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
(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)
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.
For more details, see an overview of Vera on Zygote.
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.
Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental
Biology. Third edition. Saunders College Pub. Philadelphia.
Kloc, M., G. Spohr, G. and L.D. Etkin. 1993. Translocation of repetititve
RNA sequences with the germ plasm in Xenopus oocytes. Science 262:
Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and
Meyerowitz, E. 1998. Principles of Development. Current Biology.
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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Wednesday, June 17, 1998