Gard DL et al. (1997), The organization and animal-vegetal asymmetry o...

XB-ART-16668

Dev Biol 1997 Apr 01;1841:95-114. doi: 10.1006/dbio.1997.8508.

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The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon F-actin and microtubules.

Gard DL , Cha BJ , King E .

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Confocal immunofluorescence microscopy with anti-cytokeratin antibodies revealed a continuous and polarized network of cytokeratin (CK) filaments in the cortex of stage VI Xenopus oocytes. In the animal cortex, CK filaments formed a dense meshwork that both was thicker and exhibited a finer mesh than the network of CK filaments previously observed in the vegetal cortex (Klymkowsky et al., 1987). CK filaments first appeared in association with germinal vesicle (GV) and mitochondrial mass (MM) of oocytes in early mid stage I, indicating that CK filaments are the last of the three cytoskeletal networks to be assembled. By late stage I, CK filaments formed complex networks surrounding the GV, surrounding and penetrating the MM, and linking these networks to a meshwork of CK filaments in the oocyte cortex. During stage III-early IV, CK filaments formed a highly interconnected, apparently unpolarized, radial array linking the perinuclear and cortical CK filament networks. Polarization of the CK filament network was observed during mid stage IV-stage V, as first the animal, then the vegetal CK filament networks adopted the organization characteristic of stage VI oocytes. Treatment of stage VI oocytes with cytochalasin B disrupted the organization of both cortical and cytoplasmic CK filaments, releasing CK filaments from the oocyte cortex and inducing formation of numerous cytoplasmic CK filament aggregates. CB also disrupted the organization of cytoplasmic microtubules (MTs) in stage VI oocytes. Disassembly of oocyte MTs with nocodazole resulted in loss of the characteristic A-V polarity of the cortical CK filament network. In contrast, disruption of cytoplasmic CK filaments by microinjection of anti-CK antibodies had no apparent effect on cytoplasmic or MT organization. We propose a model in which the organization and polarization of the cortical network of CK filaments in stage VI Xenopus oocytes are dependent upon a hierarchy of interactions with actin filaments and microtubules.

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Species referenced: Xenopus laevis
Genes referenced: actl6a krt12.4 noct tbx2
???displayArticle.antibodies??? Krt5.2 Ab1 Pan Cytokeratin Ab Tuba4b Ab2 Tuba4b Ab4

