mRNA transport in Drosophila
Drosophila oocytes/eggs localize several maternal mRNAs to distinct cortical domains, including bicoid (bcd) at the anterior pole, oskar (osk) and nanos (nos) at the posterior, and gurken (grk) mRNAs first at the posterior, then at the anterior, and eventually to the dorso-anterior regions (Fig. 1A–C). The localization of bcd and grk mRNAs is essential to specify antero-posterior and dorso-ventral body axes, respectively (Frohnhöfer & Nüsslein-Volhard 1986; Berleth et al. 1988; Driever & Nüsslein-Volhard 1988; Neuman-Silberberg & Schüpbach 1993; González-Reyes et al. 1995), while osk acts as a germline determinant (Lehmann & Nüsslein-Volhard 1986; Ephrussi et al. 1991; Ephrussi & Lehmann 1992), and nos is required for germline and abdominal development (Lehmann & Nüsslein-Volhard 1991; Gavis & Lehmann 1992; Wang & Lehmann 1992; Wang et al. 1994; Gavis et al. 2008). This mRNA localization pattern is established during Drosophila oogenesis through different mechanisms that mediate the transportation of these mRNAs from ovarian nurse cells to oocytes (Fig. 1A,B, left side of each panel) (Theurkauf et al. 1992; Theurkauf & Hazelrigg 1998; Cha et al. 2001; Clark et al. 2007; Mische et al. 2007), which continues until the completion of the mid-stage of oogenesis when the nurse cells start to contract and dump their contents into oocytes (Fig. 1C, thick dark blue and pink arrows), and subsequent mRNA translocation to distinct cortical domains within oocytes during the mid- and late stages of oogenesis (Fig. 1B,C, right side of each panel). mRNA localization during these stages is largely dependent on the microtubule cytoskeleton (Pokrywka & Stephenson 1991, 1995), which is re-organized several times within oocytes at different stages of oogenesis, contributing to the various paths or mechanisms that exist for mRNA transportation. For example, re-organization of microtubule distribution is triggered by Grk-dependent signaling between the oocyte and overlying follicle cells at the posterior pole for the initiation of mid-oogenesis (Fig. 1A, B) (Theurkauf et al. 1992; González-Reyes et al. 1995), and cortical microtubules that mediate a vigorous cytoplasmic flow that leads to nos mRNA localization to the posterior are generated during the late-stage of oogenesis (Fig. 1C, curved green lines) (Gutzeit & Koppa 1982; Theurkauf 1994; Palacios & St. Johnston 2002; Forrest & Gavis 2003; Weil et al. 2008).
Figure 1. Schematic representation of mRNA localization dynamics during oogenesis and early embryogenesis I. Shown is the distribution of several maternal mRNAs in Drosophila (A–C), and Xenopus (D–F). Small colored circles and stars represent localized mRNAs with the list of them shown to the right. Colored arrows indicate the movement of the same-colored mRNAs. In Drosophila, the oocyte (right side of each panel) arises from a 16 cell germline cyst, which results from four rounds of incomplete cell divisions. The other 15 cells serve as nurse cells (left side of each panel). The oocyte and the nurse cells are connected through the ring canals. The mid-oogenesis in Drosophila (B) is defined in this review to start at stage VIII, when Grk signaling at the posterior pole between the oocyte and the surrounding follicle cells triggers re-organization of microtubules. The late-oogenesis (C) is defined to begin at stage XI, when the nurse cells contract and extrude their contents into oocytes. Although grk mRNA is still localized in the dorso-anterior cortex at the late-stage of oogenesis, this is not shown in this figure.
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The four maternal mRNAs are transcribed in the ovarian nurse cells and, therefore, must be transported into the oocyte. bcd and grk mRNAs are thought to accumulate in the same RNP complex and to be transported on a dynein-mediated transport pathway toward the ring canals that connect the nurse cells to the oocyte (Fig. 1B, dark blue circles and green stars on left side) (Clark et al. 2007; Mische et al. 2007). In this pathway, concentrated microtubule arrays at the ring canals, which appear to project into the cytoplasm of nurse cells (Fig. 1B, two green lines on left side), serve to transport mRNA (Theurkauf et al. 1992). However, how the RNP complexes encounter the microtubule arrays within nurse cells is not understood, although it may occur through random movement and an entrapment process (Theurkauf & Hazelrigg 1998; Clark et al. 2007; Mische et al. 2007). Once the RNP complexes with specific mRNAs reach the ring canals, they undergo remodeling, which involves an exchange of localization factors, and sort mRNAs according to their target destination for further transportation within the oocyte (St. Johnston 2005; Mische et al. 2007; Becalska & Gavis 2009). Several osk RNP localization factors, including Tropomyosin II, Barentsz and Mago nashi, are thought to replace the dynein motors that mediate the nurse cell-to-oocyte transportation with a kinesin motor at the ring canals (Newmark & Boswell 1994; Erdélyi et al. 1995; van Eeden et al. 2001). Events that occur in nurse cells are important for correct mRNA localization within oocytes. For example, exogenously injected bcd mRNA is only properly localized to the anterior region of oocytes when injected into nurse cells, as mRNA directly injected into oocytes fails to localize (Cha et al. 2001).
