loss of activity, but can sustain internal deletions. Shorter sequences from within the bcd, nos, and Vg1 signals display no localization activity in a single copy, yet gain in vivo activity when multimerized. This behavior is consistent with the idea of recognition involving the summing of multiple low-affinity and/or low-specificity interactions, and may indicate a conserved mechanism for the recognition of many localization signals (Arn, 2003).

This study characterizes one bcd mRNA recognition complex, but this complex is unlikely to be the only one. In partially purified ovary extracts, a second complex is detected with similar binding specificity, and previous work has shown that at least two different RNA recognition events act in localization of bcd mRNA. Different recognition complexes could act redundantly or could be responsible for different phases of localization. One recognition complex might link the mRNA to transport machinery for movement, while other complexes might tether mRNAs to anchors at intermediate or final destinations. The aspect of movement or anchoring that is associated with the complex characterized in this study remains uncertain. Swa protein is highly concentrated at the anterior of the oocyte, and PABP is generally dispersed in the ooplasm but transiently enriched at the anterior cortex; these distributions might indicate a role in anchoring. However, Mod and Nod appear nuclear by immunolocalization, have known roles in nuclei, and are not concentrated at sites of bcd mRNA accumulation, although a fraction of Mod protein is cytoplasmic. This would favor an interaction necessary for an active transport step. Both Mod and Nod proteins appear to be members of a growing class of shuttling proteins affecting gene expression both through nuclear association with DNA and by cytoplasmic association with RNA (Arn, 2003).

Differences in overall steady-state localization for proteins from the same complex are not problematic, because the amount of each protein present in the recognition complex appears to be small relative to the total cellular pool. Moreover, it would not be surprising to find that individual components are shared between recognition complexes that act sequentially, since persistent association of a subset of the proteins would help ensure continuity in the localization process (Arn, 2003).

Roles for microtubules have been demonstrated in at least three different types of movement during localization of bcd mRNA. Early in oogenesis, long microtubules appear to provide tracks for minus end-directed movement of mRNAs from nurse cells into the oocyte. Later, these microtubules are dispersed, and bcd mRNA can be seen to move along short microtubules within the nurse cells. Finally, there are microtubule-dependent movements of bcd mRNA within the oocyte, and these movements are required for anterior localization. As yet, no motor has been definitively assigned to any of these movements (Arn, 2003).

An intriguing feature of the recognition complex is the presence of Nod, a member of the family of kinesin-like proteins which direct movement along microtubules. A fusion protein containing the presumptive motor domain of Nod and a part of kinesin heavy chain becomes localized to the minus ends of microtubules and has been used for marking microtubule polarity in Drosophila cells. The behavior of this fusion protein has led to the view that Nod has minus end-directed motor activity. However, the distribution of endogenous Nod is at odds with this interpretation. Recent studies on the biochemical properties of recombinant Nod argue that the protein lacks canonical motor activity and may function to transiently or stably crosslink nucleic acid complexes to microtubules. Whatever its precise role, the presence of Nod in a IV/V RNA binding complex supports a role for this complex in microtubule-based localization steps (Arn, 2003).

Bicoid mRNA localization: the role of the cytoskeleton

Anterior patterning of the Drosophila embryo depends on localization of Bicoid mRNA to the anterior pole of the developing oocyte: BCD mRNA localization requires both the exuperantia (exu) gene and an intact microtubule cytoskeleton. A GFP-Exu fusion protein supports BCD mRNA localization and complements the embryonic anterior axis defects produced by exu mutations. During mid-oogenesis, the GFP-Exu fusion protein assembles into particles that are concentrated around the nurse cell nuclei; these particles cluster at the ring canals that link the germline cells of the egg chamber, and accumulate at the anterior pole of the oocyte. At these stages, BCD mRNA also shows a perinuclear or apical distribution in nurse cells, and accumulates at the anterior cortex of the oocyte. These observations suggest that the GFP-Exu particles are transport ribonuclear proteins containing BCD mRNA, and that these particles are targeted to the anterior cortex of the developing oocyte. Although direct proof for an association of BCD mRNA with these particles has not yet been obtained, microtubule depolymerization disrupts both BCD mRNA and Exu protein localization, suggesting that these two components utilize a similar, if not identical, anterior localization pathway (Theurkauf, 1998 and references).

To gain insight into the mechanism of anterior patterning, time lapse laser scanning confocal microscopy was used to analyze transport of particles containing a Green Fluorescent Protein-Exu fusion (GFP-Exu), and to directly image microtubule organization in vivo. These observations indicate that microtubules are required for three forms of particle movement within the nurse cells, while transport through the ring canals linking the nurse cells and oocyte appears to be independent of both microtubules and actin filaments. As particles enter the oocyte, a final microtubule-dependent step directs movement to the oocyte cortex. Exu protein and BCD mRNA are synthesized in a cluster of 15 nurse cells that are linked to the oocyte by ring canal bridges. The first cytoplasmic transport steps in anterior patterning therefore take place within the nurse cells. The analysis indicates that transport within the nurse cell cytoplasm is composed of at least three microtubule-dependent steps that produce a net movement of GFP-Exu particles toward the oocyte. The majority of the individual GFP-Exu particles within the nurse cell cytoplasm move rapidly and with no apparent net directionality with respect to the egg chamber axis. These movements are reversibly inhibited by the microtubule-disrupting drug colcemid. Microtubules throughout the nurse cell cytoplasm that lack clear orientation with respect to the egg chamber axis have been directly observed. Based on these observations, it is concluded that microtubules mediate random particle movements within the nurse cells. In the absence of microtubules, no movement or redistribution of GFP-Exu particles was observed. Simple diffusion thus appears to be insufficient to efficiently disperse these large particles. It is therefore speculated that the random microtubule-dependent particle movements are essential to dispersing GFP-Exu particles. It is proposed that this particle dispersal is required for efficient net particle transport through the nurse cells (Theurkauf, 1998).

