Posttranscriptional regulation of Bicoid

In Drosophila embryos, graded activity of the posterior determinant Nanos (nos) generates abdominal segmentation by blocking protein expression from maternal transcripts of the hunchback (hb) gene. When active inappropriately at the anterior pole, nos can also block expression of the anterior determinant bicoid (bcd). Both regulatory interactions are mediated by similar sequences in the 3' untranslated region of each transcript. These NOS response elements (NREs) are both necessary and sufficient to confer nos-dependent regulation, the degree of regulation determined by the number and quality of the elements and the level of NOS in vivo. Based on these and other results, it is argued that NOS acts as a morphogen, controlling HB expression (and hence abdominal pattern) as a function of its concentration-dependent interaction with the NREs. Thus, it would seem that the requirements for NOS mRNA localization, involving 11 proteins of the posterior group are imposed by the presence of the NRE in BCD and HB mRNAs (Wharton, 1991).

Maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of Bicoid and Toll mRNAs. cortex mutant 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 mutant 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 mutant cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. cortex eggs are fertile, but embryos 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).

Pattern formation in Drosophila depends initially on the translational activation of maternal messenger RNAs (mRNAs) whose protein products determine cell fate. Three mRNAs, Bicoid, Toll, and Torso, dictate respectively the anterior, dorsoventral, and terminal specifications. They all show increases in polyadenylate [poly(A)] tail length concomitant with translation. In contrast, posteriorly localized Nanos mRNA, although also translationally activated, is not regulated by poly(A) status. These results implicate at least two mechanisms of mRNA activation in flies. Studies with Bicoid mRNA have shown that cytoplasmic polyadenylation is necessary for translation, establishing this pathway as essential for embryogenesis (Salles, 1994).

During the transition from the maternal to the zygotic developmental program, the expression of genes important for pattern formation or cell cycle regulation changes dramatically. Rapid changes in gene expression are achieved in part through the control of mRNA stability. This report focuses on bicoid, a gene essential for formation of anterior embryonic structures in Drosophila melanogaster. Bicoid mRNA is synthesized exclusively during oogenesis. Bicoid mRNA stability is regulated. Bicoid mRNA is stable for more than 12 days in oocytes that are retained in females. It is also stable in laid unfertilized eggs (even if it is translated), and during the first 2 h of embryogenesis. Specific degradation is activated at cellularization of the blastoderm. The Bicoid mRNA half-life is estimated to be less than 30 min between 2 and 3 h of development. To identify cis-acting sequences required for Bicoid mRNA's regulated stability, fusions between bicoid and other genes that produce stable mRNAs were introduced into the Drosophila germ line by P-element-mediated transformation. The analysis of the fusion mRNAs identified a bicoid instability element (BIE) contained within a 43-nucleotide sequence immediately following the stop codon. The BIE is sufficient to destabilize the otherwise-stable ribosomal protein A1 mRNA and is separable from the previously identified Bicoid mRNA localization signals and from the "nanos response element." Similar mechanisms may regulate a class of developmentally important maternal genes whose mRNA has a temporal profile similar to that of bicoid. While no similarities were found by comparing the entire BIE sequence with sequences in databases, the short sequence UUUCAUU present in the BIE was found to appear in a subset of the genes listed in the EMBL UTR database. Of about 1,800 UTRs searched, 112 (~6%) contained the UUUCAUU motif. Interestingly, a significant proportion of the genes retrieved from the UTR database (~35%) could be classified among four classes of regulatory genes whose mRNA and protein concentrations are expected to change rapidly as a function of time. In the majority of the genes (~82%), the motif was present in the 3' UTR. The presence of the UUUCAUU motif in several classes of genes suggests the existence of a common pathway for mRNA destabilization. Further research is needed to verify the validity of this hypothesis (Surdej, 1998).

Conserved signals and machinery for RNA transport in Drosophila oogenesis and embryogenesis

Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the same machinery and RNA signals drive specific accumulation of maternal RNAs in the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos; interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor complex that drives transcript localization in a variety of tissues (Bullock, 2001).

During Drosophila oogenesis, specification of the oocyte is associated with selective accumulation of RNA determinants supplied by the neighboring, interconnecting ovarian nurse cells. Subsequently, deposition of mRNA transcripts at selected sites within the oocyte leads to localized translation of the proteins that establish the prospective embryonic body axes. gurken (grk) transcripts reside first posteriorly and then anterodorsally, and sequentially establish the anteroposterior and dorsoventral axes. bicoid (bcd) and oskar (osk) transcripts localize to the anterior and posterior of the oocyte, respectively, to pattern the anteroposterior body axis (Bullock, 2001).

The injection assay was used to investigate whether any maternal transcripts that localize in the oocyte are recognized by the localization machinery of blastoderm embryos. Five such transcripts [bcd, grk, nanos (nos), osk and female sterile (1) K10 (K10)] were tested, and all accumulate in the apical cytoplasm after injection. With the exception of osk transcripts -- only a small proportion of which localize apically -- the efficiency of localization of these transcripts appears indistinguishable from that of pair-rule transcripts. Maternal transcripts also localize apically when zygotically expressed from endogenous transgenes. Preinjection with colcemid severely inhibits apical localization of the injected maternal transcripts, indicating that their localization in blastoderm embryos, like that of the pair-rule transcripts, is dependent on intact microtubules (Bullock, 2001).

The common aspect of maternal RNA localization measured in these experiments is unlikely to be transport within the oocyte, because the maternal transcripts tested are distinctly distributed in late stage oocytes by means of different motors and accessory factors. However, all the transcripts -- with the possible exception of grk -- are synthesized in adjacent nurse cells and reach the oocyte by transport along microtubules. To test whether this process is analogous to apical localization in blastoderm embryos, a bcd transcript was used containing a single nucleotide change (4496G->U). This change prevents early oocyte-specific transport (stages 4-6) without disrupting later (stage 6 onwards) import of transcripts into the oocyte or their subsequent accumulation at the anterior cortex. This mutation inhibits apical bcd localization in blastoderm embryos, suggesting that transcripts localize in this injection assay through the same machinery that transports transcripts into the early oocyte (Bullock, 2001).

These data suggest that components of the blastoderm localization machinery are also likely to function in RNA transport into the early oocyte. Genetic screens for maternal mutations that affect formation of the embryonic axis have identified egl and BicD as genes required for oocyte differentiation and for specific RNA accumulation in the oocyte. However, their exact activities are uncertain. BicD protein includes multiple heptad repeats, which may mediate oligomerization and interactions with other proteins; Egl includes a domain shared with 3'-5' exonucleases. During oogenesis, these two proteins form complexes together and colocalize at the minus ends of microtubules. The integrity of the microtubule cytoskeleton is defective in egl and BicD mutants, which has been proposed to explain subsequent defects in RNA localization. Alternatively, Egl and BicD might act directly in RNA transport. However, evidence that distinguishes between these two possibilities is lacking (Bullock, 2001).

Whether Egl and BicD are present in early embryos was examined. Both proteins are supplied maternally to the embryo. They are noticeably enriched apical to the nuclei at blastoderm stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the proteins is present in the basal cytoplasm (Bullock, 2001).

Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of protein/RNA co-transport. The complex may have an additional role in anchoring transcripts at their destination. Alternatively, maintenance of localized transcripts might not depend on an independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).

An anterior function for the Drosophila posterior determinant Pumilio

Bicoid is a key determinant of anterior Drosophila development. The prototypical Puf protein Pumilio temporally regulates bicoid (bcd) mRNA translation via evolutionarily conserved Nanos response elements (NRE) in its 3'UTR. Disruption of Pumilio-bcd mRNA interaction by either Pumilio or bcd NRE mutations causes delayed bcd mRNA deadenylation and stabilization, resulting in protracted Bicoid protein expression during embryogenesis. Phenotypically, embryos from transgenic mothers that harbor bcd NRE mutations exhibit dominant anterior patterning defects and similar head defects have been discovered in embryos from pum minus mothers. Hence, Pumilio is required for normal anterior development. Since bcd mRNA resides outside the posterior gradient of Nanos, the canonical partner of Pumilio, the data suggest that Pumilio can recruit different partners to specifically regulate distinct mRNAs (Gamberi, 2002).

To identify sequences regulating bcd mRNA expression, focus was placed on the perfect bipartite NRE sequence GUUGU-N5-AUUGUA (A box-N5-B >box) in the 3'UTR of bcd, starting 50 nucleotides downstream of the bcd translational stop codon. This bcd motif was noticed previously, but its role in normal development was unclear because it resides outside the Nanos embryonic domain. The hb 3'UTR contains two NRE motifs, while bcd has one NRE and an additional B box at position +79 (termed 1 1/2 NREs). By aligning the bcd and hb 3'UTRs from all available species, it was found that the bcd motifs are closer to the second hb NRE. Moreover, the 1 1/2 NREs was absolutely conserved in the bcd 3'UTR of eight fly species that diverged more than 60 million years ago, underscoring functional constraint. Thus, the role the NREs play in bcd expression and their contribution to normal embryonic development were analyzed (Gamberi, 2002).

Pumilio temporally regulates bcd mRNA expression: its mutation causes delayed deadenylation and stabilization of the bcd message, resulting in protracted Bicoid protein expression. Disruption of this molecular control perturbs normal Drosophila head development (Gamberi, 2002).

An intricate combination of spatial and temporal controls orchestrate expression of a gene hierarchy resulting in appropriate embryonic patterning. For bcd, initial spatial restriction in the embryo is provided by anterior localization of translationally silent bcd mRNA. The RNA is then translationally deployed over a short period, resulting in a pulse of Bicoid. This latter process of temporal control of localized bcd mRNA expression is regulated by the evolutionarily conserved bcd NRE to ensure proper head development. Either NRE or Pumilio mutation causes protracted bcd translation. Resulting Bicoid found later in development would have prolonged access to its downstream targets and/or novel access to inappropriate targets from which it is temporally segregated in wild type. Either could interfere with anterior development, ultimately causing head defects (Gamberi, 2002).

While affected Bicoid targets are presently only speculative, it was fortuitously noticed that late zygotic hb expression (hbzyg) was increased in Northern blots of bcd NRE mutants, hinting at one potential affected molecule. This is consistent with the pum- data. A second target candidate arises from the defective mouth hook (mh) base present in both bcd NRE mutant transgenics and pum- embryos. This alteration, which is suggestive of a maxillary segment defect, similarly occurs when orthodenticle (otd) is expressed ectopically. Interestingly, Bicoid activates otd transcription and resulting Orthodenticle has the same DNA-binding specificity as Bicoid. Hence, the prolonged Bicoid expression in mutant bcd NRE transgenics and pum- embryos may interfere with normal head development through a complex pattern of interactions (Gamberi, 2002).

Pum was originally characterized as a posterior group gene: Pumilio and Nanos cooperate to repress maternal hb (hbmat) in the posterior of the embryo, allowing abdominal patterning. However, ubiquitous expression of Pumilio in excess of hb implies it could possess additional function(s) elsewhere. This study demonstrates that Pumilio also participates in Drosophila anterior embryonic patterning. pum embryos exhibit head defects. The Pumilio anterior function is mediated via bcd post-transcriptional expression, since similar anterior abnormalities occur when Pumilio's presumptive bcd mRNA-binding site is mutated (Gamberi, 2002).

It was asked if bcd NRE regulation required the Pumilio canonical partner Nanos. When bcd mRNA is injected posteriorly or Nanos is expressed anteriorly by genetic means, Pumilio and Nanos can affect bcd expression because all factors co-exist. In each case, large Nanos amounts are present and head morphogenesis is inhibited (Gamberi, 2002).