In contrast to the longstanding model that microtubules are polarized along the anteroposterior axis of oocytes, with minus ends nucleated at the anterior pole and plus ends projecting towards the posterior pole, such that kinesin-associated osk mRNA is transported to the posterior pole, recent live imaging studies suggest that microtubules are largely randomly distributed (Fig. 1B, numerous green lines on the right side). It is proposed that osk mRNA is transported in a kinesin-dependent manner on randomly oriented microtubules with a slight bias towards the posterior during the mid-stage of oogenesis (Fig. 1B, red and light red circles on right side) (Zimyanin et al. 2008). The slight polarization bias is considered sufficient for osk mRNA to be enriched at the posterior region, because the transportation of mRNA requires a relatively long period (6–10 h). Localization of osk mRNA to the posterior cortex requires two steps: long-range, kinesin-dependent transportation throughout the oocyte into the posterior cytoplasm, as described above, and short-range, myosin-dependent translocation or entrapment at the posterior cortex (Krauss et al. 2009). The notion that microtubules are largely randomly distributed may be consistent with the above experimental result for bcd mRNA, in which exogenously injected bcd mRNA into oocytes accumulated at the nearest cortex in a dynein- and microtubule-dependent manner (Cha et al. 2001; Mische et al. 2007). Notably, mislocalization of bcd mRNA occurs even when it is injected into nurse cells that lack functional nurse cell-derived Exuperentia (Exu) proteins (Cha et al. 2001). Therefore, the presence of Exu allows bcd RNPs to locate and use a microtubule subpopulation with minus ends oriented toward the anterior cortex in the background of largely randomized microtubules. The discriminatory use of a microtubule subpopulation has recently been suggested for Vg1 mRNA localization in Xenopus oocytes, where Vg1 RNPs may use a microtubule subpopulation with its plus ends directed to the vegetal cortex (Fig. 1F, light blue stars and brown lines) (Messitt et al. 2008).
bcd mRNA continues to accumulate at the anterior cortex during the late-stage of Drosophila oogenesis in a dynein- and microtubule-dependent manner (Fig. 1C, dark blue circles and thin dark blue arrows). This late accumulation accounts for the majority of bcd mRNA present in the anterior region of the embryo (Weil et al. 2006) and uses distinct transport mechanisms from those involved during the mid-stage of oogenesis. Swallow and Staufen are only required for the late transportation pathway (St. Johnston et al. 1989; Weil et al. 2006, 2008). Swallow plays an indirect role in bcd mRNA localization by organizing the cytoskeleton, whereas Staufen contributes directly to transportation (Weil et al. 2010). nos, cyclin B, germ cell-less and polar granule component mRNAs, are also accumulated during the late-stage of oogenesis, but unlike bcd, these mRNAs are localized to the posterior region (Dalby & Glover 1992; Jongens et al. 1992; Wang et al. 1994; Nakamura et al. 1996).
Interestingly, nos RNPs are passively transported by diffusion and become trapped at the posterior cortex via posteriorly-localized germ plasm (Fig. 1C, pink stars and yellow circles) (Forrest & Gavis 2003). However, the localization of nos mRNA to the posterior cortex is not entirely microtubule-independent, as parallel arrays of microtubules run beneath the oocyte cortex (Fig. 1C, curved green lines) to create a cytoplasmic flow, termed ooplasmic streaming (Fig. 1C, light blue arrow), and facilitate the probability of RNPs encountering the germ plasm (Forrest & Gavis 2003; Weil et al. 2008). Recently, osk mRNA was shown to be transported to the posterior cortex not only during mid-oogenesis, as described above, but also at the late-stage of oogenesis (Fig. 1C, red circles) (Sinsimer et al. 2011). The transportation of osk and nos mRNAs during late-oogenesis requires the same localization factors Lost and Rumpelstiltskin (Sinsimer et al. 2011). Although only 4% of nos mRNA is localized to the posterior pole (Bergsten & Gavis 1999), after fertilization, nos in other locations is subject to deadenylation and degradation by the multifunctional RNA binding protein Smaug, resulting in greater than a hundred-fold enrichment of nos mRNA at the posterior pole (Zaessinger et al. 2006). The protection/degradation mechanism of localization mediated by Smaug is also used for hsp83 mRNA in the posterior pole plasm (Tadros et al. 2007).