Vectorial particle transport is observed in the region near the ring canals linking the nurse cells with the oocyte. In this region, particles tend to move directly to the cell-cell junctions. These movements, like the random movements observed in bulk nurse cell cytoplasm, are reversibly inhibited by colcemid. In addition, a dynamic population of microtubules associated with the nurse cell-oocyte ring canals is directly observed. It is therefore proposed that GFP-Exu particles approach the ring canal junctions by a microtubule-dependent random walk. The particles then associate with microtubules that are organized around the ring canals, and are transported to the nurse cell-oocyte junctions. It is this second step that imparts net directionality on particle transport through the nurse cell cytoplasm (Theurkauf, 1998).

Previous ultrastructural analysis of Exu distribution failed to identify microtubules in direct association with Exu-containing structures, termed sponge bodies. However, the current in vivo analysis indicates that at least some of the microtubules in the nurse cells turn over within 10 to 20 seconds. These microtubules are therefore likely to be difficult to preserve by standard fixation procedures. The failure to identify microtubules directly associated with sponge bodies may reflect the dynamic nature of these filaments. Perinuclear particle clustering in the nurse cells also appears to be microtubule-dependent. This process is reversibly disrupted by colcemid, and microtubules are associated with the surface of the nurse cell nuclei. The function of microtubule-dependent perinuclear clustering is not yet clear, although it seems unlikely that this process contributes directly to movement through the nurse cells. It has been proposed that Exu particles are RNPs that contain BCD mRNA, as well as other proteins. If so, these particles could form in the perinuclear region as BCD mRNA exits the nurse cell nuclei, and microtubule-dependent transport could facilitate complex formation by concentrating cytoplasmic components of the particles in this region. Consistent with this suggestion, GFP-Exu particle size is decreased by microtubule depolymerization, and particles appear to increase in size and fluorescence intensity on microtubule repolymerization (Theurkauf, 1998).

Essentially all of the GFP-Exu particle movements in the nurse cell cytoplasm are microtubule-dependent: these movements are presumably mediated by microtubule motor proteins. It is speculated that several different microtubule motors function in Exu particle motility. Alternatively, the variability in the rates and directionality of particle movements in the nurse cells could reflect complexities in the underlying microtubule cytoskeleton or particle-specific differences in the regulation of a single motor. However, the use of multiple motors for this transport process would serve to isolate axis specification from complete disruption by mutations in single motor protein genes. Mutations in known motor proteins have not yet been identified that disrupt these transport steps. Once GFP-Exu particles are localized to the nurse cell-oocyte ring canals, a distinct transport process appears to drive movement through the cell-cell junctions. These movements are uniform in direction and velocity, raising the possibility that they reflect a very local flow of cytoplasm through the ring canal junctions. The apparent absence of bulk movement through the ring canal suggests that this step in the transport pathway is not due to cytoplasmic flow, but reflects the action of a more selective mechanism. The best characterized specific transport processes require microtubules or actin filaments, yet movement through the ring canals is relatively insensitive to microtubule and actin assembly inhibitors. These observations raise the possibility that this transport step is independent of both actin filaments and microtubules. However, cytochalasin D and colcemid only affect dynamic filaments that are in equilibrium with subunits in the cytoplasm. It is therefore possible that stable actin filaments or microtubules mediate transport through the ring canals. The inhibitor data reported here, combined with previously published data, suggest that GFP-Exu transport through the nurse cell-oocyte ring canals is independent of both microtubules and actin filaments (Theurkauf, 1998).

This suggests a multi-step model for transport and anterior localization of Exu during stages 9 and 10 of oogenesis. It is speculated that Exu protein assembles into particles within the nurse cell cytoplasm, perhaps at the nuclear periphery, where these particles are localized by a microtubule-dependent process. Particles dissociate from the perinuclear regions, and random microtubule-dependent movements then distribute these large particles throughout the nurse cell cytoplasm. As particles approach the posterior of the nurse cell, they interact with microtubules originating near the nurse cell-oocyte ring canals, and are transported to the cell-cell junctions along these microtubules. Particles are then transported through the ring canals in a second vectorial process that appears to be independent of both actin filaments and microtubules. In the final transport step, particles entering the oocyte interact with microtubules originating at the anterior cortex of the oocyte, and are localized to the anterior in a microtubule-dependent step. At the cortex, particle may associate with asymmetrically localized binding sites that stabilize the asymmetric distribution. These observations and previous studies suggest that the polarity of the oocyte microtubule network is not in itself sufficient to generate anterior asymmetry, and that additional factors are required to restrict morphogens to the anterior pole. Based on these observations, a multi-step anterior localization pathway is proposed (Theurkauf, 1998).

Several observations indicate that factors in addition to egg chamber geometry and microtubule-dependent transport play a role in anterior axis specification. For example, BCD mRNA that is ectopically localized to the posterior of mago nashi and PKA mutant oocytes is dispersed as ooplasmic streaming begins at stage 10b, while the anteriorly localized transcripts in these oocytes are stable in spite of cytoplasmic streaming. Stable association of BCD mRNA with the cortex thus appears to be restricted to the anterior pole. In addition, several mRNAs are localized with BCD to the oocyte anterior during stages 9 and 10 of wild-type oogenesis. However, unlike BCD mRNA, these transcripts are dispersed upon ooplasmic streaming at stage 10b. These observations indicate that anterior transcript binding sites are specific for BCD mRNA, or a complex containing this mRNA. It has been suggested that localization of BCD mRNA depends on both microtubule-dependent movement to the cortex and transcript stabilization by a microtubule-independent mechanism. In a modified model for BCD mRNA transport, BCD mRNA binding activity is restricted to the anterior cortex, where it mediates pole-specific stabilization of transcript accumulation (Theurkauf, 1998 and references).