A major Nanos role in normal head formation seems unusual because Nanos and bcd mRNA reside at opposite ends of the embryo. Surprisingly Nanos does influence bcd expression and subsequent anterior development to some degree. This suggests undetectable Nanos amounts may regulate bcd mRNA in the anterior. Analogously, a contribution of low Nanos levels in oogenesis has been reported (Gamberi, 2002).

bcd mRNA might encounter low Nanos levels via the NRE-dependent back-up mechanism postulated to repress it when it escapes localization, diffuses posteriorly and intercepts the Nanos gradient. Alternatively, sufficient Nanos moieties might diffuse anteriorly, analogous to when enough Bicoid molecules exist in the posterior of the embryo to elicit hairy stripe 7 expression or to cooperate with Caudal in knirps activation. In a different scenario, nos mRNA translational repression throughout the embryo may be leaky, yielding low basal Nanos levels everywhere, including the anterior. How a Pumilio-bcd mRNA complex can recruit enough Nanos for action and whether this involves additional (anterior?) factors to modulate Nanos activity are questions for future studies (Gamberi, 2002).

nos- severe head involution defects occur at a significantly lower frequency than in pum- cuticles (4% versus 81%; null versus presumptive null), raising the intriguing possibility that an additional partner(s) for Pumilio exists at the anterior that affects bcd NRE function independently of Nanos. Consistently, the sequence between the A and B boxes of the bcd and hb NREs diverges at two of the four nucleotide positions known for hb recruitment of Nanos. While Pumilio and Nanos are usually thought of as functioning in concert, they have only partially overlapping roles in the Drosophila germline and may function independently in oogenesis. The alternate Pumilio partner for bcd might be an anterior Nanos paralog (although only one nos gene was found in the fly genome) or a distinct moiety. Interestingly, S. cerevisiae has five Puf proteins involved in mRNA metabolism (Olivas, 2000) but no Nanos homologs, suggesting some Puf proteins can function with novel partners (Gamberi, 2002).

Molecular data indicate the bcd NREs act temporally, repressing translation in a deadenylation dependent way. Mutating either Pumilio or the bcd NREs results in protracted Bicoid expression. Presently, it cannot be distinguish if the bcd NREs primarily constitute a translational control element with mRNA deadenylation and instability accompanying specific repression or a regulated instability element whose downstream effects are seen at the protein level. Interestingly, in addition to detecting specific Pumilio-dependent bcd NRE regulation, a second effect of pum- mutation was noticed: stabilization of maternal mRNAs devoid of NREs. While it is unclear whether this effect is direct, it may reflect a novel Pumilio function in general NRE-independent mRNA turnover (Gamberi, 2002).

Complementary phenotypic analyses of bcd NRE mutant transgenes has revealed that prolonged Bicoid expression interferes with maxillary segment determination, which may affect head involution by altering the intersegmental contacts required for appropriate head morphogenetic movements. Incomplete overlap between the highly penetrant mouth hook defect and the partially penetrant head involution defect might reflect the complexity of fly head development, which is subjected to redundancy and fail-safe mechanisms (Gamberi, 2002).

The conservation of the bcd and hb NREs, their Pumilio association, and their ability to direct translational regulation imply functional similarity between these elements. However, the hb regulatory system operates on a uniformly distributed mRNA to repress its expression in the embryonic posterior where Nanos is most concentrated. By contrast, bcd mRNA is spatially restricted to the anterior via localization, which conceivably impacts NRE action and predicts underlying functional differences between bcd and hb NREs (Gamberi, 2002).

The novel Pumilio role in anterior development documented here raises the exciting possibility that the prototypical Puf protein Pumilio operates more generally than previously thought, regulating multiple physiological pathways in different Drosophila embryonic locales. Furthermore, since Pumilio is also expressed in the adult fly and pum- flies exhibit additional uncharacterized phenotypes, Pumilio may function in mRNA metabolism throughout the life of the fly (Gamberi, 2002).

To date, NREs have been identified in three mRNA species: hb, bcd and cyclin B. For each, NRE organization differs: hb and bcd contain two and 1 1/2 copies, respectively, of the basic (A box-N5-B box) NRE motif, while cyclin B contains one NRE motif with a larger spacer. Furthermore, hbmat and hbzyg mRNA have identical NREs, but hbzyg mRNA seems relatively insensitive to regulation by Pumilio/Nanos. Differences among NREs combined with distinct distributions of NRE-containing mRNAs and their known effectors underlie a potential combinatorial model of NRE recognition in which a common factor (Pumilio) associates with the mRNA target sequence and subsequently recruits different (sets of) factors (e.g. Nanos, Brat for hbmat mRNA) to regulate ultimately and specifically unique target expression. How Pumilio functions on different NRE-containing mRNAs, what factor combinations are employed in distinct situations and whether Nanos homologs are involved in every case are experimental questions begging to be answered (Gamberi, 2002).

Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila

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

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

To address whether cytoplasmic polyadenylation could be affected by the level of PAP, PAP was overexpressed in the female germline using the UASp-hrg transgene and nos-Gal4. This overexpression does not cause gross alteration of oogenesis, but is extremely detrimental to embryogenesis, leading to 99% lethality of the progeny. These embryos stop development early, before cleavage of nuclei. Cytoplasmic polyadenylation was analysed in these embryos by measuring poly(A) tails of bicoid mRNA that is regulated by this process during embryogenesis. In the wild-type, poly(A) tails of bicoid mRNA lengthen from 80 residues in oocytes to 170 residues in 1 h embryos. This elongation of the poly(A) tails induces Bicoid protein synthesis in early embryos. Following overexpression of PAP, poly(A) tails of bicoid mRNA strongly increase in length, with a pool of mRNAs bearing a 250 residue poly(A) tail in oocytes and most mRNAs having a poly(A) tail between 300 and 600 residues in 1 h embryos. The fact that bicoid mRNA poly(A) tails lengthen in 0- 1 h embryos, at a stage when there is no transcription, shows that the process affected by PAP overexpression is cytoplasmic polyadenylation. This was confirmed by showing that when PAP is overexpressed ubiquitously in somatic cells with the da-Gal4 driver, poly(A) tails of sop mRNA are not affected. Therefore, poly(A) tail length control during nuclear polyadenylation is not altered by PAP overexpression, although the level of somatic overexpression is in the same range as that of germline overexpression. Surprisingly, although sop mRNA does not undergo cytoplasmic polyadenylation in wild-type embryos, overexpression of PAP in the female germline leads to a strong lengthening of sop mRNA poly(A) tails by cytoplasmic polyadenylation. Similar results were found for rp49 mRNA. This indicates that the increasing PAP level affects both poly(A) tail length control and specificity during cytoplasmic polyadenylation. Bicoid protein accumulation and bicoid mRNA poly(A) tail length was correlated by immunostaining of ovaries and embryos with anti-Bicoid. Poly(A) tail elongation of bicoid mRNA in oocytes, following PAP overexpression, does not induce translation since no Bicoid is detected in UASp-hrg; nos-Gal4 oocytes. Therefore, in oocytes, long poly(A) tails are not sufficient to induce bicoid mRNA translation. In embryos where PAP is overexpressed, poly(A) tail lengthening correlates with a precocious accumulation of Bicoid and with an increase in Bicoid protein level. These data demonstrate that a tight regulation of PAP level is essential to control cytoplasmic polyadenylation and to early development (Juge, 2002).

The Drosophila microtubule-associated protein Mini spindles is required for cytoplasmic microtubules in oogenesis

Drosophila mini spindles is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles. msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event. bcd RNA localization is defective in msps mutants. Defects in cytoplasmic microtubules were identified in both the germ and follicle cells of mutant ovaries, and the expression pattern of msps mRNA and protein in developing egg chambers was determined. The findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions (Moon, 2004; full text of article).

What role might Msps play in bcd mRNA localization? bcd RNA is initially localized in stages 8 and 9 mspsP/msps208 oocytes, but some egg chambers have patchy or dispersed RNA, indicating that localization is less efficient at these stages. Later in oogenesis, bcd RNA localization is completely lost in the msps mutants. This progressive loss of bcd RNA localization mirrors the temporal decline in msps expression that occurs in the mutant egg chambers. A model is favored in which msps is required to regulate the structure of more than one population of cytoplasmic microtubules, including microtubules required in mid-oogenesis for bcd RNA transport and microtubules required later for anchoring bcd RNA at the oocyte anterior. Consistent with this model, defects were obaserved in several populations of microtubules in msps egg chambers. The requirement for msps in the late maintenance of bcd, but not osk mRNA localization, is consistent with studies that show that the maintenance of osk mRNA localization is actin dependent, not microtubule dependent (Moon, 2004).

Biochemical experiments have shown that other XMAP215/TOG family members promote spindle assembly and regulate microtubule dynamics by promoting growth of microtubules at their plus ends. During oogenesis, Msps may affect the dynamics of microtubules that provide the pathway for bcd RNA transport within and between the nurse cells and oocyte; possibly, these microtubules include those observed in this study. The gradient of Msps in the nurse cells is intriguing and may serve to preferentially regulate the plus ends of these microtubules. Alternatively, Msps might affect the minus ends of these and other microtubules required for anchoring bcd RNA in the oocyte. XMAP215/TOG proteins are concentrated at the poles of spindles in dividing cells. It has been proposed that Msps may organize the poles of the female meiotic spindle by capturing microtubules at their minus ends. Msps could act similarly on cytoplasmic microtubules required for bcd RNA localization and stabilize the attachment of their minus ends at the oocyte cortex, where Msps is itself concentrated (Moon, 2004).

Given their localization to the poles and microtubules of spindles in dividing cells, members of the XMAP215 family of proteins might play a role in the association of mRNAs, or even noncoding structural RNAs, with spindles. Recently, mRNA localization to centrosomes has been shown to accompany the asymmetric distribution of patterning mRNAs to daughter cells in Ilyanassa embryos. The factors that mediate this centrosomal attachment are unknown, as is the extent of association of RNAs with spindles in general. MAPs, including the XMAP215/TOG proteins, are candidates for such events and may prove to be valuable tools for identifying spindle-associated RNAs (Moon, 2004).

Bicoid as a target of the Torso pathway

The Torso signal transduction pathway exhibits two opposite effects on the activity of the Bicoid (Bcd) morphogen: (1) Bcd function is repressed by Torso (Tor) at the anterior pole of the embryo leading to a retraction of the expression of many Bcd targets from the most anterior region of the embryo, where the Tor tyrosine kinase receptor is activated, and (2) Bcd function is strengthened by Tor in a broader anterior region, as indicated by a shift of the posterior border of Bcd targets toward the anterior pole in embryos deprived of Tor activity. Anterior repression of Bcd targets is not observed in embryos lacking maternal contribution of D-sor, which acts downstream of Tor and encodes a MAP-kinase kinase. This indicates that the Ras signaling cascade is directly involved in this process, although the known transcriptional effectors of the Tor pathway, tll and hkb, are not. Bcd is a good in vitro substrate for phosphorylation by MAP-kinase (MK); phosphorylation of the protein occurs in vivo on MK sites. Examination of Bcd sequence reveals three optimal (S165, T200 and T353) and seven weak (T188, T193, S195, T197, S343, S359 and S439) consensus sites for phosphorylation by MK. Six of these sites are concentrated in the serine/threonine (S/T)-rich domain that follows the HD. This domain also contains a PEST sequence that has been implicated in the degradation of proteins with short half-lives. The four remaining MK sites are located within the activation domain in the C-terminal part of the protein. In the presence of a Bcd mutant that can no longer be phosphorylated by MK, expression of Bcd targets remains repressed by Tor at the pole while the strengthening of Bcd activity is reduced. These experiments indicate that phosphorylation of Bcd by MK is likely to be required for the Tor pathway to induce its full positive effect on Bcd. This suggests that Tor signaling acts at a distance from the anterior pole by direct modification of the diffusing Bcd morphogen (Janody, 2000a).

The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo. This regulation does not involve the homeodomain or direct phosphorylation of Bicoid. When three copies of the yeast GCN4 activation domain were fused downstream the ST domain of Bcd (Bcd-GCN4) and expressed under the control of bcd regulatory sequences, a head phenotype was induced in the embryo. In addition, Torso-dependent repression of the Bcd target genes hb and otd is no longer observed in these embryos. This indicates that the Bcd-GCN4 protein is insensitive to downregulation by Torso at the anterior pole, and that the absence of repression of hb and otd might lead to a head phenotype. To understand whether this lack of repression is due to the site of insertion of the GCN4 activation domains or to their presence in Bcd-GCN4, these domains were fused to the C-terminal end of Bcd and the activity of the new fusion protein (Bcd-GCN4C) was analysed in vivo. The expression of Bcd-GCN4C as a maternal anterior gradient also induces a head phenotype, which is correlated with the lack of anterior repression of the Bcd target genes. This indicates that both Bcd-GCN4 and Bcd-GCN4C are insensitive to downregulation by Torso at the anterior pole, and suggests that it is unlikely that the insertion of the GCN4 activation domains in Bcd-GCN4 disrupts the domain of Bcd, mediating its downregulation by Torso. More probably, the intrinsic properties of the GCN4 activation domains render Bcd-GCN4 and Bcd-GCN4C insensitive to Torso (Janody, 2001).