mRNA anchoring in Drosophila
Recent developments in live-imaging techniques, particularly fluorescence recovery after photobleaching (FRAP), have made it possible to separate the anchoring and transport phases in the mRNA localization pathway. In FRAP, mRNA is considered to be stably anchored if the photobleaching of fluorescent-tagged mRNA at the localization site does not result in fluorescence recovery from adjacent regions. In contrast to the major role microtubule cytoskeleton plays in mRNA transportation, actin-dependent anchoring of mRNA to the oocyte cortex has been shown in several studies (Becalska & Gavis 2009; Kugler & Lasko 2009). Anchoring of mRNA is associated with two major functions: stable mRNA localization and translation. Although translation is often suppressed during mRNA transportation, upon arrival and anchoring of mRNA at the target destination, translation is initiated locally. Indeed, anchoring is required for the translation of nos and osk mRNA (Gavis & Lehmann 1994; Markussen et al. 1995; Rongo et al. 1995).
The germ cell determinant Osk is both required and sufficient for germ cell formation (Lehmann & Nüsslein-Volhard 1986; Ephrussi et al. 1991; Ephrussi & Lehmann 1992). osk mRNA is alternatively translated to produce two protein isoforms, Short Osk and Long Osk, upon its arrival at the posterior cortex (Markussen et al. 1995; Rongo et al. 1995). Short Osk recruits numerous proteins and mRNAs, including nos mRNA, which are required for germ cell development, to the germ plasm, whereas Long Osk functions to maintain osk mRNA at the posterior cortex, thus forming a feedback loop (Breitwieser et al. 1996; Vanzo & Ephrussi 2002). Actin-dependent anchoring has been suggested by the treatment of oocytes with actin de-polymerization chemicals, which results in the release of osk mRNA from the target localization site (Cha et al. 2002), and by mutant analysis of the cortical actin-binding protein Moesin, which releases osk and nos mRNAs (Jankovics et al. 2002; Polesello et al. 2002). nos mRNA is thought to be anchored to the cortical actin cytoskeleton via its association with germ plasm (Fig. 1C, pink stars, yellow circles, and purple bars) (Forrest & Gavis 2003). Recent studies suggest that Long Osk promotes endocytosis-coupled actin remodeling for cortical anchorage of the germ plasm (Vanzo et al. 2007; Tanaka & Nakamura 2008; Tanaka et al. 2011).
bcd mRNA is continuously transported to the anterior cortex in a dynein- and microtubule-dependent manner, as described above, until the completion of oogenesis, at which point the transport of bcd mRNA is shifted to a stable cortical actin-dependent anchoring process (Fig. 1C, dark blue circles and purple bars) (Weil et al. 2008). The stable anchoring of bcd mRNA is likely needed as mature oocytes can be dormant for weeks prior to fertilization. Upon egg activation, however, bcd mRNA is released from its anterior cortical tether as a result of re-organization of the actin cytoskeleton (Weil et al. 2008). The release of mRNA from a cortical tether upon egg activation is also observed in Xenopus, where Vg1 mRNA is released from the vegetal cortex (Yisraeli et al. 1990). The dispersal of Vg1 mRNA before initiating cell divisions may ensure that this developmentally important factor is inherited by the proper number of vegetal cells for establishing later embryonic patterning. Similarly, the dispersal of bcd mRNA during egg activation could be important for antero-posterior patterning of Drosophila embryos.
grk mRNA exhibits a dynamic distribution during the mid-stage of oogenesis: it is localized to the posterior at an early stage (Fig. 1A), subsequently moves to the anterior, and eventually accumulates around the oocyte nucleus in the dorso-anterior cortex (Neuman-Silberberg & Schüpbach 1993). The two steps involved in the transportation of grk mRNA towards the anterior and dorso-anterior regions are both dynein and microtubule dependent (Fig. 1B, green stars and arrows) (MacDougall et al. 2003). Of the three target sites, grk mRNA is stably anchored only in the dorso-anterior cortex within large electron-dense cytoplasmic structures known as sponge bodies (Delanoue et al. 2007; Jaramillo et al. 2008). Interestingly, the anchoring of grk mRNA is also dynein-dependent (Fig. 1B, orange rectangles) (Delanoue et al. 2007), indicating that dynein functions in two distinct roles: a motor in mRNA transportation and as an mRNA anchor. One grk mRNA localization factor, Squid (Sqd), is required for grk mRNA to enter the sponge bodies (Fig. 1B, light purple rectangles), as grk mRNA remains associated with transport particles and does not exhibit dorso-anterior localization in a sqd mutant (Delanoue et al. 2007).
mRNA transport and anchoring in Xenopus
Xenopus oocytes, like those of Drosophila, have multiple transport pathways with distinct mechanisms for mRNA localization that operate in different stages of development (Fig. 1D–F). A subset of maternal mRNAs is distributed differentially either at the animal or vegetal poles of oocytes. Whereas the mechanism(s) of mRNA enrichment in the animal hemisphere are unclear, two major distinct pathways are known to mediate mRNA transport to the vegetal cortex (Forristall et al. 1995; Kloc & Etkin 1995): the early MEssage TRansport Organizer (METRO) pathway that is activated in stage I-II oocytes (Fig. 1D,E) and the late pathway (Fig. 1F) that begins as the METRO pathway ceases to function.