A mutation in a novel gene, capulet (cap), was identified in a mosaic screen to isolate mutations that perturb actin organization in germline clones. Adenylate cyclase-associated proteins (CAPs) have been shown to inhibit actin polymerization in vitro, by sequestering monomeric actin. This actin-binding activity has been mapped to the carboxy-terminal region of CAP; however, a 'verprolin homology'-related domain has been identified in all CAPs, just carboxy-terminal of the polyproline-rich domain. In members of the verprolin/WASP family, this motif binds actin monomers in vitro, but catalyses actin polymerization in vivo. Therefore, in CAP homologues, this region of the protein may be used to facilitate actin binding. As CAP proteins have also been found associated with Abl tyrosine kinase and with adenylate cyclase, it is possible that CAP represents an intermediary in these signal transduction cascades, perhaps altering actin dynamics in response to extracellular cues (Baum, 2000).

The genetic screen also identified a mutation in the catalytic subunit of protein kinase A (PKA). Therefore, pka and cap mutant phenotypes in the Drosophila germline were compared. Like the cap mutant, pka germline clones lose nurse cell cortical actin, while simultaneously accumulating ectopic actin structures. In addition, the pka mutant phenotype is sensitive to the dosage of CAP, and actin defects are dramatically enhanced in pka;cap double germline clones. These data suggest that PKA and CAP functionally cooperate in the germline to control actin organization (Baum, 2000).

In cap germline clones, F-actin accumulates in a highly polarized fashion within the egg chamber and oocyte. Thus, whether loss of CAP perturbs other aspects of normal polarity, including the asymmetric localization of mRNAs within the oocyte was investigated. The distributions of bicoid and oskar mRNAs, which localize to anterior and posterior poles of the oocyte, respectively, were examined. Although oskar mRNA is concentrated in one region of the oocyte in over 90% of egg chambers, oskar mRNA is mislocalized in 76% of stage 8-10 cap germline clone egg chambers. Moreover, in 28% of cases, oskar transcripts are localized to the anterior or lateral part of the oocyte. In addition, in 64% of stage-10 egg chambers that maintain correct overall polarity, oskar mRNA has a diffuse distribution and is not tightly focused at the posterior pole. The localization of bicoid transcripts was also examined. bicoid mRNA accumulates at an aberrant site in 65% of cap mutant egg chambers, and is localized to the posterior pole in 36% of stage 8-10 egg chambers. Thus, cap germline clones display two related mRNA polarity defects: (1) although oocytes are able to concentrate oskar and bicoid mRNAs locally within the oocyte, they appear unable to coordinate mRNA polarity with the morphological polarity of the egg chamber; (2) in the majority of egg chambers in which oskar mRNA is correctly transported to the posterior pole of the oocyte, oskar message is not tightly localized at the cortex (Baum, 2000).

It can be concluded that CAP is a major regulator of actin dynamics in Drosophila, and that CAP is likely to function to inhibit actin polymerization in vivo, as it does in vitro. A striking feature of the cap phenotype is the accumulation of actin filaments at polar sites within the egg chamber. This cannot be explained by differences in the monomeric actin pool in nurse cells versus the oocyte, as G-actin, as measured by DNaseI staining, is equally distributed within the egg chamber, as is profilin. Thus, CAP inhibits actin filament formation at specific cellular sites, possibly in response to signaling events (Baum, 2000).

In both yeast and multicellular eukaryotes, the actin cytoskeleton responds to cell signaling events. Therefore it is interesting to note that homologs of Drosophila CAP have been shown to interact physically with an Abl tyrosine kinase and adenylate cyclase. These latter proteins transduce extracellular cues, in a way that is not fully understood, to remodel the actin cytoskeleton within the growth cones of migrating neurons to facilitate axon guidance. Thus, CAP may constitute part of the machinery that reorganizes the actin cytoskeleton in response to these signals in neurons and in other polarized cells. Interestingly, the genetic screen also identified the catalytic subunit of protein kinase A (PKA), which acts downstream of adenylate cyclase, as a gene required for proper actin organization and oocyte polarity. Since yeast, Hydra and human CAPs have been shown to facilitate the activation of adenylate cyclase, CAP and PKA may be elements of a conserved signal transduction pathway. The phenotypic similarities shared by cap and pka germline clones suggest that CAP and PKA act together in the Drosophila female germline. Given this interaction, CAP could be a substrate for PKA, or could facilitate the activation of adenylate cyclase upstream of PKA. Alternatively, because a reduction in both CAP and PKA activity leads to a more severe phenotype, the two genes may act in parallel pathways. CAP and PKA are, however, unlikely to be essential components in a common signal transduction pathway in Drosophila because no evidence is found for related CAP and PKA functions in somatic tissues (Baum, 2000).

In existing mutants known to perturb the germline actin cytoskeleton, oocyte polarity is either unaffected or completely disrupted. Therefore, whether oocyte polarity is altered in the cap mutant was investigated by examining the localization of both bicoid and oskar mRNAs. When compared to other known mutants, cap germline clones exhibit novel mRNA polarity defects (although similar defects are exhibited by pka null germline clones). First, cap mutant oocytes are able to localize mRNAs to discrete areas within the oocyte, but the sites of mRNA deposition do not respect the existing morphological axes of the egg chamber. Second, in the majority of stage-10 egg chambers with the correct polarity, oskar mRNA is observed in a shallow gradient, as if diffusing away from the cortex at the posterior pole. Thus, CAP seems to be required, both to coordinate mRNA localization with the axial polarity of the egg chamber, and to tether mRNAs to the cortex. Because microtubules are thought to mediate the transport of mRNAs to opposite poles of the oocyte in the wild type, the defect in oocyte axial polarity in the cap mutant may result from defects in the underlying microtubule cytoskeleton. cap germline clones frequently contain a misoriented microtubule array, with plus ends focused at the anterior cortex. This altered microtubule polarity is therefore probably responsible for the mislocalization of oskar and bicoid mRNAs at early stages of oogenesis. At later stages, following disassembly of the polar microtubule array, an actin-based structure at the posterior pole of the Drosophila oocyte, dependent on CAP and tropomyosin, may act as a tether to hold oskar mRNA at the cortex (Baum, 2000).