These observations suggest two hypotheses. (1) There are certain types of activation domains, such as those of Bcd, that are downregulated by Torso, while others, such as those of GCN4, remain insensitive to Torso. The presence in Bcd-GCN4 and Bcd-GCN4C of the GCN4 activation domains, which are insensitive to Torso, might overcome or mask the inhibition of the endogenous activation domains of Bcd by Torso. (2) The Bcd protein might contain a repression domain induced by Torso. This putative repression domain might reduce the activity of the endogenous activation domains of Bcd upon Torso activation, whereas it might be unable to act on the heterologous GCN4 activation domains (Janody, 2001).

The transcriptional regulation of Bicoid in response to the Torso pathway was analyzed using other Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain. Bicoid possesses three autonomous activation domains. Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not. The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso. Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso. The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso. The molecular mechanism that leads to the downregulation of the ST and acidic domains by the Torso pathway remains to be uncovered. Since the ST domain and acidic domain are large, composed of 100 and 149 amino acids, respectively, they might be bipartite and might contain, in addition to an activation domain, a repression domain stimulated by Torso. Alternatively, the ST and C domains might function with positive co-factors whose activities are directly downregulated by activation of the Torso pathway. A simple scenario might be that both the ST and C domains of Bcd function through the same co-factor. Alternatively, there might be different co-factors for each of these domains (Janody, 2001).

Regulation of Nuclear Import

The maternal transcript of the anterior segmentation gene bicoid (bcd) is localized at the anterior pole of the Drosophila egg and translated to form a gradient in the nuclei of the syncytial blastoderm embryo after fertilization. The nuclear gradient of Bcd protein (a transcription factor) leads to differential expression of zygotic segmentation genes. The rapid nuclear division in the early zygote requires that Bcd quickly enters the nuclei after each mitosis using an active nuclear import system. Nuclear transport depends on the asymmetrical distribution of two forms of the small GTPase Ran: Ran-GTP that is concentrated in the nucleus and Ran-GDP in the cytoplasm. Ran requires RanGTPase-activating protein-1 (RanGAP1) on the cytoplasmic side of nuclear pore complexes to convert Ran-GTP to Ran-GDP. In vitro studies with vertebrate proteins demonstrate that the RanGAP1 associated with the nuclear pore complex is modified with small ubiquitin related modifier-1 (SUMO-1; see Drosophila SUMO) by a ubiquitin-conjugating enzyme (E2 enzyme). Mutation of the Drosophila semushi (semi) gene, which encodes an E2 enzyme, blocks nuclear import of Bcd during early embryogenesis and results in misregulation of the segmentation genes that are Bcd targets. Consequently, semi embryos have multiple defects in anterior segmentation. This study demonstrates that an E2 enzyme is required for nuclear transport during Drosophila embryogenesis. semi could be responsible for modification of other proteins essential for Bcd nuclear transport. Nevertheless, these results indicate the possible connection of the function of an E2 enzyme of the Ubc9 family to nuclear import in Drosophila. Hunchback is accumulated in the nucleus in a normal fashion in semi mutants. In semi mutants, posterior segmentation genes function correctly (Epps, 1998).

Posttranscriptional regulation carried out by Bicoid as an RNA binding protein

Bicoid, a DNA binding homeodomain protein and the primary determinant of anterior pattern in the fly, binds RNA, and acts as a translational repressor of Caudal mRNA. The Bicoid response element maps in the Caudal mRNA 3' untranslated region, and has been localized to a discrete 342-nucleotide segment. The Bicoid response element of Caudal mRNA contains at least two distinct BCD binding sites. An in-frame deletion of 11 amino acids within the BCD homeodomain results in a protein that is unable to regulate Caudal mRNA. Substitutions in the recognition helix of the Bicoid homeodomain results in a nonfunctional translational repressor. Thus the BCD homeodomain is implicated in repression of Caudal translation. Bicoid bound to the 3' UTR of CAD mRNA blocks translational initiation at the 5' end, possibly by interfering with a step that depends on the 5' cap structure (Dubnau, 1996).

In embryos lacking BCD activity as a result of mutation, the CAD gradient fails to form and CAD becomes evenly distributed throughout the embryo. This suggests that BCD may act in the region-specific control of CAD mRNA translation. BCD binds through its homeodomain to CAD mRNA in vitro, and exerts translational control through a BCD-binding region of CAD mRNA (Rivera-Pomar, 1996b).

The Drosophila body organizer Bicoid (Bcd) is a maternal homeodomain protein. It forms a concentration gradient along the longitudinal axis of the preblastoderm embryo and activates early zygotic segmentation genes in a threshold-dependent fashion. In addition, Bcd acts as a translational repressor of maternal Caudal (CAD) mRNA in the anterior region of the embryo. This process involves a distinct Bcd-binding region in the 3' untranslated region of CAD mRNA. Using cotransfection assays, Bcd is found to repress translation in a cap-dependent manner. Bcd-dependent translational repression involves a portion of the PEST motif of Bcd, a conserved protein motif best known for its function in protein degradation. Rescue experiments with Bcd-deficient embryos expressing transgene-derived Bcd mutants indicate that amino acid replacements within the C-terminal portion of the PEST motif prevent translational repression of CAD mRNA but allow for Bcd-dependent transcriptional activation. Thus, Bcd contains separable protein domains for transcriptional and translational regulation of target genes. Maternally-derived Cad protein in the anterior region of embryos interferes with head morphogenesis, showing that CAD mRNA suppression by Bcd is an important control event during early Drosophila embryogenesis (Niessing, 1999).

While the Bcd gradient has served as a model system in understanding pattern formation in Drosophila, it is suspected that this is not the case in more ancestral insects. The long-germ mode of development as found in Drosophila is probably an adaptation to its particularly rapid embryogenesis. The ancestral type of embryogenesis in insects and arthropods is the short germ type. In these embryos, the germ rudiment forms at the posterior ventral side of the egg. In extreme cases like the grasshopper, it may be restricted to only a few percent of the total egg length - which makes it difficult to imagine how an anteriorly localized BCD mRNA could determine pattern formation at the posterior end of the egg. Moreover, classical experiments have only yielded evidence for a posteriorly localized organizing activity. Therefore, bcd could be considered a late addition during insect evolution and its pivotal function during embryogenesis could be restricted to higher dipterans. This paper is concerned with early pattern formation of the flour beetle Tribolium castaneum. Tribolium is a typical example for short germ embryogenesis, representing the ancestral type of embryogenesis in insects, albeit not in its extreme form, like the grasshopper. In contrast to Drosophila, only cephalic and thoracic segments, but not abdominal segments, are determined during the blastoderm stage. Furthermore, the most anterior 20% of the Tribolium blastoderm cells form an extra-embryonic membrane, the serosa. This structure is not found in this form in higher Dipterans like Drosophila, but is again an ancestral feature of insect embryogenesis. Prior to gastrulation, most blastoderm cells move from anterior and dorsal positions towards the posterior ventral region where they form the embryo proper. This germ rudiment then continues to grow from its posterior end to form a germ band which eventually encompasses all abdominal segments (Wolff, 1998).

Thus, in short germ embryos, the germ rudiment forms at the posterior ventral side of the egg, while the anterior-dorsal region becomes the extra-embryonic serosa. It is difficult to see how in these embryos an anterior gradient like that of Bicoid protein in Drosophila could be directly involved in patterning of the germ rudiment. Moreover, since it has not yet been possible to recover a bicoid homolog from any species outside the diptera, it has been speculated that the anterior Bicoid gradient could be a late addition during insect evolution. This question was addressed by analyzing the regulation of potential target genes of bicoid in the short germ embryo of Tribolium castaneum. Homologs of caudal and hunchback from Tribolium are regulated by Drosophila bicoid. In Drosophila, maternal Caudal mRNA is translationally repressed by Bicoid. Tribolium Caudal RNA is also translationally repressed by Bicoid, when it is transferred into Drosophila embryos under a maternal promoter. This strongly suggests that a functional bicoid homolog must exist in Tribolium. The second target gene, hunchback, is transcriptionally activated by Bicoid in Drosophila. Transfer of the regulatory region of Tribolium hunchback into Drosophila also results in regulation by early maternal factors, including Bicoid, but in a pattern that is more reminiscent of Tribolium hunchback expression, namely in two early blastoderm domains. Using enhancer mapping constructs and footprinting, it has been shown that Caudal activates the posterior of these domains via a specific promoter. These experiments suggest that a major event in the evolutionary transition from short to long germ embryogenesis was the switch from activation of the hunchback gap domain by Caudal to direct activation by Bicoid. This regulatory switch can explain how this domain shifted from a posterior location in short germ embryos to its anterior position in long germ insects, and it also suggests how an anterior gradient can pattern the germ rudiment in short germ embryos, i.e. by regulating the expression of caudal (Wolff, 1998).

The key to understanding the qualitative switch that took place in insect evolution is believed to lie in the more anterior serosa expression domain of Tribolium hb. Reporter gene data suggest that this domain may already be activated by Bcd in Tribolium. To explain the switch in the regulation of the more posterior gap domain of hb expression, one can envision an intermediate state, where the serosa domain and the embryonic (gap) domain have fused into a single domain. To achieve this, the evolution of a few additional Bcd binding sites in the hb upstream region would have been sufficient. In this intermediate stage both Bcd and Cad could have acted as activators on the gap domain of hb. Subsequent loss of Cad regulation would then have moved the posterior boundary of this combined domain towards the anterior. It is noted that the Tribolium hb gene has three known promoters, one of which appears to be specialized for mediating Cad regulation. In Drosophila, only two promoters are present, neither of which has a known responsiveness to Cad. Thus, in all likelihood, the Cad dependent promoter and its associated enhancer was lost. Since no other enhancer activity has been found for later expression patterns of hb in the cad dependent fragment, the loss of this region could have been a single step. Intriguingly, a combined serosa and gap domain is still evident in the lower dipteran Clogmia. In this fly, hb is expressed in a large anterior domain, from which at later stages also the serosa is recruited (Rohr, personal communication to Wolff, 1998). This mechanism, the modification of the way gap genes sense maternal positional information while this information itself remains constant, can explain how the blastoderm fate map changed during evolution of short germ insects to insects with long germ embryos. Moreover, it represents an intriguing example for the importance of regulatory adaptation during the evolution of developmental processes (Wolff, 1998).

Translational control plays a key role in many biological processes including pattern formation during early Drosophila embryogenesis. In this process, the anterior determinant Bicoid (BCD) acts not only as a transcriptional activator of segmentation genes but also causes specific translational repression of ubiquitously distributed caudal (cad) mRNA in the anterior region of the embryo. Translational repression of cad mRNA is dependent on a functional eIF4E-binding motif. The results suggest a novel mode of translational repression, which combines the strategy of target-specific binding to 3'-untranslated sequences and interference with 5'-cap-dependent translation initiation in one protein. The results suggest that 3'-UTR-bound BCD interferes with the assembly of the initiation complex and thereby causes repression of cad mRNA translation (Niessing, 2002).

The cap-dependent mode of translation depends on the assembly of an evolutionarily conserved protein complex that is initiated by the binding of the translation initiation factor 4E (eIF4E) to the m7GpppN-cap structure. Subsequently, the adapter protein eIF4G binds to eIF4E and allows additional factors (including eIF4A, eIF4B, eIF1, eIF1A, eIF2, eIF3, and the ribosomal subunits) to assemble into a complex that initiates translation. The cap-dependent translation initiation process can be regulated by eIF4E-binding proteins such as BP1, BP2, and Maskin. They block the eIF4E::eIF4G association through outcompeting binding to eIF4E, involving a small eIF4E-binding motif of the minimal consensus sequence YxxxxL (Niessing, 2002 and references therein).

The results show that Bcd can associate with cap-associated eIF4E in vitro and that the eIF4E-binding motif of Bcd is necessary for Bcd-dependent translational repression of cad mRNA in the embryo. These findings suggest a repression mechanism in which Bcd blocks the eIF4G::eIF4E interaction necessary for the initiation of cap-dependent cad mRNA translation. Because no interaction between recombinant eIF4E and Bcd could be detected in the absence of cad mRNA, it is concluded that the binding of Bcd to the cad 3'-UTR is most likely a prerequisite for their interaction. This interpretation is consistent with findings where a mutant Bcd, which lacks the ability to bind cad mRNA, is also unable to repress translation (Niessing, 2002).