The METRO pathway localizes several mRNAs, including nanos-related Xcat2 (Mosquera et al. 1993; Forristall et al. 1995; Kloc & Etkin 1995), Xdazl (Houston et al. 1998), and DEAD-South (MacArthur et al. 2000), many of which encode different families of RNA-binding proteins that are components of the germ plasm, and thus specify the germline cells in later development. The mRNAs within the germ plasm may not be translated for many months (MacArthur et al. 1999; Houston & King 2000). These early localized germline mRNAs are targeted to the germ plasm-forming METRO region within a subcellular structure called the mitochondrial cloud (MC) or Balbiani body (Heasman et al. 1984; Kloc et al. 1996; Zhou & King 1996a), which is enriched with endoplasmic reticulum (ER), Golgi, and mitochondrial membranes (Fig. 1D,E, green circles). The MC/Balbiani body is located asymmetrically on the future vegetal side of the nucleus (Fig. 1D). Initially, the asymmetrical distribution of the mitochondrial and organelle precursors of the MC/Balbiani body is established through the targeted growth of the organelle aggregate that contains the centrosome by dynein-dependent transport of these organelle precursors toward the centrosome (Heasman et al. 1984; Pepling et al. 1999; Kloc et al. 2002, 2004), while other organelle aggregates surrounding the nucleus remain the same size. The germline mRNAs localize at the MC/Balbiani body during stage I of oogenesis (Fig. 1D, red stars) by both diffusion and an entrapment mechanism (Kloc et al. 1996; Zhou & King 1996a,b; Chang et al. 2004), which is conserved between Drosophila and Xenopus for the localization of nos (Forrest & Gavis 2003) and Xcat2 mRNAs, respectively. The smooth ER-like membranes within the METRO region of the MC/Balbiani body are associated with these mRNAs and therefore may serve as the site for the entrapment (Chang et al. 2004). In stage II oocytes, the MC/Balbiani body associated with the localized mRNAs moves to the vegetal cortex (Fig. 1E).
In contrast to the early pathway, the late pathway localizes germ-layer determinants to the vegetal cortex in a microtubule-dependent manner (Yisraeli et al. 1990). The best characterized mRNAs that are localized through this pathway are the T-box transcription factor VegT (Lustig et al. 1996; Stennard et al. 1996; Zhang & King 1996), and the transforming growth factor-beta (TGF-β) growth factor Vg1 (Weeks & Melton 1987), which have both been shown to play roles in mesoderm and endoderm specification during early embryogenesis (Dale et al. 1993; Thomsen & Melton 1993; Horb & Thomsen 1997; Joseph & Melton 1998; Zhang et al. 1998). These two mRNAs are uniformly distributed in the cytoplasm and are excluded from the MC/Balbiani body in stage I oocytes (Fig. 1D, light blue stars) (Melton 1987; Forristall et al. 1995; Kloc & Etkin 1995), in which the early pathway is active. Whether the early or late pathway is used for the localization of a specific mRNA within the cytoplasm may be already determined in the oocyte nucleus following transcription (Kress et al. 2004), as localization factors such as Vg1RBP/Vera (Deshler et al. 1997, 1998; Havin et al. 1998), VgRBP60/hnRNP I (Cote et al. 1999), and 40LoVe (Kroll et al. 2009), among which the latter is related to Drosophila Squid (Kroll et al. 2009), bind to Vg1 mRNA in the nucleus and are excluded from the MC/Balbiani body after the export of the Vg1 RNP complex from the nucleus. The importance of nuclear events in mRNA localization has also been demonstrated in Drosophila, as the recruitment of the exon-exon junction complex upon the splicing of osk mRNA is essential for its localization to the posterior pole (Hachet & Ephrussi 2001, 2004; Kataoka et al. 2001; Le Hir et al. 2001a,b; Mohr et al. 2001; Tange et al. 2005). By stage III, during which the late pathway begins to transport mRNAs, Vg1 mRNA associates with a distinct ER subdomain in a halfway point to the vegetal pole in a microtubule-independent manner (Fig. 1E, light blue stars and arrows) (Deshler et al. 1997; Kloc & Etkin 1998). The use of ER networks for capturing mRNAs is conserved between the early and late pathways. A network of cortical ER (cER) is also associated with localized maternal mRNAs in ascidian eggs (described below), while grk mRNA of Drosophila associates with ER as well (Saunders & Cohen 1999). Whether conserved localization factors mediate the association of ER and mRNAs remains to be determined; however, evidence suggests that in addition to binding to the 3′UTR of Vg1 mRNA, Vg1RBP/Vera also weakly binds to ER (Deshler et al. 1997). During mid-oogenesis (stages III–IV), late pathway mRNAs are finally transported to the vegetal cortex in a process that requires the functions of kinesins I and II (Fig. 1F) (Betley et al. 2004; Yoon & Mowry 2004; Messitt et al. 2008).
