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The body plan of the embryo is established by a polarized source of developmental information in the oocyte. The Xenopus laevis oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including Vg1 and Xwnt-11, which might function in body plan specification, and Xcat-2, which might function in germ cell development. To identify components of the RNA anchoring mechanism, we used the manually isolated vegetal cortex (IVC) to assay loss or change in spatial arrangement of mRNAs caused by disruption of cortical elements. The role of cytoskeleton in mRNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin microfilaments and cytokeratin filaments. Treatment of oocytes with cytochalasin B caused clumping of Vg1 and Xwnt-11 as revealed by in situ hybridization of the IVC, but did not cause their release, as confirmed by RT-PCR analysis. These mRNA clumps did not match the distribution of actin microfilament clumps, but were distributed similarly to the remnant cytokeratin filaments. Treatment of oocytes with monoclonal anti-cytokeratin antibody C11 released these mRNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. These results show that Vg1 and Xwnt-11 are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depend on an intact actin meshwork. Colcemid did not disrupt Vg1 and Xwnt-11 retention in the IVC, so anchoring of these mRNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased Vg1 and Xwnt-11. Loss of these mRNAs was due mainly to ribonuclease activity released from membrane components. However, when ribonuclease activity was suppressed under cold temperature, a higher amount of Vg1 and Xwnt-11 was recovered in the supernatant. This result suggested that a fraction of these mRNAs required membranes to be retained in the cortex. By contrast, Xcat-2 mRNA was neither released nor degraded following treatments with cytochalasin B, C11, colcemid and Triton X-100 under cold temperature, so no cortical element could be implicated in its anchoring.
Fig. 1. Retention of vegetally localized mRNAs in the IVC and the effect of cytoskeletal inhibitors, CB and C11. Whole oocytes (vegetal pole
view: A,E,I) and IVCs (cytoplasmic view: B,C,D,F,G,H,J,K,L) were stained, using whole-mount in situ hybridization, for Vg1, Xwnt-11 and
Xcat-2 mRNAs. (A,E,I) In the whole oocyte, the Vg1 distribution covered the vegetal surface, extending from the vegetal pole (black asterisk)
to the region just below the animal-vegetal boundary (arrow). The white asterisk indicates the pigmented animal hemisphere. Xwnt-11 and
Xcat-2 staining was concentrated as a disc at the vegetal pole, with Xwnt-11 having a broader distribution. Portions of the vegetal surface had
peeled off from some oocytes (E,I). (B,F,J) The IVC retained mRNAs with a granular texture, and their distribution patterns resembled those of
the whole oocyte. Vg1 was distributed in the entire cortex, whereas Xwnt-11 and Xcat-2 were both concentrated as a disc at the vegetal pole.
The boundary of the Xwnt-11 disc was not sharp relative to the Xcat-2 disc. (C,G,K) Spatial arrangement of mRNAs in CB-treated IVCs.
Oocytes were treated with 25 mg/ml CB or control buffer, containing 0.02% DMSO. After 20-23 hours of incubation, IVCs were harvested and
assayed. Clumps of Vg1 were retained in the IVC (see Fig. 2B for high magnification). Clumps of Xwnt-11 were discernible in the peripheral
region of the disc. Clumps of Xcat-2 were not evident, because their staining was concentrated as a disc. (D,H,L) Retention of mRNAs in C11-
treated IVCs. Oocytes were microinjected with the monoclonal antibodies anti-cytokeratin C11 or control anti-tau. After approximately 24
hours of incubation, IVCs were harvested and assayed. Most IVCs lacked Vg1 staining, whereas Xwnt-11 staining decreased. Xcat-2 appeared
to be retained with C11 treatment as in the control IVC. Brown animal cortex (D, arrow) sometimes borders the rim of the IVC. Bar, 500 mm
(A-L).
Fig. 2. Components of the IVC of oocytes treated with cytoskeletal inhibitors, CB and C11. (A,B) At a high magnification, in situ hybridization
staining for Vg1 mRNA was relatively diffuse in the control IVC (A) and was punctate in the CB-treated IVC (B). (C,F,I) Cytoskeletal and
membrane-bound structures were distributed throughout the IVC. Immunocytochemical staining with the monoclonal antibody 1h5 revealed the
cytokeratin network (C). The boxed area in C is magnified in the inset, showing the texture of the cytokeratin filaments (compare with the inset
of E). Phalloidin labelling revealed the actin meshwork that was interspersed by the non-fluorescent cortical granules (F). Immunocytochemical
staining with the polyclonal anti-TRAPa antibody revealed thin, irregularly shaped structures of ER (I). (D,G,J) Effect of CB treatment.
Oocytes were treated with 10 mg/ml (D) and 25 mg/ml (B,G,J) of CB or control buffer, containing 0.02% DMSO. After 15-24 hours of
incubation, IVCs were harvested and assayed. The cytokeratin network was destroyed, although filaments (arrows) remained (D). The actin
meshwork was disrupted into phalloidin-labelled aggregates of actin (G). Clumps of ER were also evident (J). (E,H,K) Effect of C11 treatment.
Oocytes were microinjected with the monoclonal antibodies anti-cytokeratin C11 or control anti-tau. After approximately 24 hours of
incubation, IVCs were harvested and assayed. The cytokeratin network was retained, although it was perturbed by C11 treatment (E). The
boxed area in E is magnified in the inset, showing that the filaments were thickened (arrow) and had varicosities (small arrows).