Bcd-dependent control of translation of cad mRNA is likely to function in a manner similar to BP1, BP2, and Maskin. However, despite the intriguing similarities among BP1/BP2, Maskin, and Bcd, the modes of how they exert translational repression are distinct. BP1 and BP2 are part of a general mRNA repression system, which blocks eIF4E::eIF4G interaction in a reversible, cell-growth-dependent manner in response to insulin receptor signaling. In contrast, Maskin represses translation in an mRNA-specific manner. It binds to the cytoplasmic polyadenylation element-binding protein (CPEB), a factor that interacts with a short uridine-rich cytoplasmic polyadenylation element (CPE) of cyclin B mRNA. CPEB-tethered Maskin acts from the 3'-end of specific mRNAs by binding to eIF4E and blocking an association of eIF4E and eIF4G. In this mode of repression, target specificity of repression is provided by the interaction of CPEB with the CPE, whereas the repression of translation at the 5'-end is executed by Maskin. Bcd uses a strategy that combines these two features of CPEB and Maskin. Its homeodomain directly binds to the Bcd response element (BRE) in the 3'-UTR of cad mRNA and provides also a direct link to the 5'-cap-bound complex involving the eIF4E-interaction motif (Niessing, 2002).

The simplest model to account for Bcd-dependent repression of translation therefore involves three essential steps, which are (1) target recognition by binding to the specific target site within the 3'-UTR, a process mediated by Bcd's arginine-rich RNA-binding motif in the homeodomain, (2) looping of cad mRNA to allow for interaction of the 3'-UTR-bound Bcd with 5'-cap-bound eIF4E, which (3) causes a BP1/BP2-like blocking of the eIF4G-binding site on eIF4E to prevent the assembly of a functional translation initiation complex. The mode of Bcd-dependent repression of translation, therefore, combines the strategy of target-specific binding to 3'-UTRs as shown for a number of other translational repressors with a repression mechanism known from growth regulation and cyclin B-dependent cell cycle regulation (Niessing, 2002).

A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP

Translational control is a key genetic regulatory mechanism implicated in regulation of cell and organismal growth and early embryonic development. Initiation at the mRNA 5' cap structure recognition step is frequently targeted by translational control mechanisms. In the Drosophila embryo, cap-dependent translation of the uniformly distributed caudal (cad) mRNA is inhibited in the anterior by Bicoid (Bcd) to create an asymmetric distribution of Cad protein. d4EHP, an eIF4E-related cap binding protein, specifically interacts with Bcd to suppress cad translation. Translational inhibition depends on the Bcd binding region (BBR) present in the cad 3' untranslated region. Thus, simultaneous interactions of d4EHP with the cap structure and of Bcd with BBR renders cad mRNA translationally inactive. This example of cap-dependent translational control that is not mediated by canonical eIF4E defines a new paradigm for translational inhibition involving tethering of the mRNA 5' and 3' ends (Cho, 2005).

This study describes a new mode of mRNA-specific translational inhibition, which acts by tethering the mRNA 5′ and 3′ end via d4EHP, an eIF4E-related protein, and Bcd. d4EHP binds to the cad mRNA 5′ cap structure, while Bcd binds to BBR in its 3′ UTR. The interaction between d4EHP and Bcd is mediated through a sequence motif in Bcd that resembles, but is distinct from, the consensus eIF4E binding domain present in classical eIF4E binding proteins such as 4E-BPs and eIF4G. Inhibition of cad mRNA translation by the d4EHP:Bcd complex demonstrates for the first time the involvement of a cellular cap binding protein other than eIF4E in cap-dependent translational control. Furthermore, it provides a new molecular mechanism governing the formation of morphogenetic gradients during early Drosophila embryo development (Cho, 2005).

It was previously reported that Bcd inhibits anterior Cad synthesis through a direct interaction with eIF4E (Niessing, 2002). This conclusion was based largely on an in vitro demonstration that Bcd could be recovered from Drosophila extracts using a cap-affinity resin, which was prebound to an excess amount of recombinant eIF4E. However, under these conditions, only a small fraction of Bcd was recovered from the extracts. It is therefore a distinct possibility that Bcd actually bound to the cap-affinity resin through endogenous d4EHP that was also present in the extracts. This possibility is consistent with both the previous data and the present study. Further supporting this conclusion, endogenous deIF4E and Bcd were not shown to interact in the previous study. The data also indicate that the L73R mutation alone is sufficient to explain the previously reported bcdY68A/L73R double mutant phenotype (Cho, 2005).

The role of 4E-BPs in regulating cap-dependent translation is well documented. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E and are therefore general inhibitors of cap-dependent translation, although the degree of inhibition varies among different mRNAs. Cup and Maskin are eIF4E binding proteins that regulate translation during oogenesis and embryonic development. They inhibit the translation of specific mRNAs by a simultaneous interaction with eIF4E at the mRNA 5′ end and proteins bound to sequence elements in the 3′ UTR. Thus, Cup and Maskin have to compete with eIF4G for binding to eIF4E. While the exact binding affinities of these proteins for eIF4E have not been determined, it is known that Maskin interacts rather weakly with eIF4E (Cho, 2005).

In contrast to 4E-BP, Cup, and Maskin, Bcd does not need to compete with eIF4G to interact with d4EHP. Rather, it is d4EHP that competes with eIF4E for cap binding, which results in translation being inhibited at the level of cap recognition. As a result of bypassing the need to disrupt the very stable eIF4E:eIF4G interaction, d4EHP should interdict translation more efficiently than 4E-BPs or other eIF4E binding proteins. 4EHP-mediated translational regulation may have a particularly important role in germline development, based on these results and on a recent report that a mutant allele of C. elegans 4EHP (ife-4) shows a severe egg-laying defect (Cho, 2005).

The delineation of a d4EHP-recognition sequence in Bcd (YxxxxxxL; x denotes any amino acid) that interacts with d4EHP via its Trp85 residue highlights the similarities between the d4EHP:Bcd interaction and that of eIF4G with eIF4E (YxxxxLphi in eIF4G; Trp73 in eIF4E; phi denotes any hydrophobic amino acid). Despite these parallels, the inability of Bcd to bind to eIF4E must be explained by structural differences. The presence of two proline residues at position +3 and +6 of the Bcd d4EHP binding motif is predicted to significantly alter the α-helical structure assumed by the YxxxxLphi peptide upon binding to eIF4E and thus prevent Bcd association with deIF4E. Furthermore, the eIF4E interaction surface of eIF4G is not limited to the YxxxxLphi motif but extends over a larger interface; the N-terminal domain of eIF4E is also required for folding and tight binding to eIF4G. Indeed, the ability of d4EHP to bind specifically to Bcd, and not to deIF4G and d4E-BP, can be explained by the importance of the N-terminal KHPL sequence of eIF4E in the interaction with eIF4G and 4E-BP, since this sequence is not conserved in d4EHP (Cho, 2005).

The demonstration that cad translation is repressed through a d4EHP- and Bcd-dependent tethering mechanism adds to the diversity of translational control mechanisms operating in the early Drosophila embryo. Why are so many translational repression pathways necessary? If an individual mechanism alone can reduce translation of a specific mRNA, but not completely abrogate it, a combination of inhibitory interactions may be needed in order to accomplish strict translational control. This can be advantageous if the diversity of factors (like Bcd, which can confer mRNA specificity for a given mechanism) is relatively limited. Multiple mRNAs also have to be translationally repressed in overlapping spatial and temporal domains. Controlling these mRNAs through mechanisms that target different components of the general translational machinery, rather than through a common mechanism, might allow more precise regulation of their individual expression patterns (Cho, 2005).

It is noteworthy that although 4EHP is conserved through evolution, Bcd exists only in higher dipterans. Thus, in other organisms, 4EHP must function during development through proteins that are analogous to Bcd. In summary, this study describes a novel mode of translational control in Drosophila development. Because cap-dependent translation regulation plays such an important role in gene expression, and since 4EHP is also expressed in somatic cells, it is predicted that examples of d4EHP-mediated translational repression other than cad are most likely to exist (Cho, 2005).

Bicoid mRNA is neither fully polyadenylated nor translated in sarah mutant eggs

The Drosophila modulatory calcineurin-interacting protein (MCIP) sarah (sra) is essential for meiotic progression in oocytes. Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract; it is unknown whether calcium signaling is involved. MCIPs [also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)] are highly conserved regulators of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 1 and 2. Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity, their in vivo functions remain unclear. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrest phenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. This is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process (Takeo, 2006; Horner, 2006).

The translation of Bicoid (Bcd) upon activation organizes anterior development in embryos. During oogenesis, bcd mRNA is synthesized in nurse cells and transported to oocytes, where it remains untranslated. Upon activation, bcd mRNA is rapidly polyadenylated and translated. To test for effects of sarah on bcd mRNA polyadenylation, the length of the poly(A) tails on bcd mRNAs was measured by using the PCR poly(A) test (PAT). In the wild-type, more than 120 A's are added to these RNAs within 1 hr of egg laying and activation. In contrast, eggs laid by sra mutant females add only about 64 nucleotides of poly(A) to bcd mRNA upon egg laying, similar to the 80 base extension that occurs in cortex mutants (Horner, 2006).

The failure of sra mutant eggs to fully polyadenylate bcd transcripts predicts these eggs will be compromised in their ability to translate Bcd protein. To test this assumption, lysates from fertilized, laid eggs of sra mutant females were probed for Bcd on Western blots. As controls, ovaries from wild-type females as well as fertilized and unfertilized laid eggs from wild-type and siblings heterozygous for the same sra alleles were also examined. In wild-type and heterozygous controls, Bcd is not observed in mature, unactivated oocytes but accumulates to high levels upon activation, as expected. However, no Bcd translation was observed in eggs laid by any sra mutants (Horner, 2006).

UAP56 RNA helicase is required for axis specification and cytoplasmic mRNA localization in Drosophila

mRNA export from the nucleus requires the RNA helicase UAP56 (Helicase at 25E) and involves remodeling of ribonucleo-protein complexes in the nucleus. This study shows that UAP56 is required for bulk mRNA export from the nurse cell nuclei that supply most of the material to the growing Drosophila oocyte and for the organization of chromatin in the oocyte nucleus. Loss of UAP56 function leads to patterning defects that identify uap56 as a spindle-class gene similar to the RNA helicase Vasa. UAP56 is required for the localization of gurken, bicoid and oskar mRNA as well as post-translational modification of Osk protein. By injecting grk RNA into the oocyte cytoplasm, this study shows that UAP56 plays a role in cytoplasmic mRNA localization. It is proposed that UAP56 has two independent functions in the remodeling of ribonucleo-protein complexes. The first is in the nucleus for mRNA export of most transcripts from the nucleus. The second is in the cytoplasm for remodeling the transacting factors that decorate mRNA and dictate its cytoplasmic destination (Meignin, 2008).

UAP56 is a conserved member of the DExH/D RNA helicase superfamily implicated in many aspects of RNA metabolism including general mRNA export from the nucleus. In human cells, UAP56 is preferentially associated with spliced mRNA and has a major role in bulk mRNA export (Gatfield, 2001). Furthermore, Xenopus UAP56 and its yeast homologue Sub2p are thought to be required co-transcriptionally for the recruitment of the mRNA export factor Aly/REF (see Drosophila Aly) to mRNA. In yeast, the THO complex, which functions in transcription elongation, interacts with mRNA export factors to form the TREX (TRanscription/EXport) complex, linking transcription and export. The human THO complex becomes associated with spliced mRNA during the course of splicing. In Drosophila cells, UAP56 has been shown to be essential for the export of both spliced and intronless poly(A)+ mRNAs. Drosophila uap56 is an essential gene that was first identified as an enhancer of position effect variegation and encodes a nuclear protein named Hel25E/UAP56. UAP56 is proposed to promote an open chromatin structure by unwinding or releasing the mRNA from the site of transcription. It is also thought to be involved in regulating the spread of heterochromatin (Meignin, 2008).