Although the late pathway initiates in the nucleus, several factors are known to be recruited to Vg1 RNPs in the cytoplasm after their export from the nucleus and play roles in transportation and anchoring. For example, XStau, a Xenopus orthologue of the Drosophila RNA binding protein Staufen, which plays important roles in osk RNP localization (St. Johnston et al. 1991), interacts with Vg1 mRNA and kinesin I, and is required for proper Vg1 mRNA localization (Yoon & Mowry 2004). Another factor, Prrp, is proposed to act as a mediator for actin-based anchoring of Vg1 mRNA to the cortical cytoskeleton (Schlüter et al. 1997; Zhao et al. 2001). In addition to the two functions of anchoring described above, namely stable mRNA localization and translation, the reported anchoring of Vg1 mRNA to the vegetal cortex may indicate a third function: the bias for mRNA transportation to a particular location in the background of randomly oriented microtubules (Fig. 1F, green and brown lines), where Vg1 mRNA is thought to be transported bidirectionally between the vegetal cortex and a halfway point to the cortex in the vegetal hemisphere (Fig. 1F, light blue arrows), with mRNA anchoring at the vegetal cortex influencing the targeting localization in that direction (Messitt et al. 2008).
Despite the spatial and temporal differences and distinct mechanisms used between the early and late transportation pathways, a number of parallels have been also observed, suggesting that common cellular machinery mediates mRNA localization throughout oogenesis. For example, a few localized mRNAs classified as intermediate have been identified (e.g., fatvg [Chan et al. 2007]), which appear to have characteristics indicative of use of both the early and late pathways. In addition, a number of germline mRNAs, including Xcat2, Xpat, and Xlsirts, are capable of using the late pathway when injected into stage III/IV oocytes (Zhou & King 1996b; Hudson & Woodland 1998; Allen et al. 2003), suggesting that a fail-safe mechanism may operate to ensure that germline mRNAs, even when transcribed late, are segregated to the vegetal cortex for proper germline formation. The use of multiple localization mechanisms for a germline mRNA is observed in Drosophila, where osk mRNA is localized to the posterior cortex by means of microtubule-dependent transportation during mid-oogenesis and ooplasmic streaming at the late stage of oogenesis (Glotzer et al. 1997; Sinsimer et al. 2011), as described above.
mRNA localization in other organisms
The use of mRNA localization in oocytes/eggs as a strategy to achieve asymmetric protein distributions is not restricted to Drosophila and Xenopus, as it has been observed in a wide range of organisms, including the wasp Nasonia vitripennis (Fig. 2A–C), the cnidarian Clytia hemisphaerica (Fig. 2D–G), zebrafish (Fig. 2H–L), and several ascidian species (Fig. 2M–Q). In addition, similar mechanisms by which mRNAs are localized in the oocytes/eggs of Drosophila and Xenopus are used in other organisms as well. It is noteworthy that mRNAs for Vg1 and VegT orthologues in the directly developing frog Eleutherodactylus coqui, in contrast to Xenopus, are localized to the animal pole of oocytes (Beckham et al. 2003), indicating that widening mRNA localization studies to include other organisms is also important.
Figure 2. Schematic representation of mRNA localization dynamics during oogenesis and early embryogenesis II. Shown is distribution of several maternal mRNAs in Nasonia (A–C), Clytia (D–G), zebrafish (H–L), and ascidians (M–Q). Small colored circles and stars represent localized mRNAs with the list of them shown to the right. Colored arrows indicate the movement of the same-colored mRNAs. As in Drosophila, the Nasonia oocyte (right side of each panel) arises from a 16 cell germline cyst with 15 nurse cells (left side of each panel). The oocyte and the nurse cells are connected through the ring canals. In Nasonia, the mid-oogenesis (A) is regarded as stage III, when oocytes are in a growth phase and the oocyte nucleus becomes localized dorso-anteriorly, while the late oogenesis (B) as stage IV, when the nurse cell dumping occurs. Before the mid-oogenesis (stage II), Nasonia otd1 and nos mRNAs are located around the nucleus (Olesnicky & Desplan 2007). In zebrafish stage III oocytes (J), vasa mRNA is localized at the cell cortex uniformly along the animal-vegetal axis and nanos1 mRNA is not detected (Kosaka et al. 2007); however, in fertilized eggs, they are observed in the animal region where the embryo proper forms (Theusch et al. 2006). These mRNAs are distributed in a broad ring with a small RNA-free region at the animal pole (K), which subsequently expands towards the periphery in a microtubule-dependent manner to make the RNA-free region larger (L). Broken red arrows in (O, P) indicate that vasa mRNA of some ascidian species Phallusia mammillata and Ciona intestinalis shows this vegetal localization (Paix et al. 2009), while that of Halocynthia roretzi does not (Prodon et al. 2009). The GV in early-stage oocytes of Ciona is positioned at the center (Prodon et al. 2006) as indicated in (M, N) and subsequently moves to the cortex to define the animal pole (O, circle with broken line), while that of Halocynthia is already in an eccentric position (Prodon et al. 2006).
