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First published online 6 July 2005
doi: 10.1242/dev.01919

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Potential structural role of non-coding and coding RNAs in the organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes

¹ Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston TX 77030, USA
² Institute of Zoology, Jagiellonian University, ul. Ingardena 6, 30-060 Krakow, Poland
³ Public Health Research Institute, Department of Molecular Genetics, 225 Warren Street, Newark, NJ 07103, USA

View larger version (86K): [in a new window]   Fig. 1. Destruction of Xlsirts or VegT, but not Xpat RNA, releases Vg1 mRNA from the cortex. (A) Stage VI oocytes analyzed by whole-mount in situ hybridization with digoxigenin-labeled Vg1 probe. (1) Control oocytes injected with antisense Xpat ODN. P, pigmented animal pole; arrows indicate non-pigmented vegetal pole with localized Vg1 mRNA. (2) Oocytes injected with antisense VegT ODN or (3) antisense Xlsirts ODNs. (B) Sections showing the fragments of vegetal cortex of control non-injected (1), antisense VegT- (2) and antisense Xlsirts- (3) injected stage VI oocytes. Arrows indicate Vg1 mRNA at the vegetal cortex in control oocyte and remnants of Vg1 mRNA at the vegetal cortex in antisense Xlsirts- and antisense VegT-injected oocytes. Scale bar: 380 µm in A; 30 µm in B. (C) Northern blot of poly(A)RNA isolated from 100 control and antisense Xpat ODN-injected oocytes, hybridized to antisense Xpat and antisense Xcat2 digoxigenin-labeled probes.

View larger version (134K): [in a new window]   Fig. 2. Cytokeratin in vegetal cortex of stage VI oocytes. (A) Confocal images of the vegetal cortex of stage VI oocytes, oocytes matured in vitro and eggs activated by pricking stained with anti-pancytokeratin C11 antibody conjugated with FITC. Left panel, stage VI oocytes. Central panel, oocytes matured in vitro with progesterone. Right panel, activated eggs. Control mock-injected stage VI (1) and matured (2) oocytes (arrows indicate cytokeratin foci). (3) Control oocyte after activation. (4) Stage VI oocyte injected with antisense Xlsirts ODNs (short arrow indicates yolk platelet). (5) Matured antisense Xlsirts-injected oocyte (arrows indicate cytokeratin foci). (6) Activated egg from antisense Xlsirt-injected oocyte (short arrow indicates yolk platelet). (7) Stage VI oocyte injected with antisense VegT ODN. (8) Antisense VegT ODN-injected oocyte that has matured. (9) Activated antisense VegT-injected egg showing only separated short filaments of cytokeratin (arrows). (B) Stage VI oocyte injected with antisense Xpat (1), antisense XlCaax (2) ODNs or VegT morpholino (3). Scale bars: 12 µm.

View larger version (102K): [in a new window]   Fig. 3. Anti-pancytokeratin C11 antibody disrupts cytokeratin network in stage VI oocytes. Anti-pancytokeratin C11 antibody conjugated with FITC was injected into the cytoplasm of stage VI oocytes. At various time points, oocytes were fixed for 5 minutes in 1% formalin in PBS and vegetal cortexes were observed under fluorescent microscope. (A) Up to 30 minutes from the injection of antibody. (B) Three hours after the injection of antibody. (C) Antibody-injected progesterone matured oocyte. (D) Antibody-injected, matured and activated (by pricking) oocyte. (E) Antibody injected oocyte post-injected with rescuing VegT RNA shows partial reconstitution of the cytokeratin network. Scale bars: 12 µm.

View larger version (67K): [in a new window]   Fig. 4. Association of injected Xlsirts and VegT RNAs with the cytokeratin filaments in stage VI oocytes. (A) Texas Red-labeled synthetic RNA (red) was injected into stage VI oocytes, cytokeratin was labeled with FITC-conjugated antibody (green), and vegetal cortices were observed under a fluorescence microscope. In Xpat RNA-injected oocytes, particles of Xpat RNA were visible outside of cytokeratin filaments (1, arrows). In VegT RNA-injected (2,3) and Xlsirts RNA-injected (4) oocytes, RNA particles were concentrated around and on the cytokeratin filaments (arrows). Scale bar: 500 nm. (B) Student's t-test statistical analysis of the number of RNA aggregates associated with cytokeratin filaments showing the statistically significant difference (P=0.03) between the number of aggregates in VegT and Xlsirts versus Xpat RNA-injected samples. Each bar represents the average (with the standard deviation) number of RNA aggregates counted in eight independent samples along the 2 µm length of cytokeratin filament in each experimental group.