During Drosophila oogenesis, the antero-posterior and dorso-ventral axes of the future embryo are specified through the cytoplasmic localization and translational regulation of a large number of specific transcripts. The most extensively studied mRNAs are gurken (grk) that encodes a TGFβ signal, bicoid (bcd) that encodes the anterior morphogen, and oskar (osk), which specifies posterior structures and the future germ line. All transcripts in the oocyte are thought to be transcribed in the nurse cell nuclei and transported through actin-rich ring canals into the oocyte, where they are selectively localized by transport on microtubules (MTs) by molecular motors. Some of these mRNAs are then localized within the oocyte cytoplasm. During their complex path of localization, these transcripts are thought to be present in large RNP complexes that contain a variety of RNA binding proteins. The composition of these complexes is thought to vary during the different steps of the biosynthesis, export and localization of the transcripts, and RNA helicases play essential roles in remodeling the RNPs during RNA processing, transport, localization, anchoring and translation (Meignin, 2008).

RNA helicases are also likely to be involved in remodeling a diverse range of trans-acting factors required for mRNA localization. These include cytoplasmic determinants and motor cofactors such as BicaudalD (BicD) and Egalitarian (Egl) as well as nuclear components such as hnRNPs or splicing factors. Such trans-acting factors are thought to dictate which molecular motor the transcripts associate with and therefore their cytoplasmic destination. For example, in the Drosophila oocyte, oskar (osk) mRNA requires Kinesin 1 to transport it to the plus ends of MTs while grk mRNA requires cytoplasmic Dynein (Dynein) to transport it to the minus ends of MTs (Meignin, 2008).

In some cases, factors recruited to the RNA in the nucleus remain with the RNA and function in the cytoplasm. For example, the nuclear exon-exon junction components (EJC) Mago nashi, Y14 and eIF4AIII are recruited by osk transcripts in the nucleus and then play a role in its cytoplasmic localization. Once at its final destination at the posterior pole, osk mRNA is translated using two distinct initiation codons, resulting in two different proteins. The short form of Osk promotes the assembly of polar granules by recruiting Vasa, a member of DExH/D-box family of putative RNA helicases at the posterior pole of the oocyte. Vasa is also required for promoting translation of grk through an interaction with the translation factor eIF5B/dIF2. In contrast, grk transcripts are able to recruit in the cytoplasm all the factors required for their localization (Meignin, 2008).

In Drosophila, at least 12 genes are members of the DExH/D RNA helicase superfamily. One of the best described, vasa, was originally identified as a member of the posterior class of maternal effect genes. Vasa is required for the translation of osk and nos mRNAs during the assembly of the pole plasm and for the localization and translation of grk mRNA during oogenesis. Other RNA helicases play a role during Drosophila oogenesis; belle (bel), hel25E or uap56, spindle E (spn-E) and eIF-4AIII. In addition, the small repeat-associated siRNAs (rasiRNAs) pathway, containing spn-E, aubergine and armitage, is required for axis specification. In many of these cases, the intracellular localization and translational regulation of bcd, osk and grk mRNA are affected to varying degrees (Meignin, 2008).

This study tested whether the RNA helicase UAP56 is required in the cytoplasm for mRNA localization and post-translational modification in addition to its well-studied roles in the nucleus in splicing and mRNA export. By creating new alleles of the gene, it was shown that uap56 is a spindle-class gene. uap56 mutants have strong Dorso-ventral egg shell defects caused by a mislocalization of grk mRNA and its incorrect translational regulation. grk mRNA injected into the oocyte cytoplasm of uap56 mutants fails to localize correctly, suggesting that the uap56 phenotype is due to a lack of a factor required in the cytoplasm for mRNA localization. It was also shown that UAP56 plays a role in osk and bcd mRNA localization and Osk post-translational modification. Thus UAP56 plays multiple roles in mRNA localization and post-translational modification in the oocyte cytoplasm. It is proposed that UAP56 is required for remodeling of cytoplasmic RNP complexes required for mRNA localization and post-translational modification (Meignin, 2008).

UAP56 is an RNA helicase that has been shown to play important roles in mRNA metabolism in the nucleus. In Drosophila, UAP56 is a component of the Exon Junction Complex (EJC) (Gatfield, 2001) and is required in tissue culture cells for bulk mRNA export from the nucleus. Using new mutations in the gene, this study has shown that UAP56 is also required for bulk mRNA export during Drosophila oogenesis. UAP56 also has a novel and unexpected role in mRNA localization and post-translational modification in the cytoplasm since uap56 mutants have defects in grk, osk and bcd mRNA localization and Osk post-translational modification. uap56 mutants show strong dorso-ventral egg shell defects due to disruption in cytoplasmic transport of grk mRNA, in addition to other phenotypes that display phenotypes that define uap56 as a spindle-class gene, like the RNA helicase and posterior group gene vasa. Therefore, these data have uncovered a new cytoplasmic role for UAP56 in mRNA transport and post-translational modification. It is proposed that UAP56 is required in the oocyte cytoplasm to remodel RNP complexes involved in mRNA localization and post-translational modification (Meignin, 2008).

The analysis of new alleles of the uap56 gene has revealed that UAP56 is required for general nuclear mRNA export from the nurse cell nuclei. This explains why it was impossible to study the uap56 null mutants during oogenesis since a total block in mRNA export causes cell lethality. The current observations are in good agreement with the previous work showing that UAP56 is essential for bulk mRNA export in Drosophila tissue culture cells and other model systems. In wild-type, the intracellular distribution of bulk polyadenylated RNAs reveals a cytoplasmic localization with an accumulation in the Nuage. In contrast, this distribution is disrupted in uap56 mutants. During Drosophila oogenesis, the Nuage is characterized by electron dense germ line specific structures that form around the nurse cell nuclei. It is suggested that the Nuage someway facilitates the assembly of mRNP particles that mediate the transport, translational regulation and storage of specific transcripts. The Nuage contains Vasa, Maelstrom, Aubergine and Belle, all thought to be required for axis specification in the oocyte. Most components of the Nuage are also localized at the posterior pole of the Drosophila oocyte. This study found that poly(A)+ RNA is present at high levels in particles in the Nuage and that UAP56 is involved in the formation of the Nuage since Vasa protein is absent from the Nuage in uap56 mutants. However, UAP56 is not specifically localized in the Nuage nor at the posterior pole, so UAP56 is neither a posterior group gene nor a component of the Nuage. These observations are interpreted as indicating that UAP56 is required upstream of Nuage and pole plasm formation. It is proposed that UAP56 is required to facilitate the correct assembly of different mRNAs, including grk and osk with the appropriate RNA binding proteins in RNPs, before, during and possibly after export from the nurse cell nuclei. This assembly is crucial for the downstream events responsible for the transport and assembly of the RNPs into the Nuage (Meignin, 2008).

Interestingly, a small fraction of poly(A)+ RNA was found in the oocyte nucleus, an observation that could be explained in two possible ways. Either a sub-population of RNA is transcribed in the oocyte nucleus or a sub-population of poly(A)+ RNA is transported from nurse cells to oocyte and then imported into the oocyte nucleus. The second explanation is favored for the following reasons. First, it is thought that the oocyte nucleus is transcriptionally inactive. Second, the I factor mRNA is known to be synthesized in the nurse cells, transported into the oocyte and then imported into the oocyte nucleus. While the genetic background that was studied is not very active for I factor transcription, it is possible that the poly(A)+ RNA detected in the oocyte nucleus represents transcripts of other transposable elements or some endogenous transcripts that follow the same pattern of biogenesis and import into the oocyte nucleus (Meignin, 2008).

During oogenesis and early embryogenesis, the majority of UAP56 protein is present in the nucleus and is probably associated with DNA. These results are consistent with previous work showing that UAP56 is closely associated with salivary gland chromosomes and localized to the nuclei of Drosophila embryos and ovaries as well as acting as an enhancer of position affect variegation in Drosophila and affecting heterochromatic gene expression in yeast. These data together with the conclusion that uap56 is a spindle-group gene strongly suggest that the gene is involved in chromatin organization (Meignin, 2008).

In yeast, Sub2p associates with the TREX (TRansport-EXport) complex, which is required directly for transcription elongation, splicing and export and is recruited during transcription. In contrast, the mammalian TREX complex is recruited during splicing. It is proposed that, in Drosophila, UAP56 has an intermediate role between yeast and human cells. It is likely to bind mRNA during transcription and be involved in splicing in a similar manner to nonsense-mediated mRNA decay (NMD) (Meignin, 2008).

The new alleles of uap56 reveal a role of UAP56 in mRNA localization and translational modification in the cytoplasm, key processes in axis specification. A reduction in the intensity of tau GFP staining in the oocyte was found, although the antero-posterior gradient, which is essential for mRNA localization, was unaffected. While the possibility cannot be excluded that the reduction in efficiency of localization of injected grk mRNA is due to a lower density of MTs in the oocyte, the alternative interpretation is favored, in which UAP56 plays a more direct cytoplasmic role in promoting remodeling of factors required for grk RNA localization (Meignin, 2008).

The observations in the context of the previous work on UAP56 suggest that, as well as being a general RNA export factor in Drosophila, the protein has a specific role in the localization and post-translational modification of a sub-population of key transcripts. In this respect, UAP56 is similar to some other components of the EJC, such as Mago nashi, Y14 and eIF4AIII, which are involved in mRNA localization and post-translation control of a subset of Drosophila transcripts. Interestingly, mutants in the small repeat-associated siRNAs (rasiRNAs) pathway also show similar defects in axis specification to uap56 mutants, raising the possibility that UAP56 is a component of the rasiRNA pathway. Nevertheless, the results are surprising, given that UAP56 is thought of as a ubiquitous house keeping gene with an essential function in the export of all mRNAs. A partial loss-of-function of an equivalent essential general mRNA export factor, NXF1, does not give rise to specific developmental defects, such as the ones observed with uap56 alleles. rasiRNA mutants show an accumulation of double strand breaks in egg chambers. However, no differences were found in the accumulation of H2A staining of double strand breaks between wild type and mutant germaria, in contrast to armi, aub and spn-D mutants. It is concluded that the defects observe in axis specification in uap56 mutants are not due to double strand breaks perturbing signaling in the germ line. Therefore, it is proposed that, unlike NXF1, UAP56 is likely to remain on the RNA after export and has a role in the remodeling of RNP complexes in the cytoplasm. This idea is supported by the fact that UAP56 is detected in the cytoplasm as well as the nucleus. However, no UAP56 colocalized is observed with mRNA in the cytoplasm or recruited by injected RNA. These observation are interpreted as indicating that UAP56 may only associate with RNA transiently in the cytoplasm, while acting as a cytoplasmic RNA remodeling factor. Such a role is novel for UAP56, and it remains to be discovered whether it is a general feature of this RNA dependent helicase in a variety of model systems (Meignin, 2008).

Formation of the bicoid morphogen gradient: an mRNA gradient dictates the protein gradient

The Bicoid (Bcd) protein gradient is generally believed to be established in pre-blastoderm Drosophila embryos by the diffusion of Bcd protein after translation of maternal mRNA, which serves as a strictly localized source of Bcd at the anterior pole. However, evidence suggests the Bcd gradient is preceded by a bcd mRNA gradient. This study has revisited and extended this observation by showing that the bcd mRNA and Bcd protein gradient profiles are virtually identical at all times. This confirms a previous conclusion that the Bcd gradient is produced by a bcd mRNA gradient rather than by diffusion. Based on the observation that bcd mRNA colocalizes with Staufen (Stau), it is proposed that the bcd mRNA gradient forms by a novel mechanism involving quasi-random active transport of a Stau-bcd mRNA complex through a nonpolar microtubular network, which confines the bcd mRNA to the cortex of the embryo (Spirov, 2009).

Revisiting the formation of the morphogenetic bcd gradient, published results (Frigerio, 1986) have been corroborated and extended by demonstrating a gradient of bcd mRNA that is very similar to that of the Bcd protein. Therefore, the results contradict the SDD model (refering to the localized synthesis, diffusion and spatially uniform degradation of the Bcd protein). Although the SDD model correctly predicts an exponential Bcd protein gradient, the diffusion coefficient of Bcd, as measured in syncytial-blastoderm embryos, is two orders of magnitude too low to account for the fact that the steady state of its gradient is reached at syncytial blastoderm, a finding that is in serious conflict with this model. The results strongly suggest that the bcd mRNA gradient forms the protein gradient and is established by transport of the mRNA along the cortex of the embryo (Spirov, 2009).