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The study of mRNA localization in the wasp Nasonia provides an interesting opportunity to investigate the mechanism by which localized mRNAs pattern the embryo along the anterior-posterior axis, as well as specify the germline cells, in an arthropod model with a long-germ type of embryogenesis other than Drosophila (Fonseca et al. 2009), where the bcd gene is absent. In Nasonia oocytes, caudal (cad) and nanos (Nv-nos) mRNAs are localized to the posterior pole and direct posterior patterning (Olesnicky et al. 2006; Lynch & Desplan 2010), while giant mRNA is targeted to the anterior pole and mimics a part of bcd function with respect to anterior patterning (Brent et al. 2007). Further, orthodenticle1 (otd1) mRNA is localized to both the anterior and posterior poles and is required for anterior patterning in a manner reminiscent of the Bcd gradient, and for posterior development (Lynch et al. 2006). otd1 mRNA is first targeted to the posterior at the mid-stage of oogenesis (Fig. 2A, dark blue circles) and then localized also to the anterior cortex at the late-stage (Fig. 2B, dark blue circles) (Lynch et al. 2006). The localization of these four maternal mRNAs in Nasonia is achieved using at least two distinct mechanisms (Olesnicky & Desplan 2007). Anterior otd1 and giant, as well as posterior cad mRNA localization, requires microtubules (Lynch & Desplan 2010). The localization of giant and cad mRNAs is transient and they diffuse at later stages and form a gradient: a gradient distribution of cad is observed in the late stage of oogenesis (Fig. 2B, green stars) and that of giant in the freshly laid embryos (Fig. 2C, red circles) (Olesnicky et al. 2006; Brent et al. 2007). In addition, anterior localization of otd1 and giant mRNAs differs in that giant is localized in a perinuclear manner (Fig. 2A) but that otd1 is not (Olesnicky & Desplan 2007). In contrast to the anteriorly localized mRNAs, posterior localization of Nv-nos and otd1 mRNAs is actin-dependent (Lynch & Desplan 2010). The localization of Nv-nos mRNA to the posterior pole occurs at an early stage of oogenesis (mid-oogenesis) (Fig. 2A, pink stars) independently of ooplsamic streaming and this early localization does not appear to depend on the assembly of the germ plasm (Lynch & Desplan 2010). This mode of localization distinctly differs from that used for nos in Drosophila oocytes (described above). However, in the freshly laid eggs/embryos, Nv-nos as well as otd1 mRNAs accumulate at the oosome, a structure that is analogous to the germ plasm in Drosophila, in the posterior region (Fig. 2C, dark blue circles, pink stars, and yellow circle) (Lynch et al. 2006; Lynch & Desplan 2010).
Due to its phylogenetic position, the study of mRNA localization in the cnidarian Clytia also provides a unique opportunity to evaluate the extent to which the mechanisms of egg and embryo polarization via mRNA localization are conserved across metazoans. In Clytia, the two antagonizing Wnt receptor mRNAs, Frizzled 1 (CheFz1) and 3 (CheFz3) (Momose & Houliston 2007), and the Wnt ligand mRNA, CheWnt3 (Momose et al. 2008), are localized to distinct regions along the animal-vegetal (oral-aboral) axis in growing and maturing oocytes. All these mRNAs are located throughout the cytoplasm and most are concentrated around the GV at an early stage of oogenesis (Fig. 2D, dark blue circles, and pink and yellow stars). CheFz1 mRNA, then, adopts a graded distribution in the cytoplasm of the animal hemisphere by a mechanism that does not involve anchoring, but might involve preferential stabilization around the animally localized germinal vesicle (GV) (Fig. 2E, dark blue circles), while CheFz3 and CheWnt3 mRNAs are distributed at the periphery of the oocyte (Fig. 2E, pink and yellow stars) (Amiel & Houliston 2009). The polarized cytoplasmic distribution of CheFz1 directs the development of oral fate (Momose & Houliston 2007). During meiotic maturation, CheFz3 mRNA is localized and anchored underneath the vegetal cortex in a microtubule-dependent manner (Fig. 2F, pink stars), although not likely involving microtubule-directed transport but maybe involving mRNA degradation (Amiel & Houliston 2009), and opposes the function of CheFz1 to define an aboral region (Momose & Houliston 2007; Amiel & Houliston 2009). In contrast, CheWnt3 mRNA is concentrated in the animal cortex in a microtubule-independent manner (Fig. 2G, yellow stars), possibly via cortical/cytoplasmic flow during maturation, and induces oral fate (Momose et al. 2008; Amiel & Houliston 2009).