There are two g-tubulin genes in Drosophila. The gTubulin23C isoform is essentially ubiquitous and is required for viability and microtubule organization during mitosis and male meiosis. In contrast, the expression of gTubulin37C (gTub37C) is restricted to ovaries and early embryos. Drosophila females homozygous for mutations in the gTub37C gene display abnormal meiotic spindles and the embryos derived from them have nuclear proliferation defects. The meiotic figures observed in females homozygous for lack-of-function alleles of gTub37C lack the bilateral symmetry and linear arrangement of the chromatin masses which characterize wild-type female meiotic figures during metaphase-I. The meiotic spindle is also severely disrupted in these mutants. gTub37C is also essential for nuclear proliferation in the early Drosophila embryo. The eggs produced by mutant mothers show an arrest of nuclear divisions during early embryogenesis because both microtubule polymerization and (as a consequence) spindle formation are blocked (Schnorrer, 2002).

gTub37C and g-tubulin ring complex protein 75 are essential for bicoid RNA localization during Drosophila oogenesis. bicoid mRNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, two novel genes essential for bcd RNA localization were identified. They encode maternal g-Tubulin37C and g-tubulin ring complex protein 75 (Grip75), both of which are g-tubulin ring complex components. Mutations in these genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the g-tubulin ring complex is essential for localization of bcd RNA. At stage 10b, gTub37C and Grip75 are found anteriorly concentrated; the formation of a microtubule-organizing center at the anterior pole of the oocyte is proposed (Schnorrer, 2002).

The screening procedure for novel genes essential for bcd RNA localization has identified mutations in gTub37C and Grip75, which affect bcd RNA localization at late stage 10b of oogenesis. These mutants are cytoskeletal factors that are essential for bcd RNA localization. They are both components of the same molecular complex, the g-tubulin ring complex, and show the same bcd RNA mislocalization and early embryonic arrest phenotype. In the early embryo, the g-tubulin ring complex is concentrated at the centrosomes, which organize the spindle microtubules. Other mutants in maternally expressed centrosomal proteins, which cause nuclear division problems during early embryogenesis, do not result in an aberrant bcd RNA distribution. centrosomin (cnn) or abnormal spindle (asp) mutants were tested and no difference was detected in bcd RNA localization. This demonstrates that the screening procedure is stringent enough to identify specific factors, and, since it covered only the left arm of the second chromosome, it is likely that more genes with a function in bcd RNA localization exist in the genome (Schnorrer, 2002).

gTub37C and Grip75 mutants are required for microtubule assembly during embryogenesis and, therefore, are necessary for starting the nuclear divisions (Llamazares, 1999; Tavosanis, 1997). However, both mutants are viable and, hence, not essential for the microtubule-dependent processes during later embryonic and larval development. For gTub37C this is less surprising because it is only expressed maternally and a second g-tubulin, gTub23C, fulfils zygotic functions. In contrast, Drosophila has no second paralog of Grip75 in the genome. Consequently, Grip75 plays an essential role only in certain microtubule-dependent processes, which appear to be those that also require gTub37C. However, both Grip75 alleles are sterile not only in females, but also in males, a phenotype described for mutants in centrosomin, but not in gTub37C or swa (Schnorrer, 2002).

gTub37C and Grip75 mutants display a specific loss of microtubule function during oogenesis. Several microtubule-dependent processes, such as oocyte specification, nuclear migration, and osk and bcd RNA transport at stage 9, are functional. bcd RNA mislocalization starts at late stage 10b. This phenotype can be explained in two ways. Either gTub37C and Grip75 affect all microtubules of the oocyte at a specific time point or they eliminate the function of only a subset of microtubules, while others are unaffected. The latter explanation, which proposes specialized microtubules, is supported by the fact that a maternal g-tubulin exists that has a relatively divergent primary sequence and comprises about 20% of the g-tubulin pool in oocytes. Furthermore, tubulin modifications such as acetylation or polyglycylation may distinguish certain microtubules from others. Attempts were made to address this problem by analyzing microtubule-dependent transport to the posterior pole. Posterior transport is normal in gTub37C mutants; however, it is possible that this transport is already completed at stage 10b, since Kin:ß-gal is no longer concentrated at the posterior at late stage 10b. Hence, posterior transport might not take place anymore and does not allow the two possibilities proposed above to be distinguished. Therefore, ooplasmic streaming, a microtubule-dependent process that occurs at the same time as the bcd RNA mislocalization in gTub37C, Grip75, and swa mutants, was analyzed. The fact that ooplasmic streaming is unaffected in all the mutants suggests that only a subset of microtubules, which are required for bcd RNA localization from stage 10b onward, but not for ooplasmic streaming, are affected. This conclusion is directly supported by the g-tubulin analysis in gTub37C and Grip75 mutants, which shows the presence of microtubules in these mutant oocytes at stage 10b (Schnorrer, 2002).

So far, no microtubule-organizing center (MTOC) has been described at the anterior pole at any stage of oogenesis that would allow directed microtubule-dependent transport to the anterior pole and demonstrate the polarity of the microtubule network. The g-tubulin ring complex is capable of nucleating microtubules in a controlled manner and, in addition, can act as a cap that stabilizes their minus ends. gTub37C and Grip75 are essential for these functions of the ring complex during embryogenesis and, most likely, also during oogenesis. Since Grip75GFP is a functional component of the gTuRC in the embryo and stably binds to gTub37C during oogenesis, the colocalization of Grip75GFP and gTub37C at the anterior pole of a stage 10b wild-type oocyte suggests that Grip75 and gTub37C form a distinct microtubule organizer at the anterior pole at stage 10b. Importantly, grk mutants show an ectopic posterior gTub37C focus, consistent with the model of microtubule minus ends at both poles in grk oocytes. Furthermore, this posterior gTub37C focus supports the idea that the gTub37C focus at the anterior pole in wild-type oocytes is not simply a consequence of dumping the nurse cell cytoplasm into the oocyte, but a distinct MTOC. This is further supported by the anterior enrichment of gTub37C in dumpless mutants. The proposed anterior MTOC might have a similar molecular composition as the gTuRC during embryogenesis and interacts with Swa protein. The disrupted bcd RNA and Nod:ß-gal localization in Grip75 and gTub37C mutants shows the functional significance of the proposed MTOC (Schnorrer, 2002).