View larger version (94K): [in a new window]   Fig. 5. Association of endogenous Xlsirts and VegT RNAs with cytokeratin network in the oocyte cortex. (A) Texas Red-labeled molecular beacons (red) that hybridize to VegT, Xlsirts and Xpat RNAs were used to detect the association of endogenous RNAs with the cytokeratin network that was detected with FITC-conjugated antibody (green). Xlsirts RNA (1, 2) and VegT mRNA (3, 4) particles were intimately associated with the cytokeratin filaments (arrows). VegT mRNA particles were often visible uniformly spaced along the cytokeratin filament (4, arrows). Xpat mRNA was not associated with the cytokeratin (5). Scale bars: 2 µm in 1, 3 and 5; 1.2 µm in 2 and 4. (B) Student's t-test statistical analysis of the number of endogenous RNA aggregates associated with cytokeratin filaments. Graph shows that the difference in the number of RNA aggregates in VegT and Xlsirts samples versus Xpat samples is statistically highly significant (P=0.002). Each bar represents the average (with the standard deviation) number of RNA aggregates counted in eight independent samples along the 2 µm length of cytokeratin filament in each experimental group.

View larger version (129K): [in a new window]   Fig. 6. Injected VegT RNA rescues and reconstitutes the cytokeratin network disintegrated by antisense VegT oligonucleotides. Cytokeratin network in control stage IV oocytes (A) and stage VI oocytes (B). (C) antisense VegT ODN-injected stage IV and (D) stage VI oocytes. After the injection of synthetic VegT RNA, the cytokeratin network reconstitutes as a more complex network in stage IV oocytes (E) and as a normal (comparable with control) network in stage VI (F) oocytes. The injection of synthetic VegT RNA into control stage IV (G) and stage VI (H) oocytes does not affect the appearance of the cytokeratin network. Scale bars: 12 µm.

View larger version (100K): [in a new window]   Fig. 7. Effect of antisense oligonucleotides injection on the ultrastructure of germ plasm in mature oocytes. Electron microscopy images of germ plasm islands in the vegetal cortex of matured oocytes. In control (A), antisense XlCaax ODN (B), antisense Xlsirts ODN (C) and VegT morpholino (D) -injected oocytes, germ plasm islands contained tiny electron-dense germinal granules (arrows) located between mitochondria (m). In oocytes injected with antisense VegT ODN (E), the germinal granules coalesced precociously, forming stringy large aggregates (arrows). These aggregates resembled germinal granule aggregates, which normally occur in four- to eight-cell embryos. Y, yolk; CG, cortical granules. Scale bars: 400 nm.

View larger version (75K): [in a new window]   Fig. 8. Effect of antisense oligonucleotides and anti-pancytokeratin C11 antibody injection on germ plasm and germinal granules in two-cell embryos. (A) Whole-mount in situ hybridization with digoxigenin-labeled antisense Xcat2 RNA probe showing the Xcat2 RNA present in the germinal granules within the germ plasm (arrows). Germ plasm of control (1), antisense Xlsirts ODNs (2), antisense VegT ODN (3) and anti-pancytokeratin C11 antibody (4) -injected embryos. (B) Higher magnification of germ plasm regions showing Xcat2 labeled germinal granules in control embryos (1, arrow); unlabelled or slightly labeled granules in antisense Xlsirts embryos (2, arrows); and labeled large stringy aggregates of germinal granules in antisense VegT (3, arrows) and anti-pancytokeratin C11 antibody (4, arrows) embryos. Scale bars: 340 µm in A; 25 µm in B.

View larger version (164K): [in a new window]   Fig. 9. Oocyte cytokeratin network disintegrated by the injection of antisense oligonucleotides or anti-pancytokeratin C11 antibody, reconstitutes spontaneously in cleaving embryos. Cleaving 2-cell stage embryos originating from control mock-injected oocytes (1), or oocytes injected with antisense Xlsirts ODNs (2), antisense VegT ODN (3), or anti-pancytokeratin C11 antibody (4) were produced by host transfer. Fixed embryos were stained with anti-pancytokeratin C11-FITC antibody and cortices of vegetal blastomeres were observed under fluorescent microscope. Intricate cytokeratin network comparable with control (1) reconstitutes in antisense Xlsirts (2) and antisense VegT embryos (3). In anti-pancytokeratin antibody embryos (4), the cytokeratin network also reconstitutes but looks different from control. Scale bars: 10 µm.