During early oogenesis in zebrafish, mRNAs of the germ plasm components vasa, nanos 1, and dazl localize to the MC/Balbiani body and are transported to the vegetal cortex (Fig. 2H, pink and yellow stars, dark blue and green circles) (Kosaka et al. 2007). The distribution pattern of these mRNAs in stage I oocytes is reminiscent of the Xenopus early METRO pathway. In fact, the zebrafish vasa 3′UTR directs GFP mRNA to the germ plasm in Xenopus oocytes, indicating that these animals share conserved localization machinery (Knaut et al. 2002). The localization of these three mRNAs to the MC/Balbiani body is abrogated in a bucky ball (buc) zebrafish mutant (Marlow & Mullins 2008; Bontems et al. 2009). buc mRNA itself is localized to the MC/Balbiani body and is considered to be a germ-cell determinant, as it is required and sufficient for germ cell formation (Bontems et al. 2009), similar to the factor osk in Drosophila. In the buc mutant, animal localization of several mRNAs, such as pou2 and Vg1, is also disrupted during the later stages of oogenesis (stages III and IV) (Marlow & Mullins 2008). An additional localization pathway may be operated during oogenesis as bruno-like (brul) mRNA localizes at the vegetal cortex in a timing later than vasa/nanos1/dazl mRNA localization (Fig. 2I, light blue stars and arrows), similar to the late pathway in Xenopus (Suzuki et al. 2000).
Vegetally-localized mRNAs in zebrafish, such as buc, vasa, and nanos 1, change their distribution in stage II oocytes (Fig. 2I, dark blue circles and arrows, and yellow stars and arrows) and eventually localize at the animal pole in late-stage oocytes or in freshly laid eggs (Theusch et al. 2006; Kosaka et al. 2007; Bontems et al. 2009). After fertilization, vasa and nanos1 mRNAs, which are distributed in a broad ring that encircles the animal pole of the egg (Fig. 2K, dark blue circles and yellow stars) and appear to be bound to a cortical actin network, move towards the periphery, leaving a large mRNA-free region at the animal pole, in a microtubule-dependent manner (Fig. 2L, dark blue and yellow vertical arrows), and further migrate laterally to accumulate at the forming cleavage furrows (Fig. 2L, dark blue and yellow horizontal arrows) (Theusch et al. 2006), where the germ plasm forms. In contrast, other vegetally-localized mRNAs such as dazl and brul, remain at the vegetal cortex until the late-stage of oogenesis (Fig. 2J, light blue and pink stars); however, following fertilization, they translocate along the plane of the cortex towards the animal pole and are incorporated in the germ plasm (Fig. 2L, pink stars and arrows) (Theusch et al. 2006). Due to the use of the different localization pathways after vegetal localization, these two classes of germline mRNAs (i.e., vasa and nanos 1 vs. dazl) are localized in distinct regions within the germ plasm (Theusch et al. 2006).
Differential distribution of different classes of germline mRNAs within the germ plasm has also been observed in other organisms. In Xenopus, Xcat2, Xpat, and DEAD-South mRNAs are associated with germinal granules, while mRNAs such as Xlsirts and Xdazl reside in the matrix between germinal granules (Kloc et al. 2002). These mRNAs all use the same METRO pathway early to localize at the vegetal cortex (described above). Likewise, non-coding RNAs of Drosophila, including mitochondria large ribosomal RNAs, which are required for germ cell formation (Kobayashi et al. 1993), are often detected in association with polar granules (Kashikawa et al. 1999), while other RNAs are found in the matrix. In addition to the active cytoplasmic transportation of mRNAs, a protection/degradation mechanism operates for zebrafish vasa mRNA to ensure its germline-specific expression during the cleavage stages (Wolke et al. 2002).