The formation of a g-tubulin ring complex-based MTOC in the middle of the anterior pole at stage 10b may explain the 'ring to disc' transition of the localization pattern of bcd RNA and Nod:ß-gal and is consistent with the anterior colocalization of gTub37C and g-tubulin. However, g-tubulin is not restricted to the anterior cortex at stages 10b–11 but extends along the whole oocyte cortex. It is likely that the g-TuRC functions as a template to nucleate certain microtubules at the anterior pole in a controlled direction (Schnorrer, 2002).

An interesting aspect of the gTub37C and Grip75 mutant phenotypes is that they uncouple Swa and bcd RNA localization. Since the defect in bcd RNA localization starts slightly later in gTub37C and Grip75 than in swa mutants, it was expected that Swa would be localized, which is the case. But the colocalization of Swa and bcd RNA is lost at late stage 10b, arguing against the hypothesis that Swa binds directly to bcd RNA in order to transport it to, or anchor it at, the anterior pole. Furthermore, it is unlikely that Swa itself acts as molecular anchor to trap bcd RNA, which is imported from the nurse cells. The situation at the anterior pole appears to be more complicated, and the following model is proposed (Schnorrer, 2002).

In a first phase lasting until stage 10a, bcd RNA is localized in an exu- and microtubule-dependent process to the anterior pole into a ring-shaped pattern. This requires neither gTub37C and Grip75 nor swa to function. In a second phase, a change in microtubule organization occurs, microtubules are mainly assembled subcortically, and transport to the posterior pole stops. Swa protein localization starts at the entire anterior cortex of the oocyte, not only in its corners, possibly in a dynein-dependent manner. The bcd RNA localization pattern changes into a disc or a cap-like pattern at the anterior pole. This is likely to be an active process, which initially does not depend on gTub37C and Grip75 but does depend on swa. Swa might use gTub23C and other Grips in order to partially reorganize the microtubule cytoskeleton. In a third phase, starting at late stage 10b, gTub37C and Grip75 are essential to keep bcd RNA anteriorly and to complete the ring- to disc-shape transition. During this time the localized amount of bcd RNA increases continuously. Since Grip75GFP and gTub37C are enriched at the anterior pole from stage 10b onward, it was proposed that an MTOC, which might organize a subset of microtubules with distinct polarity, is established there at this time. In gTub37C and Grip75 mutants, this MTOC is disrupted and bcd RNA diffuses into the oocyte. This is presumably promoted by large amounts of nurse cell cytoplasm entering the oocyte through the ring canals at the anterior pole. Therefore, bcd RNA either requires a stable anchor or continuous transport back to the anterior pole during phases two and three. The hypothesis of continuous transport along microtubules is favored, considering the molecular nature of gTub37C and Grip75 (Schnorrer, 2002).

In contrast to bcd RNA, Swa stays localized at the anterior cortex in gTub37C and Grip75 mutants. This argues that Swa itself is less sensitive to microtubule disruption than bcd RNA and might be anchored at the anterior cortex after its initial localization. The localized amount of Swa does not increase further after stage 10b. Swa binds to the g-TuRC and might regulate microtubule turnover or dynein motor recycling. This binding is lost in swa mutants, demonstrating that the C terminus of Swa is required for the interaction with the g-TuRC. The possibility that Swa itself has the capacity to change properties or location of a MTOC remains to be tested. In the future, proteins like Exu and Swa, which seem to have specialized functions for only a few processes and are poorly conserved during evolution, might be possible to integrate into the cellular machinery required for asymmetric localization of determinants. This machinery requires a variety of conserved cytoskeletal components, which can be used for mRNA transport at different developmental stages, such as Drosophila oogenesis and embryogenesis (Schnorrer, 2002).

In the Drosophila oocyte, microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. mRNA localization and nuclear positioning at mid-oogenesis is shown to depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. It is concluded that the microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole (Januschke, 2002).

The localization of bcd mRNA in the oocyte occurs in multiple steps. Several of these involve active transport along microtubules. bcd mRNA coassembles into particles with Exuperantia (Exu) in the nurse cells and in the oocyte. This complex is essential for the correct localization of bcd to the anterior cortex in a microtubule-dependent manner. During mid-oogenesis, bcd maintenance at the anterior cortex is dependent on Swallow (Swa). This protein harbors a putative double-strand-RNA binding motif and a coiled-coil domain, which interacts with the Dynein light chain (Dlc-1). Swa has been proposed to act as an adaptor between bcd mRNA and the Dynein motor. Swa itself localizes to the anterior cortex of stage-10 oocytes, and this localization requires the coiled-coil domain, suggesting that polar transport of Swa and its cargo, bcd mRNA, occurs in a Dynein-dependent manner. The observation that bcd mRNA is delocalized in oocytes overexpressing p50 Dynamitin (Dmn), a component of the Dynactin complex provides further support for this suggestion (Januschke, 2002).

Surprisingly, in khc mutant oocytes, bcd mRNA is not tightly concentrated to the anterior cortex but is diffusely spread out in a wide cortical ring that expands toward the posterior. Thus, correct bcd localization depends not only on minus end-directed, but also on plus end-directed, motors. Kinesin I might be directly involved in anchoring of bcd mRNA to the anterior cortex. Alternatively, Kinesin I might be required for efficient Dynein-dependent transport of bcd. The observation that Dynein is mislocalized in khc mutant oocytes supports the latter hypothesis. Dhc fails to accumulate at the posterior pole of khc mutant oocytes and instead is enriched at the anterior cortex. Thus, Kinesin I appears to be necessary to relocate Dynein to the posterior pole after it has moved together with its cargo to the anterior pole. This would allow for renewed rounds of cargo loading and transport to the anterior cortex. Without sustained posterior-to-anterior transport, the bcd mRNA/adaptor complexes might become delocalized by diffusion. This scenario indicates that sustained transport could be an alternative to an independent anchorage step (Januschke, 2002).

Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia

The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals. During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid. To elucidate the molecular pathways triggered by Par-1, a genome-wide, high-throughput screen for Par-1 targets was carried out. Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation. Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo. Two motifs were identifed in Exu that are phosphorylated by Par-1; their mutation abolishes bicoid mRNA localisation during mid-oogenesis. Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis. These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation. Furthermore, this analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent (Riechmann, 2004).

Par-1 has two distinct functions in bicoid mRNA localisation. Par-1 is necessary for the release of bicoid mRNA from the oocyte cortex. Genetic epistasis experiments indicate that the exu independent function of par-1 acts at a step upstream of exu in bicoid mRNA localisation. By generating mutants that abolish Exu phosphorylation, two phases of Exu dependent bicoid mRNA localisation could be further distinguished; an early phase, in which bicoid mRNA localisation is abolished when Exu is unphosphorylated and a late phase, in which the requirement for Exu phosphorylation is less stringent. Thus, these results show that bicoid mRNA localisation is a multi-step process, and that redundant mechanisms are used to ensure the anterior accumulation of bicoid mRNA (Riechmann, 2004).

Exu protein is an essential mediator of bicoid mRNA localisation. Par-1 kinase phosphorylates Exu, and this phosphorylation is necessary for anterior localisation of bicoid mRNA during mid-oogenesis. Exu phosphorylation does not affect Exu localisation, its ability to form mobile particles, or its colocalisation with bicoid mRNA. How then might Par-1 phosphorylation enable Exu to mediate bicoid mRNA localisation? Experiments in which fluorescently labelled bicoid mRNA was microinjected into living egg chambers have revealed that Exu is required in the nurse cells for anterior localisation of bicoid mRNA within the oocyte. These experiments have led to a model whereby Exu associates in the nurse cells with bicoid mRNA and mediates the recruitment of additional nurse cell factors required for targeting of bicoid mRNA to the anterior of the oocyte. The finding that mutation of Exu phosphorylation sites results in a phenotype that is, during mid-oogenesis, indistinguishable from that of exu-null mutants suggests that Exu phosphorylation is involved in the recruitment of these anterior-targeting factors in the nurse cells. Phosphorylation might increase the binding affinity of Exu for these nurse cell factors, promoting their association with bicoid mRNA. The colocalisation of Exu-GFP, Par-1 and bicoid mRNA in patches in the nurse cells suggests that this is where the bicoid RNP complexes assemble (Riechmann, 2004).

The consequences of exu and par-1 mutations on bicoid mRNA localisation are distinct. Although loss of exu function results in diffuse bicoid mRNA distribution in the ooplasm, a reduction in par-1 function causes cortical localisation of the mRNA. An Exu protein has been generated that localises bicoid mRNA independent of phosphorylation by Par-1 and rescues exu mutants, but that is unable to rescue bicoid mRNA localisation in par-1 mutants. Therefore, the cortical mislocalisation of bicoid mRNA in par-1 mutant oocytes is independent of Exu function. What might be the other function of Par-1 in localisation of bicoid mRNA? The fact that bicoid localisation requires the microtubule cytoskeleton, together with the report that oocyte microtubules are improperly polarised in par-1 mutants, suggests that cortical localisation in the mutants is caused by a microtubule defect. It has been proposed that microtubules of different qualities may nucleate from different regions of the oocyte cortex. A simple explanation for the aberrant localisation of bicoid mRNA in par-1 oocytes would be that the subset of microtubules nucleating from the anterior corners of the oocyte and serving as tracks for anterior transport of bicoid mRNA are not restricted to the anterior corners, but spread along the cortex, resulting in the lateral cortical localisation of bicoid mRNA. However, this model is not supported by the genetic epistasis experiments, which indicate that the exu independent function of par-1 acts at a step upstream of exu in bicoid mRNA localisation. Therefore, a different model is favored, in which in wild-type oocytes bicoid mRNA first localises cortically preceding its targeted transport along microtubules. In this model, most of the bicoid mRNA entering the oocyte moves in a nonpolar fashion, either passively or by active transport, to the oocyte cortex. Only after this cortical localisation does the targeted transport of bicoid mRNA to the anterior corners of the oocyte commence. In par-1 mutants, the improperly organised microtubule cytoskeleton prevents release of the mRNA from the cortex to the (anterior-targeting) microtubules and the mRNA remains cortically localised. In exu mutants, the polarity of the microtubules is normal and bicoid mRNA is released from the cortex. However, its targeted transport to the anterior is impaired and the mRNA is diffusely distributed in the ooplasm (Riechmann, 2004).

