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We found that there are two major pathways by which RNAs are localized at the vegetal cortex during oogenesis of Xenopus laevis. One of these, through which Xlsirts, Xcat2 and Xwnt11 are localized, involves transport during stages 1 and 2 of oogenesis via a region of the mitochondrial cloud that we call the message transport organizer or METRO. This pathway involved three steps, transport of RNA from the GV to the mitochondrial cloud, sorting of the RNAs to specific regions of the METRO, and translocation to and anchoring at the vegetal cortex. These three RNAs exhibit a distinct pattern of spatial localization within the METRO when they approach the vegetal cortex. The other pathway is used by Vg1. We detected Vg1 throughout the oocytecytoplasm during stages 1 and 2. During stage 3 it was translocated to the vegetal cortex and associated with the cortex overlapping the region at which the Xlsirt, Xcat2, and Xwnt11 RNAs are anchored. Our results also showed that anchoring of these RNAs was dependent in part on actin microfilaments, but was independent of microtubules. These results demonstrate a novel mechanism of translocation and RNA sorting used by RNAs several of which may be involved in the establishment of the embryonic body axis.
Fig. 1. Localization of Vg1, Xlsirts, Xcat2 and Xwnt11 at the vegetal cortex in stage 4 oocytes. Stage 4
oocytes were analyzed by whole-mount in situ hybridization for the presence of Vg1, Xlsirts, Xcat2 and
Xwnt11 RNAs according to the procedures described in methods. (A) Oocyte hybridized with the Vg1
probe; (B) oocyte hybridized with the Xlsirt probe; (C) oocyte hybridized with the Xcat2 probe;
(D) oocyte hybridized with the Xwnt11 probe. The bar in D represents 200 um.
Fig. 2. (A) Whole-mount in situ hybridization to different stage oocytes showing the patterns of Xlsirts, Xcat2 and Xwnt11 RNAs within the
mitochondrial cloud. Panels a,d and g are oocytes in the size range of 90-110 mm in diameter (Dumont stage 1) hybridized with the Xlsirt,
Xcat2 and Xwnt11 probes, respectively. Panels b, e and h are larger oocytes (size range 230-300 mm in diameter; Dumont mid to late stage 1)
hybridized with the same probes. Panels c, f and i are oocytes in the size range 275-400 mm (Dumont stage 2) hybridized with the same probes.
The arrows point to the mitochondrial cloud. GV, germinal vesicle. The bars represent 100 mm in g and 125 mm in i. (B) Sections of different
stage oocytes hybridized with Xlsirts, Xwnt11 and Xcat2 probes. Panels a, c and e are sections of whole-mount late stage 1 oocytes hybridized
with the Xlsirt, Xwnt11 and Xcat2 probes, respectively. Panels b, d and f are sections of stage 3 oocytes. GV, germinal vesicle.
Fig. 3. (A) Localization of Vg1 mRNA during oogenesis. Different stage
Xenopus oocytes were analyzed by whole-mount in situ hybridization using
the Vg1 probe. (a) Stage 1 oocytes. The arrow points to the mitochondrial
cloud; (b) Stage 2 oocyte. The arrow points to the position of the
translocating Vg1 RNA that is accumulating over the localized Xlsirts,
Xcat2, and Xwnt11 RNAs at the vegetal cortex. (c) Stage 4 oocyte showing
the distribution of the Vg1 RNA from the apex of the vegetal pole region to
the future marginal zone. A, animal pole, V, vegetal pole. Bars represent
100 um.
(B) Sections showing the localization of Vg1 RNA in different stage
oocytes. (a) Section through a late stage 1 oocyte showing the distribution
of Vg1 RNA throughout the entire cytoplasm. (b) Section through stage 2
oocyte showing the accumulation of Vg1 RNA at the vegetal cortex at the
position of the METRO localized RNAs. The arrows show the apparent
movement of the Vg1 mRNA along the cortical layer towards the future
marginal zone. (c) Section through a stage 3 oocyte showing the continued
streaming of the Vg1 mRNA toward the vegetal cortex.
Fig. 4. Detection of Xwnt11, Xcat2, Xlsirts and Vg1 RNAs by double in situ
hybridization. (A) Section of a stage 2 oocyte hybridized with probes for Xlsirts and
Xcat2 RNA. The solid arrow represents the Xlsirt RNA while the open arrow
represents the Xcat2 mRNA. These were distinguished using two different color
detection systems that differentiated each RNA. (B) Section of a stage 2 oocyte
hybridized with the Xlsirt probe (solid arrow) and the Xwnt11 probe (open arrow).
(C) Section of a stage 3 oocyte hybridized with the Xlsirt probe (solid arrow) and the
Vg1 probe (open arrow). (D) Lower magnification view of a similar section as in C
showing the accumulation of the Vg1 transcripts overlapping the Xlsirt RNA that was
localized in a disk-like pattern at the apex of the vegetal pole. The colors of the two
probes were computer enhanced using Adobe photoshop.
Fig. 5. The effect of cytochalasin B and
nocodazole on the Xlsirt RNA in the migrating
mitochondrial cloud. (A) Control untreated stage
2 oocytes that were cultured for 2 days. (B) Stage
2 oocytes that were cultured for 2 days in the
presence of cytochalasin B (25 mg/ml). (C) Stage
2 oocytes that were cultured in nocodazole
(1 ug/ml) for 2 days.
Fig. 6. The effect of cytochalasin B and nocodazole on the anchoring of Xlsirts, Xcat2, Xwnt11 and Vg1 RNAs. Stage 3 oocytes were treated
with either cytochalasin or with nocodazole for 2 days in culture. The treated oocytes were analyzed using whole-mount in situ hybridization
and sectioned to determine the effect of the drugs on the localized RNAs. Panels a,d,g and j are untreated control oocytes cultured for 2 days
and hybridized with the Xlsirt, Xcat, Vg1 and Xwnt11 probes respectively. Panels b, e, h and k are oocytes treated with cytochalasin B for 2
days and analyzed with the same probes. Panels c, f, i and l are oocytes treated with nocodazole for 2 days and also analyzed with the same
probes as above.
Fig. 7. Model for the potential interaction
between the two different pathways used to
localize RNAs at the vegetal cortex in Xenopus
oocytes. Maternal RNAs are localized at the
vegetal cortex using two pathways. The first (the
METRO pathway) localizes Xlsirts, Xcat2 and
Xwnt11. These RNAs utilize the message
transport organizer (METRO) which is a region
of the mitochondrial cloud at which these RNAs
accumulate after leaving the GV. The METROassociated
RNAs translocate to the apex of the
vegetal pole region during stage 1 and 2. RNAs
such as Vg1 that are localized using the second
pathway (Vg1 pathway) are distributed evenly
throughout the cytoplasm during stage 2. During
stage 3 they appear to translocate through a
region where the mitochondrial cloud was
located earlier. The Vg1 transcripts associate
with the cortex overlapping the localized Xlsirts.
They then appear to spread along the inner
cortical layer until they reach the marginal zone
region.