View larger version (97K): [in a new window]   Fig. 10. Effect of antisense oligonucleotide and anti-pancytokeratin C11 antibody injection on the ultrastructure and molecular composition of germinal granules in four-cell embryos. (A) Electron microscopy of sections of germ plasm islands from four-cell embryos hybridized in situ, as whole mounts, with digoxigenin-labeled Xcat2 RNA probe. Hybridization signal was detected using nanogold-conjugated anti-digoxigenin antibody and silver enhancement (see Kloc et al., 2002a). In control uninjected embryos (1, inset 1') and embryos injected with antisense Xpat ODN (2, inset 2'), the germinal granules (arrows) labeled with black silver grains (representing Xcat2 mRNA) are visible between mitochondria (m) and yolk platelets (Y). In embryos injected with antisense Xlsirts ODNs (3, inset 3') the germinal granules (small arrows) are devoid of label, and Xcat2 mRNA (black silver grains, large arrows) is located outside the germinal granules. In embryos injected with antisense VegT ODN (4), the germinal granules (small arrows) form huge aggregates containing low level of Xcat2 mRNA (small black silver grains, large arrows). In embryos injected with anti-pancytokeratin C11 antibody (5) the germinal granules form huge aggregates (small arrows), similar to aggregates in antisense VegT embryos, but heavily labeled with black silver grains (large arrows). Scale bars: 320 nm in 1-5, and 160 nm in 1'-3'. (B) Student's t-test statistical analysis of the number of silver grains (Xcat2 mRNA) associated with germinal granules. Each bar represents the average (with the standard deviation) number of silver grains counted in 10 independent samples in 0.25 µm2 area of germinal granule in each experimental group. Number above the graph represents statistically significant (P<0.05) P value. The graph shows that the decrease in the number of silver grains (Xcat2 mRNA) present in the germinal granules of antisense Xlsirts and antisense VegT embryos is statistically highly significant (P=0.00001 and P=0.001, respectively).

View larger version (24K): [in a new window]   Fig. 11. Effect of antisense oligonucleotide injection on primordial germ cells (PGCs) in blastula. Whole-mount in situ hybridization with digoxigenin-labeled Xpat RNA probe. Embryos were acquired by host transfer and cleared to visualize their interior. In all embryos, the side view of blastulae is shown with the animal pole at the top and the vegetal pole at the bottom. In control embryo (A), the Xpat labeled islands of germ plasm (arrows) are visible in the compact group at the vegetal tip of the blastula. In antisense VegT-injected embryos (B,C), the germ plasm is visible as the large aggregates (double arrow, B and C) and also as small aggregates dispersed within the vegetal blastomeres (arrows, B). In antisense Xlsirts injected embryos (D,E), the germ plasm was either barely visible (arrow, D) or was dispersed over the larger surface of vegetal region of the blastula (arrows, E). Scale bars: 300 µm. The experiment was repeated three times with 20 embryos in each group.

View larger version (27K): [in a new window]   Fig. 12. Models of the effect of depletion of Xlsirts and VegT RNAs on the cytokeratin network and germinal granules. (A) The effect of Xlsirts RNA and VegT mRNA depletion, and of anti-cytokeratin antibody on the cytokeratin network and the germinal granules. In the cortex of stage VI oocytes, the cytokeratin (green) forms a complex, multi-layer network. The particles of VegT mRNA (red) and Xlsirts RNA (blue) are integrated into the network in RNA-specific pattern (1). The ablation of VegT mRNA or injection of anti-pancytokeratin antibody causes a severance of the network (2), which in turn, results in a premature aggregation of the germinal granules (3). The ablation of Xlsirts RNA results in the collapse of the network into a flattened, less complex sheet (2). The means of association of cytokeratin with germinal granules are unknown, but one possibility is that cytokeratin anchors germinal granules (pink spheres) indirectly, via an unknown binding protein(s) (yellow, 3). (B) The effect of VegT mRNA depletion and anti-cytokeratin antibody on the cytokeratin network and the germinal granule aggregation in oocytes and embryos. Control (left panel), and antisense VegT ODN or anti-cytokeratin C11 antibody injected (right panel) oocytes and embryos. Part of the vegetal cortex is shown. For simplicity, the cytokeratin is shown only in part of the cortex. In stage VI control oocytes, the germinal granules (pink spheres) are located in germ plasm islands (yellow) within the cortical network of cytokeratin filaments (green). In antisense VegT and anti-pancytokeratin antibody injected stage VI oocytes, the cytokeratin network is severed. In control mature oocytes, the cytokeratin network is replaced by cytokeratin foci with normal germinal granules and germ plasm. However, in mature antisense VegT- and anti-pancytokeratin antibody-injected oocytes, the germinal granules coalesce into long stringy aggregates. Upon egg activation, the cytokeratin network reconstitutes in control but not in antisense VegT-injected eggs, and only partially in anti-pancytokeratin antibody-injected eggs. In addition, the germ plasm islands aggregate into larger islands. In activated eggs, the germinal granules remain as small, separate entities in control eggs, and as stringy aggregates in antisense VegT and anti-pancytokeratin antibody-injected eggs. During early cleavage, the cytokeratin network is present both in control and in antisense VegT or anti-pancytokeratin antibody-injected embryos, and the germ plasm islands segregate to the vegetal apex of vegetal blastomeres. In control embryos, the germinal granules aggregate into larger, more complex germinal granules, and in antisense VegT or anti-pancytokeratin antibody-injected embryos, the stringy aggregates of germinal granules that were present in matured and activated eggs aggregate even further, resulting in the formation of extremely large aggregates.

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