Immunocytochemistry was also performed, using C11 as the primary antibody. The same immunostaining pattern was revealed as when 1h5
was used as the primary antibody, or when the primary antibody was omitted and the secondary antibody, DTAF-goat anti-mouse, was used
alone (not shown). The actin meshwork was unperturbed by C11 treatment (H). There was less immunostaining for ER with C11 treatment (K).
Bar, 100 mm (A-K).
Fig. 3. ER disruption by detergent treatment of the IVC. IVCs were
incubated in P10EM buffer with or without 0.5% TX for 30 minutes
at 21°C (A,B) or at 4°C (C-F). ER was visualized by
immunocytochemically staining for TRAPa (A-D), an integral
membrane protein of ER, and Vg1 RBP (E,F), a protein that interacts
with ER. (A,B) TRAPa immunostaining revealed irregularly shaped
structures in the control IVC. TRAPa-immunostained ER was
reduced and fragmented (arrows) after TX treatment of the IVC at
21°C. (C,D) When TX treatment was performed at 4°C, TRAPa
immunostaining appeared reduced and clumped (arrows).
(E,F) Remnants of ER (arrows) were present after TX treatment at
4°C, because Vg1 RBP immunostaining was present. Bar, 100 mm
(A-F).
Fig. 4. RT-PCR analysis of the detergent-treated IVC. (A) Fractions
of detergent-treated IVCs. In each experiment, IVCs were incubated
in P10EM buffer with or without 0.5% TX for 30 minutes at 21°C or
at 4°C. After incubation, the IVCs were separated into the vegetal
cortex (V) and supernatant (S) fractions. Yeast RNA was added to
the fractions, which were homogenized, and total RNA was
extracted. The amount of total RNA per fraction was equalized based
on yeast RNA, and RNAs were reverse-transcribed into cDNA. The
target molecules, Vg1, Xwnt-11 and Xcat-2 in the cDNA samples,
were PCR amplified and the PCR products were examined by gel
electrophoresis and autoradiography. With TX treatment at 21°C,
both Vg1 and Xwnt-11 were present in the V fraction at less than
25% of the control. Vg1 and Xwnt-11 were not detected in the S
fraction. Xcat-2 decreased in the V fraction by approximately 50% of
the control. Very little Xcat-2 was recovered in the S fraction. These
results are representative of seven independent experiments that were
carried out at 21°C. With TX treatment at 4°C, most of the Vg1 and
Xwnt-11 were retained in the V fraction, with a small but repeatable
amount released to the S fraction. The level of Xcat-2 in the V
fraction treated with TX at 4°C was similar to the control V fraction.
These results are representative of three independent experiments
that were carried out at 4°C. (B) mRNAs are degraded by TX in the
presence of IVCs. IVCs were incubated in P10EM buffer with or
without 0.5% TX for 30 minutes at 21°C. The vegetal cortex (V) and
supernatant (S) fractions of the IVCs were kept together as a sample.
Yeast RNA was added to the samples, and total RNA was extracted
and subjected to RT-PCR. In the TX-treated sample, Vg1, Xwnt-11
and Xcat-2 signals decreased significantly. These results are
representative of four independent experiments. Xbra, which is first
expressed at the gastrula stage, was used as a marker to test the effect
of TX treatment on exogenous RNA in the presence of IVCs. One mg
of total RNA from stage 10 early gastrula was added to the IVCs
during incubation in TX. In two independent experiments, the Xbra
signal decreased with TX treatment in the presence of IVCs.
Fig. 5. Model of Vg1 and Xwnt-11 mRNA anchoring.
Schematic sagittal views of the IVC harvested from control
oocytes (A), from oocytes treated with CB (B) or C11 (C), and
after treatment with TX (D). (A) A single filament of actin and
of cytokeratin is shown for clarity. Actin microfilaments are
located immediately internal to the plasma membrane and
form a meshwork. Cytokeratin filaments are internal to the
actin meshwork and form a network whose integrity depends
on the actin meshwork. These two filament systems are linked
by a mediating protein(s). Vg1 and Xwnt-11 mRNAs are
retained in the IVC through associations with the cytokeratin
filaments. However, a fraction of Vg1 and Xwnt-11 is
associated with membranes, which may include ER. ER may
be associated with the cytokeratin filaments. (B) CB treatment
causes the actin microfilament to clump. The mediating
proteins that link the cytokeratin filament and actin
microfilament are partially freed when actin clumps.
Consequently, the cytokeratin filament breaks apart and is no
longer fixed in an extended position. The ends of the cytokeratin filament are free to move, and when the filament clumps, so do the associated
Vg1 and Xwnt-11 mRNAs, and ER. (C) Monoclonal anti-cytokeratin antibody C11 binds along the length of the cytokeratin filament. The
cytokeratin filament may be physically distorted by C11 antibody binding, resulting in mRNA release. The actin filament remains intact with
C11 treatment. C11 might affect retention of ER in the cortex, as ER was lost in two of four experiments. (D) TX solubilizes the IVC, although
both the cytokeratin filament and actin microfilament remain intact. Most of Vg1 and Xwnt-11 mRNAs remain associated with the IVC.