Two classes of localized maternal mRNAs have been identified in ascidian species, an immediate sister group to vertebrates. Type I postplasmic/PEM RNAs, such as PEM1, macho1, PEM3, and POPK1, are concentrated at the vegetal cortex of fertilized eggs by microfilament-driven cortical contractions (Fig. 2P, light blue stars and arrows, and green stars and arrows), and are then relocated towards the future posterior pole by sperm-aster microtubule-driven translocations and microfilament-dependent relaxation (Fig. 2Q, light blue stars and arrows, and green stars and arrows) (Roegiers et al. 1999; Sasakura et al. 2000; Yamada et al. 2005; Kumano & Nishida 2007; Prodon et al. 2007; Sardet et al. 2007; Paix et al. 2009). The second class, type II postplasmic/PEM RNAs, which include vasa and PET1, are distributed evenly throughout the cytoplasm (Fig. 2P, red circles), and progressively localize to the posterior pole during embryogenesis (Fig. 2Q, red broken arrows) (Prodon et al. 2007; Sardet et al. 2007). This class, however, may be better categorized as type I in that in some ascidian species, numerous postplasmic/PEM RNAs including those previously regarded as type II exhibit significant accumulation at the vegetal cortex of zygotes (Fig. 2P, red broken arrows), as well as uniform distribution throughout the cytoplasm (Paix et al. 2009). A cortical structure called the centrosome attracting body (CAB) (Hibino et al. 1998), which comprises three distinct parts: the sub-membranous polarity plot layer (Patalano et al. 2006), the cER-mRNA domain (Sardet et al. 2003), and vasa-positive granules (Paix et al. 2009), forms in association with type I and type II postplasmic/PEM RNAs in the posterior pole as development proceeds, and is inherited by the posterior-most blastomere during the cleavage stages. The cER-mRNA domain and vasa-positive granules are already present but dispersed in oocytes (Paix et al. 2009), and after fertilization they get compacted together to form an ascidian equivalent to germ plasm within the CAB (Iseto & Nishida 1999; Fujimura & Takamura 2000; Takamura et al. 2002; Shirae-Kurabayashi et al. 2006; Paix et al. 2009).
In stage I oocytes, Ciona intestinalis PEM3 (Ci-PEM3) mRNA is found located around the GV (Fig. 2M, light blue stars) (Prodon et al. 2006, 2007). However, in full-grown stage III oocytes, Ci-PEM3 and Ci-PEM1 mRNAs are both distributed as cortical patches, which are found uniformly at the entire cell cortex (Fig. 2N, light blue and green stars) (Prodon et al. 2006), suggesting that Ci-PEM3 mRNA is transported from the GV periphery to the cell cortex (Fig. 2N, light blue arrows) as oocytes grow. Although the mechanisms underlying this transportation are not known, the MC/Balbiani body is not observed in ascidian oocytes (Prodon et al. 2006). Prior to germinal vesicle break down (GVBD), Ci-PEM1, Halocynthia roretzi PEM1 (Hr-PEM1), Phallusia mammillata PEM1 (Pm-PEM1) and Pm-macho1 mRNAs are distributed as patches, which are located uniformly at the cell periphery in full-grown oocytes (Prodon et al. 2008; Paix et al. 2011). Ci- and Hr-PEM1 mRNAs are anchored to a network of rough cER in a microfilament-dependent manner (Prodon et al. 2005, 2008). After GVBD, these mRNAs are excluded from the animal pole by vegetally directed and microfilament-driven surface, cortical, and cytoplasmic flows and exhibit a graded distribution along the animal-vegetal axis (Fig. 2O, light blue and green stars) (Prodon et al. 2008). Following fertilization, the mRNAs are further concentrated vegetally (Fig. 2P) and subsequently translocated to the future posterior pole (Fig. 2Q) as described above, while maintaining their association with cER. To date, many localized mRNAs that are associated with cER have been identified in addition to Ci- and Hr-PEM1, and include Hr-macho1, Hr-POPK1, Hr-ZF1, Hr-Wnt5, Pm-PEM1, and Pm-macho1 (Sardet et al. 2003; Nakamura et al. 2005; Paix et al. 2009). A recent study suggests that localized mRNAs are translated on or near the cER (Paix et al. 2011). In contrast to type I postplasmic/PEM RNAs, Hr-vasa mRNA (type II) is located around the GV in full-grown stage-III oocytes (Fig. 2N, red circles) and distributed uniformly throughout the cytoplasm after GVBD (Fig. 2O, red arrows) (Prodon et al. 2009). Hr-vasa mRNA is then thought to accumulate at the future posterior pole after fertilization. Pm-vasa mRNA, in contrast to Hr-vasa mRNA, accumulates at the vegetal cortex (Fig. 2O, red broken arrows) and also displays uniform distribution throughout the cytoplasm of non-fertilized eggs (Paix et al. 2009). Interestingly, Pm-vasa, Pm-POPK1, and Pm-PEM3 mRNAs are localized in granules in non-fertilized eggs (Fig. 2O, two adjacent red circles), which represents a different distribution from that of Pm-PEM1 and Pm-macho1 mRNAs on the cER network (Paix et al. 2009), as described above. Therefore, ascidian germline mRNAs also appear to reside in distinct subdomains within the germ plasm, a conclusion that is supported by the recent findings that PEM1 plays a role in germline transcriptional repression (Kumano et al. 2011; Shirae-Kurabayashi et al. 2011), and POPK1 regulates CAB/germ plasm formation (Nakamura et al. 2005).