The following model was used to explain the formation of the bcd mRNA gradient. The establishment of the bcd RNA gradient by nuclear cycle 10 and its disappearance during early nuclear cycle 14 occur in five phases. (1) The bcd RNA, which is associated with Stau and presumably many other proteins and anchored to the actin cytoskeleton at the anterior cortex of the mature oocyte, is released upon fertilization by calcium signaling. (2) This Stau-bcd mRNA complex is transported posteriorly along microtubules emanating from numerous microtubule-organizing centers (MTOCs) that are closely spaced and distributed throughout the cortex of the embryo. The posterior transport of bcd mRNA is driven by its concentration gradient. (3) This transport is arrested by the breakdown of the cortical microtubular network when the nuclei reach the cortex. At this time, a new microtubular network of astral microtubules forms. These extend from the centrosomes, located between the plasma membrane and each nucleus, and surround each nucleus with apical-basal polarity. Thus, the bcd mRNA gradient is established by nuclear cycle 10 and does not change until the end of nuclear cycle 13. (4) During syncytial blastoderm, the bcd mRNP particles are transported apically on astral microtubules by the minus end-directed dynein/dynactin motor complex, a process that depends on the maternal proteins Bicaudal D (BicD) and Egalitarian (Egl). (5) Whereas bcd mRNA is stable until nuclear cycle 13, it is rapidly degraded during early nuclear cycle 14. This degradation is assumed to be mediated by Stau and is triggered by apical factors and signals (Spirov, 2009).

This model combines the current results with those reported by others. The evidence that led to this ARTS (active RNA transport and protein synthesis) model will be discussed in detail. Despite its speculative aspects, it should serve as a useful hypothesis for future experiments that test its predictions. In addition, the model postulates a new principle to explain the formation of the bcd mRNA gradient: a quasi-random transport through a cortical microtubular network that is driven by a high initial concentration of bcd mRNA at the anterior pole (Spirov, 2009).

Stau protein binds to the 3'UTR of bcd mRNA in oocytes and colocalizes with bcd mRNA at the anterior pole of freshly laid eggs. Additional proteins probably stabilize the interaction of Stau with bcd mRNA in the embryo, as in oocytes. Localization of bcd mRNA to the anterior pole is established by continual active transport of the Stau-bcd mRNA complex on microtubules, mediated by the minus end-directed motor dynein, when nurse cells empty their content into stage 10B-13 oocytes. Subsequent anchoring of the Stau-bcd mRNA complex to the actin cytoskeleton stabilizes its anterior localization in mature oocytes. This anchoring step depends on swallow (swa), the product of which interacts with the dynein light chain and γTub37C, which is part of the MTOC at the anterior end of oocytes. Upon fertilization, calcium signaling releases the Stau-bcd mRNA complex from the actin cytoskeleton, which depends on the product of the sarah (sra) gene, an inhibitor of the calcium-dependent phosphatase calcineurin. Swa protein is no longer required and is degraded (Spirov, 2009).

A network of microtubules, in which the MTOCs are closely spaced (separated by a few microns), occupies the cortical region of embryos during nuclear cycles 1-9. Consistent with this observation is the pattern of cortical staining of early embryos for several Dgrips, proteins of the γ-tubulin ring complex that caps the minus ends of microtubules at MTOCs. Evidently, these microtubules nucleate from MTOCs that are established in late oocytes. No function has been reported previously for this cortical microtubular network, which breaks down at the end of nuclear cycle 9. It is proposed that its existence is crucial for the formation of the bcd mRNA gradient (Spirov, 2009).

A mechanism based on diffusion of the bcd mRNA cannot explain the gradient observed because bcd mRNA is restricted to the cortex of the embryo. Diffusion of bcd mRNA to the interior would dramatically reduce its concentration along the cortex, where its function is required, because unlike for Bcd protein in the SDD model, there is no source replenishing the lost bcd mRNA. Active transport of a Stau-bcd mRNA complex on microtubules, similar to that observed in late-stage oocytes, is suggested by the striking colocalization of Stau and bcd mRNA until the latter disappears. However, the microtubules with MTOCs located at the anterior pole are disassembled in late oocytes. Indeed, the cortical microtubular network in embryos at nuclear cycles 1-9 shows no sign of an overall polarity, but appears to be nonpolar, with its plus ends growing in all directions from MTOCs closely spaced throughout the cortex (Spirov, 2009).

How can such a nonpolar microtubular network establish a bcd mRNA gradient by active transport of the Stau-bcd mRNP particles? Because the network exhibits no polarity, it supports only random transport as would occur by diffusion. The only restriction to the random transport is its confinement to the cortex of the embryo. Like diffusion, it is driven by the concentration gradient of the transported molecules, here by the high initial concentration of bcd mRNA at the anterior pole. Average transport velocities of Stau-bcd mRNA complexes on microtubules, as measured in stage 10B-13 oocytes, range from 0.36 to 2.15 µm/second. Such a non-random transport in the embryo would move bcd mRNA molecules within minutes from the anterior to the posterior pole and thus destroy its function as an anterior morphogen. Therefore, it seems crucial that bcd mRNA transport in the embryo occurs through a nonpolar microtubular network. It is additionally important that the time of 90 minutes that is required to establish the bcd mRNA gradient at 25°C is tuned finely with the time required for the first nine nuclear divisions, after which the nuclei reach the cortex (Spirov, 2009).

The efficiency of a system employing random transport can be estimated from the average posterior drift velocity of bcd mRNAs along the cortex. When, 90 minutes after fertilization, syncytial blastoderm is reached, bcd mRNA has moved posteriorly on average by ~50 µm (from 5% EL at fertilization to 15% EL), which corresponds to an average drift velocity of ~0.01 µm/second. This is 100 times slower than the average transport velocity on a microtubule in the oocyte and is thus rather inefficient. Since this transport of bcd mRNA occurs on a microtubular network with randomly oriented microtubules, it is irrelevant whether transport is mediated by the minus end-directed dynein/dynactin or the plus end-directed kinesin motors. In the oocyte, Stau-bcd mRNP particles are transported exclusively by dynein in a process that depends on the presence of Exuperantia (Exu) in nurse cells. Since Exu disappears from late oocytes, this might permit Stau-bcd mRNPs to interact with dynein or kinesin upon their release from the actin cytoskeleton (Spirov, 2009).

Just before the present study was submitted, transport in oocytes through a microtubular network exhibiting only a slight directional bias (57% of plus ends oriented posteriorly) was shown to localize Stau-oskar (osk) mRNA particles to the posterior pole (Zimyanin, 2008). Although it is conceivable that the bcd mRNA gradient is established through such a biased microtubular network, the net average posterior velocity in oocytes of 0.03 µm/second (Zimyanin, 2008) would displace the bcd mRNA on average by 162 µm towards the posterior pole of the embryo by the time the bcd RNA gradient is established. This is more than twice the observed average posterior displacement of bcd mRNA. Nevertheless, the bcd mRNA gradient might be established through such a biased microtubular network if transport is mediated by both minus- and plus-end motors. In such a case, however, the average posterior drift velocity would also depend on the availability of both motors. If the probability of Stau-bcd mRNP interacting with either motor is the same, transport by the microtubular network becomes independent of its directional bias, and the network behaves like the nonpolar microtubular network. However, a nonpolar microtubular network is favored in the embryo because it seems more robust to disturbances (Spirov, 2009).

An intriguing feature of the bcd mRNA gradient during nuclear cycles 10-13 is the maintenance of a constant apical gradient similar to the basal gradient. It has been noted that bcd transcripts are localized to the narrow apical periplasm at late syncytial blastoderm, and that this localization depends on a signal in their 3'UTR. Apical transport of bcd mRNA becomes evident during nuclear cycle 14, when the excess of basal bcd mRNA disappears more rapidly than its apical counterpart (Spirov, 2009).

Although no net apical transport of bcd mRNA is apparent before its degradation during nuclear cycle 14, the establishment of an astral microtubular network during nuclear cycle 9 suggests that it might occur as early as nuclear cycle 10. Such a system, capable of transporting Stau-bcd mRNA particles to the apical periplasm, might be important to stabilize the bcd mRNA gradient against disturbances by the strong periplasmic flow that is observed in the cortex during nuclear cycles 10-13. Nevertheless, if Stau-bcd mRNA complexes detach when they reach the minus ends at the apical MTOCs, some apically localized bcd mRNAs might be subject to the periplasmic flow. Such a disturbance would be minor, as it would be corrected immediately by rapid apical transport of Stau-bcd mRNA particles, which occurs at a velocity of 0.5 µm/second (Spirov, 2009).

Why is it important to localize bcd mRNA not only to the basal, but also to the apical, nuclear periplasm? An answer is probably provided by elegant studies that have demonstrated that the nuclear concentration of Bcd protein remains approximately constant at a certain position along the anteroposterior axis during syncytial blastoderm (Gregor, 2007). This finding was surprising in view of the fact that the number of nuclei double after each nuclear division, their volume increases by 30% during interphase of each nuclear cycle, and the Bcd concentration drops fourfold when nuclear membranes disappear during mitosis. It was explained by measurements revealing that nuclear import of Bcd is sufficiently rapid to maintain a high and constant nuclear Bcd concentration. Hence, it might be crucial that Bcd can be imported through the entire nuclear surface (Gregor, 2007). Consistent with an accelerated nuclear import of Bcd by the product of the lesswright (lwr) gene (Epps, 1998), Lwr was found in cleavage-stage and syncytial-blastoderm nuclei (Spirov, 2009).

Whereas bcd mRNA is stable before nuclear cycle 14, basal bcd mRNA disappears owing to its degradation and transport to the apical periplasm within ~10 minutes of early nuclear cycle 14. Thus, the estimated half-life of basal bcd mRNA is ~2 minutes. Apical bcd mRNA decreases only when basal bcd mRNA becomes limiting. At this time, the estimated half-life of apical bcd mRNA is also ~2 minutes. Therefore, bcd mRNA is degraded in the basal and apical periplasm, or only in the latter. This degradation is presumably mediated by a bcd instability element (BIE) located within a 43-nucleotide sequence following the stop codon. In mammals, Stau may trigger the degradation of an mRNA by binding to its 3'UTR and to the nonsense-mediated decay (NMD) factor Upf1, in a process that is different from NMD and is called Staufen-mediated mRNA decay (SMD) (Kim, 2005). As the Drosophila genome encodes a Upf1 homolog, Stau might well function not only in the transport of bcd mRNA but also in its degradation (Spirov, 2009).

Since Bcd protein disappears ~25 minutes after bcd mRNA, a lag during which its level decreases at least tenfold, its half-life is less than 8 minutes at this time. The presence of a conserved PEST sequence in Bcd might be responsible for its short half-life, presumably also during earlier stages, a hypothesis that is consistent with the similarity between the slopes of the bcd mRNA and protein gradients (Spirov, 2009).

There are many ways to generate a morphogenetic gradient. The original proposal of how the Bcd protein gradient forms closely followed Wolpert's model of generating a morphogenetic gradient by a localized source synthesizing the morphogenetic molecules that are subject to diffusion and spatially uniform degradation. This model predicts a steady state at which the Bcd concentration decays exponentially along the anteroposterior axis. This study now shows that the Bcd protein gradient is generated by an entirely different mechanism. Since there is no source of bcd mRNA, its posterior transport from the anterior pole must be arrested when the optimal gradient is reached. This arrest is triggered by the breakdown of the cortical microtubular network and is well timed with the arrival of the nuclei at the cortex, when gap genes are activated by the Bcd protein. At this time, the gradient is established and remains constant until nuclear cycle 14, when bcd mRNA is rapidly degraded. Thus, the bcd mRNA gradient is not established as a steady state, but by a process that is terminated by the breakdown of the microtubular network required for its formation (Spirov, 2009).

Compared with the diffusion-based mechanism of the SDD model, a random active transport system has several advantages for the formation of the bcd mRNA gradient. The microtubular network is able to confine the movement of the bcd mRNA to the space where its function is required. The final shape of the gradient depends on several parameters: the initial concentration of bcd mRNA at the anterior pole, the transport velocity along microtubules, the average travel time per microtubule, the time between discharge from one and reloading onto another microtubule, and the time when transport is arrested by the breakdown of the microtubular network that supports the random transport. In addition, the availability of minus end- and plus end-directed motors might further influence the generation of the bcd mRNA gradient by random transport. Therefore, perhaps the greatest advantage of random active transport is that variations in these parameters during evolution permit the adaptation of the gradient to its optimal shape at the time when its function is required during development. For these reasons, it is suspected that random active transport represents a general mechanism that might have found wide application during evolution (Spirov, 2009).

