CONTENTS Main Page Dynamic Development The Foundations of Developmental Biology Gametogenesis From Sperm and Egg to Embryo Genetic Regulation of Development Organizing the Multicellular Embryo Generating Cell Diversity Dynamic Development at a Glance Learning Resources Research Resources The Developmental Biology Journal Club Developmental Biology Tutorial

Patterns of Messenger RNA Localization in Xenopus Oocytes How does a messenger know where to go? Most RNAs are not localized to particular sites in the oocyte. An example is histone messenger, which is uniformly distributed early in oogenesis and its presence in the vegetal hemisphere becomes diluted by yolk platelets during vitellogenesis. However, certain classes of transcripts are localized. Localization has been detected by identifying cDNA that is representative of RNA that is enriched in either the animal or vegetal hemisphere. Labeled cDNAs from either hemisphere were used to screen an oocyte cDNA library. Then, in situ hybridization is used to determine transcript localization. One of these screens identified Vg1, which forms a crescent at the vegetal pole of the fully-grown oocyte . (See Browder et al., 1991, Figs. 3.34, 3.35; Gilbert, 1997, Fig. 12.15; Kalthoff, 1996, Fig. 8.8; Wolpert et al., Fig. 3.2) How does Vg1 become localized during oogenesis? It is initially uniformly distributed and during late oogenesis is translocated into the vegetal hemisphere. Translocation is inhibited by inhibitors of microtubules, whereas cytochalasin B (an inhibitor of microfilaments) prevents anchorage at the vegetal pole. Vg1 has a sequence in the 3' UTR that is required for localization (Mowry and Melton, 1992). That sequence is analogous to a postal code. The code must be read by a recognition system, perhaps by a protein that recognizes it and facilitates its transport to the correct address and keeps it there. A candidate protein, called Vera, which binds Vg1 to vesicles of the endoplasmic reticulum, has recently been identified. So what? Why is it important to identify transcripts that localize to the vegetal hemisphere of oocytes? This messenger is localized to cells of the vegetal hemisphere after fertilization. As we shall discuss later, signals emanating from cells in the vegetal hemisphere are important in establishment of the embryonic body plan. Significantly, the Vg1 protein is a member of the TGF-ß family of proteins. Why might that be a significant characteristic? As we mentioned above, Vg1 is localized late in oogenesis. Other mRNAs that are reported to be localized in the vegetal cortex are Xwnt11 and Xcat2 and nontranslatable RNAs called Xlsirts. They are localized early in oogenesis. The oocyte cytoskeleton and cis-acting elements on the RNA have been implicated in transport/localization of these transcripts. The cis-acting elements that provide the "postal code" for Xlsirts are in the 79-nucleotide repeat sequences, whereas elements in the 3' UTR of Xcat2 specify its localization. Kloc et al. (1993) have reported that these messengers are translocated to the vegetal cortex via the mitochondrial cloud. Recently, Kloc and Etkin (1995) have shown that Xlsirts share a similar pattern of translocation to the vegetal cortex with a number of transcripts, but that Vg1 follows a distinct pathway. Figure 1 (Kloc and Etkin, 1995) and Figure 5 (Forristall et al., 1995) show the distribution of four transcripts in the vegetal cortex of vitellogenic Xenopus oocytes. Vg1 was broadly distributed in the vegetal hemisphere, whereas the others (Xlsirt, Xcat2, and Xwnt11) are localized to a disk-shaped region at the apex of the vegetal pole. Confocal micrograph of a live stage 1 Xenopus laevis oocyte injected with Texas red-labeled Xlsirt mRNA (red) and counterstained for mitochondria (green), 36 hours post-injection. Xlsirt mRNA is localized in the disc at the vegetal pole (bottom). (M. Kloc, C. Larabell and L.D. Etkin, unpublished. Copyright © 1996, M. Kloc). Because Xlsirt, Xcat2, and Xwnt11 share a common site within the vegetal pole, Kloc and Etkin asked whether their translocation patterns were also similar. As shown in Figure 2 (Kloc and Etkin, 1995), all three of these transcripts can be found within the mitochondrial cloud. The cloud is initially found adjacent to the germinal vesicle and later near the vegetal cortex. By stage 3, the cloud-associated transcripts are anchored at the cortex. The portion of the cloud containing these transcripts has been coined the METRO (messenger transport organizer). Localization of RNAs through the METRO involves three steps: 1. movement of transcripts from the GV to the mitochondrial cloud; 2. sorting of transcripts within the cloud; and 3. translocation to the cortex. Vg1, on the other hand, is distributed throughout the cytoplasm during early oogenesis and is absent from the mitochondrial cloud (Kloc and Etkin, 1995, Fig. 3A,a). Later, when Xlsirt, Xcat2, and Xwnt11are localized to the vegetal pole, Vg1 was detected in a wedge-shaped pattern at the apex of the vegetal pole, overlapping with Xlsirt, Xcat2, and Xwnt11 (Fig. 3A,b). The Vg1 is associated with a subdomain of the endoplasmic reticulum. This specialized subdomain of the endoplasmic reticulum first appears above the mitochondrial cloud as the METRO and its associated mRNAs move toward the vegetal cortex. When the METRO RNAs are anchored, the wedge-shaped subdomain of the ER is fully elaborated, and the Vg1 is associated with