The requirement for Exu phosphorylation in bicoid mRNA localisation decreases during the later stages of oogenesis. This is revealed by the partial recovery of bicoid mRNA localisation in exu mutants that abolish phosphorylation. These mutants are indistinguishable from exu-null mutants through stage 10b of oogenesis, but during early embryogenesis two-thirds of the mutants localise enough bicoid mRNA at the anterior to support formation of a Bicoid protein. This indicates that the mechanism of bicoid mRNA localisation changes after stage 10b of oogenesis, from an early phase that is strictly dependent on Exu phosphorylation, to a late phase that is less dependent on phosphorylation. Stage 10b is the stage at which ooplasmic streaming commences, providing a possible mechanism for localisation of bicoid mRNA in mutants in which Exu phosphorylation cannot occur. Before stage 10b, anterior targeting of bicoid mRNA could be mediated solely by directed transport of bicoid mRNA complexes along microtubules, a process that is strictly dependent on Exu phosphorylation. After stage 10b, this directed transport might be complemented or replaced by a passive trapping mechanism, which has also been postulated for the localisation of oskar and nanos mRNAs during late oogenesis. This mechanism relies on the movements generated by ooplasmic streaming, which could bring bicoid mRNA complexes into contact with the anterior cortex of the oocyte, where the mRNA could be trapped by localised anchoring molecules. This change in the mechanism of bicoid mRNA localisation would occur at the time of assembly of the anterior MTOC that is essential in the late phase of bicoid mRNA localisation, suggesting that the MTOC might be involved in the trapping mechanism. Such a trapping mechanism would be differentially affected in Exu-null mutants and in mutants that specifically abolish Exu phosphorylation. It is possible that Exu provides bicoid mRNA not only with factors required for anterior targeting, but also with factors required for anchoring of bicoid mRNA. Unphosphorylated Exu might be inactive in recruiting the factors for anterior targeting, but be competent for binding of factors required for anchoring (Riechmann, 2004).

It is proposed that in the first phase of bicoid mRNA localisation, the mRNA is transported to the anterior corners of the oocyte, resulting in a ring-like distribution. This targeted transport requires the formation of RNP complexes that contain bicoid mRNA and specific anterior-targeting factors that allow the RNPs to identify those microtubules that nucleate from the anterior corners of the oocyte. Assembly of this complex takes place in the nurse cells and requires the phosphorylation of Exu by Par-1. Upon entry of the complex into the oocyte, a specific proportion of the RNP complexes encounter the microtubules that nucleate from the anterior corners, and these complexes are directly transported to their final destination. However, a large proportion of the complexes does not find these microtubules directly, and moves first to the oocyte cortex. The transfer of these cortically localised complexes to microtubules nucleating from the anterior corners solely requires a properly polarised microtubule network. Only at this stage can the nurse cell factors assembled on the mRNA act to transport the cortically localised complexes to the anterior corners of the oocyte. During the second phase of bicoid mRNA localisation, the ring-shaped distribution changes to a disc-shaped distribution and a MTOC forms at the anterior of the oocyte. The third phase of bicoid mRNA localisation begins after the onset of ooplasmic streaming. In this late phase, the mechanism of bicoid mRNA localisation changes from targeted transport to passive trapping, mediated by ooplasmic streaming, and the mRNA is anchored at the anterior margin. The generation of exu mutants that abolish phosphorylation allows distinguishing between early and the late mechanisms of bicoid mRNA localisation, since the two mechanisms differ in their sensitivity to Exu phosphorylation (Riechmann, 2004).

It has also been shown that Par-1 controls posterior patterning by phosphorylating Oskar. In addition, Par-1 regulates anterior patterning by phosphorylating Exu. Although Oskar is an intrinsically unstable protein whose stability is increased by Par-1 phosphorylation, Par-1 phosphorylation does not affect Exu stability but does affect its ability to mediate bicoid mRNA localisation. Thus, Par-1 uses at least two different mechanisms to generate polarity within the same cell. Interestingly, these two Par-1 substrates, Oskar and Exu, are unique to Diptera, showing that during evolution Par-1 gained fly-specific mediators of cell polarisation as substrates. Par-1 is therefore flexible in the mechanisms and in the targets by which it mediates cell polarisation. This is in striking contrast to the PDZ-containing proteins Par-3 and Par-6, which appear to establish polarity by the assembly of a conserved protein complex (Riechmann, 2004).

Localization of bicoid mRNA in late oocytes is maintained by continual active transport

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential to produce the Bicoid protein gradient that patterns the anterior-posterior axis of the embryo. Previous studies have characterized a microtubule-dependent pathway for bicoid mRNA localization during midoogenesis, when bicoid first accumulates at the anterior. The majority of bicoid is actually localized later in oogenesis, when the only known mechanism for mRNA localization is based on passive trapping. Through live imaging of fluorescently tagged endogenous bicoid mRNA, a temporally distinct pathway has been identified for bicoid localization in late oocytes that utilizes a specialized subpopulation of anterior microtubules and dynein. The directional movement of bicoid RNA particles within the oocyte observed in this study is consistent with dynein-mediated transport. Furthermore, the results indicate that association of bicoid with the anterior oocyte cortex is dynamic and support a model for maintenance of bicoid localization by continual active transport on microtubules (Weil, 2006).

Bcd patterning activity in the early embryo depends on the efficacy of bcd mRNA localization during oogenesis. Through live imaging of bcd mRNA fluorescently labeled in vivo, direct evidence is provided for a distinct, late bcd localization pathway that initiates with nurse cell dumping and is responsible for the majority of bcd present at the anterior of the embryo. Since specification of cell fates along the anterior-posterior axis is sensitive to single changes in bcd gene dosage, the predominant late source of localized bcd mRNA most likely makes the primary contribution to the Bcd protein gradient. Thus, it is proposed that although bcd localization also occurs during midoogenesis, the late pathway is the relevant one for anterior-posterior patterning. A new role has been identified for Stau as a component of this pathway and it is shown that localization of bcd/Stau complexes during late stages of oogenesis depends on the integrity of a subpopulation of microtubules that are anchored at the anterior cortex by the actin cytoskeleton. Movement of bcd RNA particles within the oocyte is consistent with anteriorly directed transport along these microtubules. Moreover, the results reveal dynamic behavior of bcd that does not fit the prevailing two-step transport and anchoring paradigm for mRNA localization. Rather, they support a model for steady-state localization of bcd at the anterior cortex by continual active transport (Weil, 2006).