Distinguishing direct from indirect roles for bicoid mRNA localization factors

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. This study used live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. Swallow and bicoid mRNA are shown to be transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, evidence is presented that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. These results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead a model is proposed for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport (Weil, 2010).

A direct role for Swa in either transport or anchoring of bcd mRNA predicts and requires that the protein be colocalized with bcd mRNA during transport or anchoring. This study tested this prediction conclusively in three ways. First, an OMX microscope with highly sensitive and rapid multi-channel imaging was used to study to movement of Swa and bcd RNA particles simultaneously. Second, advantage was taken of the increased resolution of the OMX microscope with fixed material to analyze the precise locations of Swa and bcd at the anterior oocyte cortex. Importantly, the results show conclusively that Swa and bcd mRNA move independently to the anterior and occupy distinct domains at the anterior cortex. Third, the subcellular distributions of Swa and bcd was determined at EM resolution, demonstrating that bcd is mostly in particles near the anterior cortex whereas Swa is mostly confined to the plasma membrane of the entire oocyte. How the membrane association of Swa, which does not contain a transmembrane domain and is not predicted to harbor lipid modifications, is mediated remains to be investigated (Weil, 2010).

At the plasma membrane, Swa is found in close proximity to the cortical actin cytoskeleton. Together with the defects in the cortical actin cytoskeleton observed in swa mutants, this indicates a role for Swa in organization of the actin cytoskeleton. Whereas the actin cytoskeleton is not required for anchoring bcd at the anterior cortex until the very end of oogenesis (Weil, 2008), it plays an indirect role in bcd localization by anchoring the anterior microtubules required for the continual transport of bcd during stages 11-13. The results indicate that, in swa mutants, these microtubules remain intact but are not properly attached to or organized at the cortex, such that bcd transport is non-productive (Weil, 2010).

Swa is not limited to the anterior, however, and can be detected along the entire cortex of the oocyte. Moreover, in swa mutants, the cortical actin cytoskeleton is disrupted throughout the entire oocyte. Accordingly, some swa alleles show defects in posterior localization of osk mRNA at late stages of oogenesis (Pokrywka, 2004). Since actin is required for osk mRNA anchoring, this phenotype could be a result of disruption of the actin cytoskeleton. Clues as to how Swa regulates the actin cytoskeleton are not readily apparent from the Swa protein sequence, and an understanding of this mechanism awaits further biochemical analysis of Swa (Weil, 2010).

IEM results provide direct evidence that bcd mRNA is packaged with Stau and dynein into RNPs that are enriched at, and presumably transported to, the anterior after stage 10b. Indeed, live imaging using the OMX system showed co-transport of bcd and Stau in the same dynamic particles. In addition to its role in bcd localization, Stau is a key component of osk RNPs and is required for kinesin-dependent transport of osk to the oocyte posterior. Thus, Stau is a component of two independent transport RNPs, each associated with a different motor protein for transport to distinct locations within the oocyte. Whether Stau, which contains five double-stranded RNA-binding domains, interacts directly with bcd and osk mRNAs or indirectly by association with sequence-specific mRNA-binding proteins, remains to be determined. However, structure/function analysis of Stau suggests that different Stau double-stranded RNA-binding domains may determine which transport factors are linked to each mRNA. Furthermore, as Stau functions in osk localization during stages 8-9 but is required for bcd localization only after stage 10b, the assembly of Stau/bcd transport complexes must be temporally regulated. Stau is also required during the oocyte-to-embryo transition, for the redistribution of bcd from its tight cortical distribution in the oocyte to its more diffuse anterior distribution in the early embryo. Stau has been shown to colocalize with bcd mRNA when it is anchored to the actin cytoskeleton at the latest stages of oogenesis and is retained by bcd particles in the early embryo (Weil, 2008). Thus, Stau remains an integral component of bcd RNPs as they are remodeled from transport to anchoring complexes and finally to their translationally active state in the embryo. In the future, detailed biochemical analysis combined with advanced imaging methods that permit detection of in vivo RNA-protein and protein-protein interactions will be necessary to ascertain how specificity is conferred on localization and anchoring (Weil, 2010).

Drosophila javelin-like encodes a novel microtubule-associated protein and is required for mRNA localization during oogenesis

Asymmetrical localization of mRNA transcripts during Drosophila oogenesis determines the anteroposterior and dorsoventral axes of the Drosophila embryo. Correct localization of these mRNAs requires both microtubule (MT) and actin networks. This study identified a novel gene, CG43162, that regulates mRNA localization during oogenesis and also affects bristle development. The Drosophila gene javelin-like, which was identified based on its bristle phenotype, is an allele of the CG43162 gene. Female mutants for jvl produce ventralized eggs owing to the defects in the localization and translation of gurken mRNA during mid-oogenesis. Mutations in jvl also affect oskar and bicoid mRNA localization. Analysis of cytoskeleton organization in the mutants reveal defects in both MT and actin networks. Jvl protein colocalizes with MT network in Schneider cells, in mammalian cells and in the Drosophila oocyte. Both in the oocyte and in the bristle cells, the protein localizes to a region where MT minus-ends are enriched. Jvl physically interacts with SpnF and is required for its localization. Overexpression of Jvl in the germline affects MT-dependent processes: oocyte growth and oocyte nucleus anchoring. Thus, these results show that a novel MT-associated protein affects mRNA localization in the oocyte by regulating MT organization (Dubin-Bar, 2011).

In order to investigate further the role of Spn-F in MT organization, new proteins that interact with Spn-F or Ik2 were sought. This study led to identification of the gene CG43162 as a novel MT-associated protein, which is part of this complex. Moreover, the study showed that CG43162 encodes the javelin-like (jvl) gene. Several lines of evidence suggest that that jvl encodes the CG43162 gene: (1) Using fine deficiency mapping of jvl mutants showed that jvl is found in CG43162 region; (2) it was shown that downregulation of CG43162 specifically in the bristles led to defects in bristle morphology, similar to the defects found in jvl mutants; 3) furthermore, a mutation in CG43162 (CG43162D590) failed to complement jvl in both ovarian and bristle phenotypes, suggesting that CG43162D590 and jvl are two different alleles of the same gene; and (4) expression of CG43162 protein in oocytes was found to rescue jvl female sterility. Considering all of these results, it is concluded that the CG43162 gene encodes jvl (Dubin-Bar, 2011).

Moreover, it is suggested that Jvl is part of the Spn-F and Ik2 complex, based on the following evidence: (1) Spn-F physically interacts with Jvl (yeast two hybrid and GST pull-down assays); (2) Spn-F physically interacts with Ik2 (Dubin-Bar, 2008); (3) jvl shares similar mRNA localization and bristle defects to spn-F and ik2; (4) Spn-F and Ik2 colocalize with Jvl to MT, where Jvl determines this localization pattern (Dubin-Bar, 2011).

For further analysis of the jvl gene, Jvl protein localization was characterized. For this purpose, the localization of Jvl protein in S2R+ cells and human cells was analyzed. GFP-Jvl fusion protein was localized to the MT network. Next, the localization pattern of Jvl during oogenesis was analyzed. Using an antibody raised against the Jvl protein, it was found that Jvl is localized to the region where the MT minus-ends reside. At early stages of oogenesis, Jvl protein localizes as a tight crescent in the posterior pole of the oocytes. During mid-oogenesis, Jvl protein is localized all around the cortex, with enrichment at the anterior pole. It was also demonstrated that GFP-Jvl colocalizes with MTs in the nurse cells. Moreover, in the bristles, GFP-Jvl is localized asymmetrically, accumulating at the bristle tip, where other MT minus-end markers are found. Considering these results, indicating that Jvl localizes with the MT network in S2R+ and human cells along with its localization in the egg chamber and developing bristle, it is concluded that Jvl protein is associated with the MT network, specifically with the MT minus-ends (Dubin-Bar, 2011).

jvl1 mutants are female fertile. However, flies hemizygous for jvl1 and flies transheterozygous for jvl (jvl1/jvl2) are female sterile. Beside sterility, it was noticed that the jvl mutant females laid eggs with dorsal-ventral defects. Determination of dorsal-ventral polarity of the eggshell depends on Grk protein signaling. In the hemizygous mutants, grk mRNA localizes in the anterior margins of the oocyte and in ectopic sites inside the oocyte. It has been suggested that grk mRNA moves in two distinct steps, both of which require MT and the motor protein Dynein. Each step depends on a different MT network. First grk mRNA moves towards the anterior of the oocyte, where it localizes transiently, and then to its final localization in the dorsal anterior corner of the oocyte. In jvl mutants, grk mRNA does not reach its final localization in the dorsal anterior corner of the oocyte, suggesting that the MT network upon which this step depends might be impaired in jvl mutants. This MT network is specifically associated with the oocyte nucleus and the minus-end in the dorsal-anterior corner of the oocyte. Next, it was found that Grk protein in jvl mutants is also mislocalized. Grk protein is colocalized with ectopic actin puncta close to the anterior of the oocyte. This localization pattern is also observed in Bicaudal-C and trailer-hitch mutants. It has been suggested that the sequestration of Grk in the actin cages interfered with the signaling to the follicle cells; therefore, it is suggested that sequestration of Grk in the actin cages in jvl mutant females similarly led to the dorsal-ventral polarity defects of the eggshell. In addition to the effect on grk mRNA and protein localization, jvl also affects bcd and osk mRNA localization. In wild-type, bcd mRNA is localized to the anterior pole of the oocyte facing the nurse cells, whereas osk mRNA is localized to the opposite posterior pole. The polar localization of these two mRNAs is maintained throughout the rest of oogenesis and well into early embryogenesis. The anterior localization of bcd requires both intact MTs and dynein motor protein function. osk localization to the posterior pole is achieved by two phases of transport: long-range MT-dependent transport by kinesin to the posterior, followed by actomyosin V-dependent positioning at the oocyte cortex (Dubin-Bar, 2011).

What could be the function of Jvl protein during oogenesis? The effects of jvl on grk and bcd mRNA localization, along with the particular changes affecting cytoskeletal organization close to the oocyte nuclear membrane as evident for Nod:KHC:β-gal localization and Tau mislocalization, suggest that jvl might be involved in either transport to the minus-end of MTs or in the organization of the minus-ends of the microtubule around the oocyte nucleus, as been suggested for its interactor, Spn-F (Abdu, 2006). However, it was also noticed that in jvl mutants, osk mRNA and protein are mislocalized. These phenotypes are probably not due to defects in either transport or organization of the MT plus-end, as the plus-end motor protein Kinesin I was properly localized as in the wild type. Examination of the cytoskeleton components of the oocyte shows that both actin and MTs are misorganized in jvl mutants. The MT levels along the anterior cortex of the oocyte were reduced with specific effects on the MT that surrounds the oocyte nucleus. However, ectopic aggregations of the actin cages were found in the middle of the oocyte. The defects in the organization of both actin and MT network, together with the defects in osk mRNA and protein localization, suggest that jvl could provide a connection between the actin and MT network. In summary, these results suggest that jvl plays a role in organization of the MT in the oocyte or in the stabilization of the connection between MT and actin cytoskeleton in the oocyte (Dubin-Bar, 2011).

This study also examined the effects of overexpression of Jvl in the germline. Overexpression of Jvl with different germline-specific Gal 4 affects oocyte growth, oocyte localization and, in later stages, oocyte nucleus localization. Interestingly enough, all of these phenotypes could arise from effects on MT network function (Dubin-Bar, 2011).

Oocyte growth depends on several processes: early in oogenesis, until stage 7, the oocyte grows at approximately the same rate as a single nurse cell. At these stages, oocyte growth is due to the transport of mRNAs and proteins, including products of early pattern-formation genes from the nurse cells to the oocyte. This transport is a microtubule-dependent process. Later in oogenesis, after stage 7, oocyte growth depends on the transport of components such as lipid droplets, mitochondria and other single particles from the nurse cells into the oocyte. This transport is an actin-dependent process. Beginning in stage 8, the oocyte expands through the uptake of yolk from the surrounding follicle cells and hemolymph. Consequently, oocyte growth is more rapid than nurse cell growth. During stage 11, the remaining nurse cell cytoplasm is rapidly transferred to the oocyte, resulting in doubling the oocyte volume. Overexpression of Jvl affects oocyte growth during stage 6 to stage 8, although the egg chamber size seems to be similar to that of wild-type stage 6 to 8 egg chambers. In these stages, oocyte growth depends on the transport of nutrients from the nurse cells to the oocyte, suggesting that overexpression of Jvl disrupted this transport. The fact that Orb protein is not detected in Jvl-overexpressing small oocytes strengthens this possibility (Dubin-Bar, 2011).