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	FIG. 1. Confocal immunofluorescence microscopy of stage VI oocytes reveals a complex network of cytokeratin filaments in the animal and vegetal cortices. (A and B) Grazing views (projections of four and five sections at 1-mm intervals, respectively) of the vegetal (A) and animal (B) cortices of stage VI oocytes stained with C11 antibodies reveal complex networks of CK filaments in the cortices of both hemispheres. Note the characteristic difference in complexity and apparent mesh size of CK networks in the vegetal and animal cortices. (C and D) Optical cross sections (projections of five serial sections collected at 1-mmintervals) of the vegetal (C) and animal (D) hemispheres revealed the cortical CK filament networks, as well as a substantial network of CK filaments in the underlying subcortical cytoplasm. Note the difference in thickness of the cortical CK network in the vegetal and animal cortices and the radial organization of CK filaments in the animal hemisphere. Arrowheads in D denote transverse CK filaments (see text). (E) A low-magnification view (a single section) of an oocyte stained with C11 antibodies reveals an extensive network of CK filaments extending throughout the cytoplasm and surrounding the germinal vesicle (GV) and perinuclear cap of yolk-free cytoplasm (PNC). (F) Numerous CK filaments are apparent in this highmagnification image of the perinuclear cap of yolk-free cytoplasm (PNC) (a projection of five sections collected at 1-mm intervals). Scale bars are 25 mm in A–D, and F and 250 mm in E.
	FIG. 2. Cytokeratin filaments are first apparent during mid stage I of oogenesis. (A) A nest of early stage 0 oocytes isolated from a juvenile frog and stained with C11 antibodies (green) and EH (red). Cytokeratin staining is apparent only in the follicle cells surrounding the oocytes. A projection of 3 optical sections. (B) Asingle late-stage 0 oocyte (35-mmdiameter) isolated from a juvenile frog and stained with C11 antibodies (green) and YP (red). Cytokeratin staining is apparent only in the surrounding follicle cells (arrowheads denote follicle cell nuclei). Note the characteristic ‘‘bouquet’’ organization of a chromatin. A projection of 3 optical sections. (C) A stereo view of a mid stage I (125-mm diameter) oocyte from a juvenile frog stained with C11, showing the assembly of a perinuclear CK filament network and the association of CK filaments with two mitochondrial aggregates (M). Note the lack of cytoplasmic and cortical CK filaments. Reconstructed from 53 optical sections. (D) A stereo view of a mid–late-stage I (150 mm) oocyte from a juvenile frog stained with C11 antibodies. Note the extensive perinuclear network of CK filaments, the CK filaments associated with the mitochondrial mass (M), and individual filaments extending from the perinuclear network into the surrounding cytoplasm. Reconstructed from 70 optical sections. Scale bars are 10 mm in A and B and 25 mm in C and D.
	FIG. 3. Cytokeratin filaments form perinuclear, cytoplasmic, and cortical networks during stages I and II of oogenesis. (A) A stereo view of a late stage I (185-mm diameter) oocyte isolated from an adult frog stained with C11 antibodies, showing a meshwork of CK filaments surrounding the GV and linking the GV and mitochondrial mass (M) to the oocyte cortex. Reconstructed from 76 optical sections. (B) A stereo pair of the mitochondrial mass of a stage I oocyte (approximately 200 mm in diameter) isolated from an adult frog and stained with C11 antibodies (reconstructed from 25 optical sections). Note the extensive network of CK filaments surrounding and filling the mitochondrial mass. (C) A stereo view of a late stage II or early stage III (350–400 mm in diameter) oocyte. Little radial order is seen in the cytoplasmic CK network. The dense, subcortical CK network (arrowheads) lies approx. 10 mm below the oocyte surface. Reconstructed from 39 optical sections. Scale bars are 25 mm.
	FIG. 4. The CK filament network of early stage IV oocytes is not detectably polarized along the A–V axis. (A) A stereo view of a stage III oocyte (approx. 400 mm in diameter). Radially oriented CK filaments (arrowheads) link the cortical CK network to a less-ordered CK network surrounding the GV (outlined in red). Reconstructed from 20 optical sections. (B,C) Stereo views (both reconstructed from 15 optical sections) of the animal (B) and vegetal (C) regions of a single early stage IV oocyte (450–500 mm in diameter). No evidence of polarity is seen in the organization of cytoplasmic or cortical CK filaments in these views of the same oocyte. Note the numerous transverse connections between the radially oriented CK filaments in all three views. Asterisks in B denote obscuration of the CK filaments in the subcortical region of the animal hemisphere due to accumulation of pigment. Scale bars are 25 mm.
	FIG. 5. Polarization of the CK filament network is evident in mid stage IV. (A) A low-magnification view of a mid stage IV (600 –650 mmin diameter) oocyte. Note the difference in thickness of the cortical CK network in the animal and vegetal hemispheres.(B) A composite of five images (each a projection of 5 optical sections) spanning the equatorial region of a mid stage IV (800 –850 mm in diameter) oocyte. Note the pronounced difference in cytoplasmic and cortical cytokeratin organization in the animal (upper) and vegetal (lower) regions. CK organization in the animal cortex and cytoplasm is similar to that of fully grown stage VI oocytes. The CK network of the vegetal cortex remains much thicker than that of fully grown stage VI oocytes (compare to Fig. 1). (C) A grazing view (projection of 6 optical sections) of the animal cortex of a mid stage IV (600–650 mm) oocyte. The organization of CK filaments is similar to that of a fully grown stage VI oocytes. (D) A grazing view (projection of 13 optical sections) of the vegetal cortex of a mid stage IV (600 –650 mm) oocyte. The cortical CK network is much thicker than that observed in fully grown stage VI oocytes. Scale bar is 100 mm in A and 25 mm in B and C.