Evidence for microtubule-directed transport in late-stage oocytes, when the cytoskeletal polarity thought to underlie transport along the anterior-posterior axis is no longer apparent, has been lacking. Indeed, previous studies have shown that posterior localization of mRNAs like nos and osk after stage 10 does not depend directly on microtubules, but occurs by a diffusion/trapping mechanism that is facilitated by ooplasmic streaming. In contrast, results presented in this study suggest that microtubules emanating from the anterior cortex support transport of bcd mRNA to the anterior. Since it has not been possible to follow the movement of bcd particles as they enter the oocyte during nurse cell dumping, the possibility cannot be excluded that some bcd is simply trapped by anterior microtubules as it enters, in a manner analogous to trapping of nos mRNA by germ plasm at the posterior. However, the requirement for microtubules, dynein, and ATP to maintain bcd mRNA localization after nurse cell dumping and ooplasmic streaming, together with the anteriorly directed movement of bcd particles near the anterior cortex, implicates these microtubules in active transport of bcd. Although ooplasmic streaming is capable of distributing mRNA localization complexes to the posterior pole, no bcd particles are detected in the posterior half of the oocyte. Thus, bcd must associate rapidly with these anterior microtubules upon entry into the oocyte. In the early embryo, nearly all bcd mRNA resides at the anterior cortex, whereas only a small fraction of nos mRNA is localized at the posterior pole. The existence of a microtubule-dependent pathway specific for anterior transport in late-stage oocytes could account for the dramatic difference in the efficiencies with which bcd and nos mRNAs are localized (Weil, 2006).

Anterior microtubules that mediate bcd mRNA localization in late-stage oocytes are most likely nucleated by a MTOC formed at the anterior cortex during stage 10. Analysis of mutants γ-Tub37C and Dgrip-75 implicate this MTOC in the transition of bcd mRNA from its ring-like distribution in stages 8–9 to a disc-like distribution in stage 10. Previously localized bcd mRNA is subsequently released from the anterior cortex in γ-Tub37C and Dgrip-75 mutants during nurse cell dumping, whereas other microtubule-dependent processes, such as ooplasmic streaming, are unaffected. These defects suggest that a specific reorganization of microtubules at the anterior cortex is responsible for maintaining bcd localization, while the majority of microtubules are reorganized for ooplasmic streaming (Weil, 2006).

Microtubules present at the anterior cortex in late-stage oocytes are distinct from microtubules that mediate bcd mRNA localization during midoogenesis and from cortical microtubules that mediate ooplasmic streaming by their dependence on the actin cytoskeleton. The results suggest that association with the actin cytoskeleton enables microtubules nucleated from the anterior MTOC during stage 10 to survive the dramatic changes in the oocyte that occur with nurse cell dumping and ooplasmic streaming and persist to later stages. These microtubules serve multiple functions in late-stage oocytes, as their selective perturbation disrupts both bcd mRNA localization and oocyte nucleus positioning (Weil, 2006).

Localization of bcd mRNA during mid- and late oogenesis can also be distinguished by a requirement for Stau. Stau participates in both the transport and anchoring of osk mRNA during midoogenesis, and Stau homologs have been implicated in microtubule-dependent transport of mRNAs in mammalian hippocampal neurons and Xenopus oocytes. This evidence, together with the presence of Stau in neuronal RNA granules, suggests a common function for Stau in coupling mRNAs to motor proteins for transport. Stau's function in bcd mRNA localization is not limited to anchoring bcd during the transition from oogenesis to embryogenesis as previously thought; rather, Stau plays an important role from the onset of nurse cell dumping. Although the stau mutant used in these experiments is a null allele, bcd mRNA localization is not completely eliminated in all stau mutant oocytes. Similarly, posterior localization of osk mRNA is greatly reduced, but it is not abolished in stau null mutant oocytes. It is possible that bcd mRNA localized during midoogenesis can persist at the anterior cortex in the absence of Stau. Alternatively, an as yet unidentified factor could act redundantly with Stau in late bcd localization. Redundant recognition elements are indeed present within the bcd mRNA localization signal (Weil, 2006).

Colocalization of Stau in particles with bcd mRNA suggests that it is an integral component of a bcd localization RNP. Individual bcd RNA particles that exhibit directional movement range in size from 0.3 to 1 μm, indicating that they consist of multiple mRNA and protein molecules. These particles are similar in size to the particles that form after injection of synthetic bcd 3′UTR RNA into embryos and become associated with astral microtubules. Formation of bcd 3′UTR particles requires Stau as well as intermolecular interactions between two or more bcd 3′UTRs. Similar assembly of large particles through interactions among bcd mRNA molecules during oogenesis would enable Stau, or another factor, to couple many bcd molecules to a single dynein motor. Concurrent transport of multiple mRNAs may contribute to the efficiency of bcd localization (Weil, 2006).

Current models for mRNA localization invoke independent transport and anchoring steps. Evidence for distinct steps comes from the differential effects of cytoskeletal inhibitors applied during and after translocation of RNAs to their destinations. In this manner, a kinesin- and microtubule-dependent transport step is paired with an actin-dependent anchoring step for localization of Vg1 and osk mRNAs, whereas a dynein- and microtubule-dependent transport step is paired with a dynein-dependent anchoring step for Drosophila pair-rule transcripts. The results indicate that bcd localization does not fit neatly into this two-step model. FRAP and FLIP experiments show turnover in the population of bcd mRNA at the anterior cortex even after dumping and streaming have ended and accumulation is maximal. This turnover suggests that bcd/Stau RNPs transported to the anterior cortex are unable to make stable associations with cortical components. Upon release from dynein, bcd/Stau RNPs may interact transiently with the cortex or may be released directly into the ooplasm, where they are reloaded onto dynein for another round of transport. Continual active transport may be critical for anterior localization of mRNAs like bcd, which occurs at a time when the rapid growth and movement of the oocyte cortex may inhibit or delay the establishment of a static anchoring mechanism. It will now be of interest to determine whether other localized mRNAs, particularly those in cell types that undergo rapid morphological changes such as migrating growth cones, behave similarly to bcd (Weil, 2006).

Posttranscriptional regulation of Bicoid and Posttranscriptional regulation carried out by Bicoid as an RNA binding protein

Continued: Bicoid Post-transcriptional regulation part 2/2

bicoid: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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