Another phenotype that was obtained in moderate overexpression of Jvl is mislocalization of the oocyte nucleus in 15% of stage 9 egg chambers. During early stages of oogenesis, the oocyte nucleus localizes to the posterior pole of the oocyte. After stage 7, following Grk signal and reorganization of the MT network, the nucleus migrates towards the anterodorsal corner of the oocyte. Positioning of the oocyte nucleus involves two anchoring steps: first anchoring to the lateral membrane, which requires dynein but not kinesin motor protein; and, second, after it localizes to the anterodorsal corner, anchoring to the anterior cortex of the oocyte, which requires both dynein and kinesin motor proteins. Moreover, nucleus anchoring also requires correct organization of the MT scaffold that surrounds the oocyte nucleus. Moderate expression of Jvl did not affect nucleus position in stage 8 egg chambers. At this stage, the nucleus was always at the dorsal anterior corner, as in the wild type. This finding implies that anchoring to the lateral cortex and migration of the oocyte nucleus is not affected in Jvl-overexpressing ovaries. However, the anchoring of the nucleus to the anterior membrane was affected. This could be due to misorganization of the MT scaffold that surrounds the nucleus. Thus, these results demonstrate that overexpression of Jvl protein affects MT-dependent processes such as transport of determinants from the nurse cells to the oocyte, and anchoring of oocyte nucleus to the anterior cortex of the oocyte. Taking into account the phenotypes detected in jvl mutants, the finding that Jvl is an MT-associated protein, together with the effects of Jvl overexpression on MT-dependent processes during oogenesis, it seems likely that jvl has a role in MT organization during oogenesis (Dubin-Bar, 2011).

Most importantly, although jvl encodes for a protein with no homology beside insects, its association with MT network in mammalian cells, along with its effect on MT network in Drosophila, may suggest the existence of mammalian protein(s) with a function analogous to Jvl (Dubin-Bar, 2011).

αTubulin 67C and Ncd are essential for establishing a cortical microtubular network and formation of the Bicoid mRNA gradient in Drosophila

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS (active RNA transport and synthesis) model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localizes at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfill the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. This study reports hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (1) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (2) long MTs up to 50 microm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the alphaTubulin67C mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. These data strongly corroborate the ARTS model and explain the occurrence of the alphaTubulin67C mRNA gradient (Fahmy, 2014).

Scaling of the Bicoid morphogen gradient by a volume-dependent production rate

An important feature of development is the formation of patterns that are proportional to the overall size of the embryo. But how such proportionality, or scaling, is achieved mechanistically remains poorly understood. Furthermore, it is currently unclear whether organisms utilize similar or distinct mechanisms to achieve scaling within a species and between species. This study investigated within-species scaling mechanisms for anterior-posterior (A-P) patterning in Drosophila melanogaster, focusing specifically on the properties of the Bicoid (Bcd) morphogen gradient. Using embryos from lines artificially selected for large and small egg volume, it was shown that large embryos have higher nuclear Bcd concentrations in the anterior than small embryos. This anterior difference leads to scaling properties of the Bcd gradient profiles: in broad regions of the large and small embryos along the A-P axis, normalizing their positions to embryo length reduces the differences in both the nuclear Bcd concentrations and Bcd-encoded positional information. The origin of Bcd gradient scaling was further traced by showing directly that large embryos have more maternally deposited bcd mRNA than small embryos. The results suggest a simple model for how within-species Bcd gradient scaling can be achieved. In this model, the Bcd production rate, which is dependent on the total number of bcd mRNA molecules in the anterior, is scaled with embryo volume (Cheung, 2011).

Embryonic patterning is a robust process that is insensitive to embryo size. The results show that the Bcd gradient profiles in D. melanogaster embryos exhibit scaling properties. The enhanced size differences between the embryos from the selected Drosophila lines have enabled investigation, in greater depth and clarity, Bcd gradient scaling and its origin. Large embryos have more maternally deposited bcd mRNA than small embryos. The differences between these embryos in both B0 (or Bmax), B being the nuclear concentration of Bicoid, and the amount of bcd mRNA are better explained by the differences in embryo volume than embryo length. In an idealized simple diffusion model, the steady state morphogen concentration at the source is a function of the morphogen production rate. If the number of Bcd molecules produced per unit time is proportional to the total number of bcd mRNA molecules in the anterior of an embryo, then the aggregate Bcd production rate for the embryo should be proportional to the total amount of maternally deposited bcd mRNA. During oogenesis, the majority of bcd mRNA is deposited when nurse cells 'dump' their cytoplasmic contents into the oocyte. If the bcd mRNA concentration in these cytoplasmic contents is similar across different egg chambers at the time of dumping, then the total number of bcd mRNA molecules in an egg should be approximately proportional to its volume, on average. Based on these considerations and the experimental results, a simple model is proposed for how Bcd gradient scaling within a species can be achieved. In this model, deposition of bcd mRNA is dependent on egg volume, which leads to a volume-dependent adjustment of the Bcd production rate in the anterior, allowing the Bcd gradient to achieve scaling in broad regions of the embryo. Several attributes of this model are discussed (Cheung, 2011).

The proposed Bcd gradient scaling model, i.e. a volume-dependent Bcd production rate, represents an early-acting scaling mechanism along the A-P axis in D. melanogaster embryos. In addition, since Bcd acts as a direct and sustained input for target gene transcription, the scaling properties of the Bcd gradient also represent a critical means by which scaled A-P patterning is achieved in broad regions of the embryo (Cheung, 2011).

The proposed volume-dependent production rate mechanism for within-species Bcd gradient scaling differs from the previously identified between-species scaling mechanism. Whereas between-species scaling is achieved by evolutionary adjustment of lambda to scale it with average length L, large and small D. melanogaster embryos have comparable lambda values in absolute length. In a simple diffusion model, lambda2=D/omega, suggesting that between-species scaling is achieved by adjusting the effective diffusion constant D and/or the effective decay rate omega, species-specific properties that are manifested throughout the embryos. In this model, Bcd gradient scaling within a species is achieved through adjusting the Bcd production rate in the anterior (via the adjustment of bcd mRNA deposition). Although the models for within-species and between-species Bcd gradient scaling are distinct from each other, they are not mutually exclusive. For example, it is possible (although untested) that the Bcd production rate also differs in embryos from different species (Cheung, 2011).

The proposed adjustment of the Bcd production rate represents a 'passive', self-correcting mechanism in the sense that it may arise as a unidirectional physiological consequence of how nurse cells provision the egg. Although FISH data clearly demonstrate that large embryos have more bcd mRNA than small embryos, precisely how this is achieved remains unknown. One could imagine that the rate of bcd transcription in nurse cells may be regulated by the cytoplasmic volume through some unknown feedback mechanism(s). Alternatively, the duration of bcd transcription (i.e. bcd mRNA accumulation) is tightly but passively related to the time that it takes for nurse cells to mature and accumulate their cytoplasmic contents (i.e. cytoplasmic volume) prior to dumping. These studies have focused on embryo size variation that arises from either genetic differences or stochastic fluctuations. However, environmental factors may represent an even more important determinant of egg size variation. Since the volume-dependent deposition of bcd mRNA, and thus the adjustment of Bcd production rate, could take place passively, the proposed Bcd gradient scaling mechanism may also play a role in correcting environmental factor-induced egg size variation. If this is true, this mechanism might represent a general means for scaling (at the level of the Bcd gradient) that is broadly utilized across species (Cheung, 2011).

Bcd gradient scaling achieved by the proposed mechanism is imperfect with respect to certain positions. Although there is good scaling in broad regions of the embryo, there are large differences in B in the most anterior parts. Such differences lead to large differences in Bcd-encoded positional information. How scaled patterning in these parts of the embryo is achieved remains unknown. It is possible that the terminal system may provide positional information in a manner that is also dependent on embryo size. This could be achieved either through a direct, but passive, mechanism, in which the amount of a rate-limiting component(s) of the terminal system is - like bcd mRNA - also determined in a volume-dependent manner, or through an indirect feedback mechanism in which the relevant action of this system is regulated by the amount of Bcd. It was also noted that, although the eve expression boundary positions in the selected large and small embryos have good scaling properties, they do exhibit an allometric shift along the A-P axis. This shift is not consistent with the shift in the positional information provided by Bcd in this region (the posterior) of the embryo. In addition, when evaluating Bcd gradient properties using the scaling coefficient, slight hyposcaling was observed in broad regions of the embryo. Together, these results suggest that additional mechanisms, such as the terminal system and/or gene regulatory networks, work with Bcd to achieve the observed eve expression patterns for pattern flow during embryogenesis] (Cheung, 2011).

The deployment of two different mechanisms for achieving Bcd gradient scaling within species versus between species is not surprising. Under sustained evolutionary pressure to maintain Bcd scaling in the face of egg size divergence across species (that require Bcd for A-P patterning), biophysical properties of Bcd gradient formation (i.e. the diffusibility or stability of Bcd) are expected to evolve in concert with embryo size. Over time, and with sufficient fine-tuning of diffusion and degradation rates by natural selection, scaling at all positions can be achieved. By contrast, for the same scaling mechanism to be utilized by embryos of different sizes (of genetic origin) within a species, alleles that control the diffusion or degradation rates of Bcd would have to be tightly associated with the alleles that control embryo size. For a trait such as egg size, with its multifactorial genetic basis, a tight association between egg size and gradient scaling alleles would be difficult, if not impossible, to achieve. Instead, a scaling mechanism achieved through passively adjusting the Bcd production rate provides an effective (though imperfect) means to correct within-species differences in egg size arising from both genetic and stochastic (and possibly environmental) origins (Cheung, 2011).

How a Bcd concentration gradient is formed remains highly controversial, even whether the positional information provided by Bcd is decoded before the gradient reaches its steady state . Although this study has not specifically investigated the process of Bcd gradient formation, the observed scaling properties of the Bcd gradient may be readily explained by a diffusion model if the Bcd production rate is scaled with embryo volume. It is interesting to note that the observed S profile exhibits a broad similarity to the profiles obtained in a model that assumes a volume-dependent Bcd production rate. Besides the difference in the amount of bcd mRNA (and consequently B0) in the large and small embryos, it is currently unknown whether these embryos exhibit other differences that are relevant to Bcd gradient formation. It is emphasized that, despite an incomplete knowledge - as a field - of how Bcd gradient formation is controlled, the current study represents a step forward in understanding scaling. Most importantly, the volume-dependent deposition of bcd mRNA provides a passive (i.e. self-correcting), early-acting mechanism for producing Bcd scaling that contributes to robust pattern formation. Going beyond the early Drosophila embryo, which does not change in overall size as a function of time, morphogen gradients also play roles in patterning tissues that grow in size. A correlation between the amplitude and L (achieved by adjusting the morphogen production rate or by other mechanisms) may also provide a simple means to maintain scaled patterning as a tissue grows in size (Cheung, 2011).

The role of regulated mRNA stability in establishing bicoid morphogen gradient in Drosophila embryonic development

The Bicoid morphogen is amongst the earliest triggers of differential spatial pattern of gene expression and subsequent cell fate determination in the embryonic development of Drosophila. This maternally deposited morphogen is thought to diffuse in the embryo, establishing a concentration gradient which is sensed by downstream genes. In most model based analyses of this process, the translation of the bicoid mRNA is thought to take place at a fixed rate from the anterior pole of the embryo and a supply of the resulting protein at a constant rate is assumed. Is this process of morphogen generation a passive one as assumed in the modelling literature so far, or would available data support an alternate hypothesis that the stability of the mRNA is regulated by active processes? A model is introduced in which the stability of the maternal mRNA is regulated by being held constant for a length of time, followed by rapid degradation. With this more realistic model of the source, three computational models of spatial morphogen propagation along the anterior-posterior axis were analyzed: (1) passive diffusion modelled as a deterministic differential equation, (2) diffusion enhanced by a cytoplasmic flow term; and (3) diffusion modelled by stochastic simulation of the corresponding chemical reactions. Parameter estimation on these models by matching to publicly available data on spatio-temporal Bicoid profiles suggests strong support for regulated stability over either a constant supply rate or one where the maternal mRNA is permitted to degrade in a passive manner (Liu, 2011; full text of article).

return to Bicoid Post-transcriptional regulation part 1/2

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

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