it (Figs. 3 and 4, Deshler et al., 1997; Etkin, 1997). Still later, Vg 1 dissociates from the METRO and extends up toward the marginal zone (Kloc and Etkin, 1995, Fig. 3A,c). Sections of oocytes reveal images that appear to show Vg1 streaming away from the METRO toward the cortex and up toward the marginal zone (Kloc and Etkin, 1995, Fig. 3B). When double in situ hybridization was conducted on oocyte sections, the locations of each transcript relative to the others could be determined. As shown in Kloc and Etkin (1995), Figure 4, during stages 1 and 2, Xcat2 was outermost, followed by Xlsirts and Xwnt11. The layering of transcripts may be indicative of a hierarchy in which Xcat2 RNA associates with the cortex first, followed by Xlsirts, then by Xwnt11. During stage 3, Vg1 transcripts appeared to associate with the cortex at the same sites where Xlsrts localized. By stage 4, Vg1 had formed a thin layer in the cortex throughout the vegetal hemisphere, and the other transcripts were situated in the disk. The METRO may be establishing a pathway that is used by Vg1 (and presumably other transcripts) during their translocation to the cortex by facilitating the formation of the wedge-shaped ER subdomain. Translocation of Vg1 to the cortex is sensitive to inhibitors of microtubules. Thus, microtubules are also involved. Perhaps the wedge-shaped ER structure serves as a substrate to orient microtubules, or the ER and associated Vg1 are translocated along microtubules (Etkin, 1997). To determine whether the association of transcripts with the mitochondrial cloud is dependent upon the cytoskeleton, nocodazole was used to destroy microtubules and cytochalasin was used to destroy microfilaments. Neither inhibitor alone nor both in combination had any effect on association of Xlsirt, Xcat2, and Xwnt11 with the cloud. Thus, the association of these transcripts with the cloud is not dependent upon either microtubules or microfilaments. Kloc and Etkin had previously shown that destruction of Xlsirt RNA with antisense oligos caused the release of Vg1 mRNA from the cortex of stage 4 oocytes. This result suggested that Xlsirt may facilitate the anchoring of Vg1 to the cortex via the cytoskeleton. Treatment of stage 3 and stage 4 oocytes with cytochalasin B caused detachment of all four of these transcripts from the outer cortical shell, although they weren't released into the cytoplasm (Kloc and Etkin, 1995, Fig. 6). Nocodazole, on the other hand, had no effect on Xlsirt, Xcat or Xwnt11 in stage 3 oocytes, but most of the Vg1 mRNA was released from the cortical shell, forming blebs, although some remained attached to the cortical shell. At stage 4, all of the Vg1 remained with the cortical shell. The authors suggest that Xlsirt is a structural component of the cortex and that components associated with the cytoskeleton link Vg1 and Xlsirt. The sensitivity of Vg1 to nocodazole during stage 3 is thought to be due to the migration of Vg1 through the cortex at that stage. Learning Objectives How was Vg1 first identified? What does it encode? Trace the movement of Vg1 during oogenesis and correlate its movement with that of Xwnt11, Xcat2 and Xlsirts. What is Vera, and what does it do? Reconstruct the formation and movement of the METRO. How are messengers localized through the METRO? What is the relationship between the METRO and the subdomain of the endoplasmic reticulum to which Vg1 is attached? How is the cytoskeleton involved in these processes? What is the evidence? What elements in these transcripts specify their localization? Digging Deeper: Vera For more details, see an overview of Vera on Zygote. Recent Literature Gard, D.L., Cha, B.J. and King, E. 1997. The organization and animal­vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon F-actin and microtubules. Develop. Biol. 184: 95-114. References Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology. Third edition. Saunders College Pub. Philadelphia. Deshler, J.O., Highett, M.I. and Schnapp, B.J. 1997. Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. Science 276: 1128-1131. Etkin, L.D. 1997. A new face for the endoplasmic reticulum: RNA localization. Science 276: 1092-1093. Forristall, C., M. Pondel, L. Chen and M.L. King. 1995. Patterns of localization and cytoskeletal association of two vegetally localized RNAs, Vg1 and Xcat-2. Development 121: 201-208. Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer. Sunderland, MA. Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill. New York. Kloc, M., G. Spohr, G. and L.D. Etkin. 1993. Translocation of repetititve RNA sequences with the germ plasm in Xenopus oocytes. Science 262: 1712-1714. Kloc, M. and Etkin, L. D. (1995) Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes. Development 121: 287-297. Mowry, K. and Melton, D. 1992. Vegetal messenger RNA localization directed by a 340nt sequence element in Xenopus oocytes. Science 255: 991-993. Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. 1998. Principles of Development. Current Biology. London. Yisraeli, J.K., Sokol, S. and Melton, D.A.. 1990. A two step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in translocation and anchoring of Vg1 mRNA. Development 18: 289-298.

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This material may be reproduced for educational purposes only provided credit is given to the original source. Leon Browder & Laurie Iten (Ed.) Dynamic Development Last revised Wednesday, June 17, 1998