	FIG. 6. Cytochalasin B disrupts CK filament and MT organization in stage VI Xenopus oocytes. (A) Stretching of the cortical CK network into the subcortical cytoplasm (arrowheads) is apparent in the animal hemisphere of an oocyte treated with 20 mg/ml CB for 20 hr (a projection of 5 optical sections). Arrowheads denote the inner margin of the stretched CK network and also point out aggregates of tangled CK filaments. (B) A projection of 13 optical sections of the animal cortex and subcortical cytoplasm of a CB-treated oocyte (20 hr in 20 mg/ml) reveals a large fibrous aggregate (large arrow) and numerous smaller aggregates of tangled CK filaments (small arrowheads). (C) Numerous brightly stained CK aggregates (black arrowheads) are apparent below the GV of this CB-treated oocyte (20 mg/ml CB for 20 hr) stained with C11 antibodies. Note the thickened cortical CK filament network in the animal hemisphere (white arrowheads; compare to Fig.1E). (D) A view of two fibrous CK aggregates in a CB-treated oocyte (20 mg/ml for 41 hr) viewed at higher magnification (a projection of 38 optical sections collected at 1-mmintervals). (E) Similar aggregates in a CB-treated oocytes (20 mg/ml for 20 hr) stained with fluoresceinconjugated phalloidin (a projection of 23 optical sections at 1.5-mm intervals). (F and G) Cross-sectional views of untreated (F) and CBtreated (G) oocytes (20 mg/ml for 40 hr) stained with 6-11B-1 (anti-acetylated a-TB). Arrows in (F) denote radially oriented MT bundles in untreated oocytes. Note the loss of radially oriented MT bundles (arrows in F) and the appearance of a broad cortical region containing disordered MTs (arrows in G) in the CB-treated oocytes. Scale bars are 25 mm in A and B and E and G, 250 mm in C, and 10 mm in F.
	FIG. 7. Nocodazole disrupts the A–V polarization of the cortical cytokeratin filament network. (A and B) Grazing views (projections of two and three optical sections, respectively) of the vegetal (A) and animal (B) cortices of oocytes treated for 48 hr with 20 mg/ml NOC, stained with C11 antibodies. Note the fineness of the CK meshwork in the vegetal cortex of NOC-treated cells, relative to the coarser meshwork found in untreated oocytes (compare with Fig. 1A). (C and D) Cross-sectional views (projections of five optical sections) of the vegetal (C) and animal (D) hemispheres of oocytes treated with 20 mg/ml NOC for 48 hr and stained with C11 antibodies. Note the nearly equal thickness of the CK network in the vegetal and animal cortices. (E and F) Cross-sectional views (single optical sections) of the vegetal (E) and animal (F) hemispheres of oocytes treated with a combination of 5 mg/ml NOC and 5 mg/ml CB for 12 hr. Inclusion of NOC inhibits the broadening or stretching of the cortical CK network seen in oocytes treated with CB alone (compare to Figs. 5C and 5D). All scale bars are 25 mm.
	FIG. 8. Microinjection of anti-cytokeratin antibodies disrupts cytoplasmic cytokeratin filaments in stage VI Xenopus. (A) Low-magnification view (a projection of 5 optical sections at 10-mm intervals) of an oocyte fixed 2 hr after injection of 50 ng of C11 anti-CK IgG, stained with C11 antibodies. Note the zone of CK filament disruption (dotted outline). Few CK filaments were observed within this zone, while CK filament organization in the surrounding cytoplasm appeared normal. (B) Low-magnification view (a projection of 4 optical sections at 10- mm intervals) of an oocyte fixed 18 hr after injection of 100 ng of C11 IgG, stained with C11 antibodies. Cytoplasmic CK filaments were eliminated nearly completely. (C and D) High-magnification views (C is a single optical section, D is a projection of 5 optical sections; note the difference in scale) of animal (C) and vegetal (D) regions fixed 18 hr after injection of 100 ng of C11 IgG, stained with C11 antibodies. Note the numerous small CK aggregates and remnants of the cortical CK network (denoted by arrowheads in C and D). (E) A view of the vegetal cortex (a projection of 10 optical sections collected at 1-mm intervals) of an oocyte injected with 100 ng of C11 IgG and stained with Texas red-conjugated anti-mouse IgG. Injected antibody is bound to individual CK filaments in the severely distorted cortical CK network. (F) Cortical and cytoplasmic CK filaments were not affected by injection of 50 nl of K glutamate injection buffer (a projections of 10 optical sections). (G) MT organization (stained with anti-a-TB) appears normal 24 hr after injection of 100 ng of C11 IgG. Note the characteristic radial bundles of MTs in the animal hemisphere. Scale bars are 250 mm in A and B, 50 mm in C, and 25 mm in D–G.
	FIG. 9. A summary of cytokeratin filament and cytoskeletal organization and polarization during oogenesis in Xenopus laevis. (A) During stage 0 and early stage I, oocytes lack CK filaments, which are found in the surrounding follicle cells. By early–mid stage I, CK filaments form a network surrounding the GV, surround and penetrate the mitochondrial mass, and begin to extend into the cytoplasm. By mid– late-stage I, an extensive network of cytoplasmic CK filaments links the perinuclear network to the mitochondrial mass and cortex. (B) CK filaments are symmetrically distributed during early stage IV. Polarization of the cortical and cytoplasmic CK filament networks is apparent during mid stage IV– stage V, as first the animal (during mid stage IV) and then the vegetal (during stage V) CK filament networks adopt the organization characteristic of stage VI. Summarized from Franz et al. (1983); Godsave et al. (1984), Klymkowsky et al. (1987); Torpey et al. (1992), Ryabova et al. (1993), and this report. (C) Organization and polarization of the CK filament cytoskeleton in stage VI Xenopus oocytes are dependent upon both F-actin and MTs: (1) CK filament organization is dependent upon cortical and cytoplasmic Factin; (2) MT organization is dependent upon F-actin; (3) polarization of the cortical cytokeratin network is dependent upon MTs; but (4) neither MT nor actin organization is dependent upon cytoplasmic cytokeratin filaments. (D) A model for the organization of the cytoskeleton of stage VI Xenopus oocytes. MTs (blue lines) are anchored to cortical and perinuclear actin (green) via the centrosomal protein g-TB (blue dots). Cytokeratin filaments (red lines) are anchored to F-actin in the cortical and perinuclear cytoplasm by an unidentified protein complex (red *). Cytokeratins are also linked to MTs, possibly by a MT-dependent motor protein. Cytoplasmic actin cables have been omitted for clarity. In this model, the A–V polarity of the MT cytoskeleton is specified by the asymmetric distribution of the centrosomal protein g-TB in the oocyte cortex, and the A–V polarity of the cortical cytokeratin network is dependent upon the asymmetric organization of MTs in the animal and vegetal hemispheres (see text).