Klymkowsky MW et al. (1987), Polar asymmetry in the organization of the cort...

XB-ART-28087

Development 1987 Jul 01;1003:543-57.

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Polar asymmetry in the organization of the cortical cytokeratin system of Xenopus laevis oocytes and embryos.

Klymkowsky MW , Maynell LA , Polson AG .

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We have used whole-mount immunofluorescence microscopy of late-stage Xenopus laevis oocytes and early embryos to examine the organization of their cortical cytokeratin systems. In both mature oocytes and early embryos, there is a distinct animal-vegetal polarity in cytokeratin organization. In mature (stage-VI) oocytes, the cytokeratin filaments of the vegetal region form a unique, almost geodesic network; in the animal region, cytokeratin organization appears much more variable and irregular. In unfertilized, postgerminal vesicle breakdown eggs, the cortical cytokeratin system is disorganized throughout both animal and vegetal hemispheres. After fertilization, cytokeratin organization reappears first in a punctate pattern that is transformed into an array of oriented filaments. These cytokeratin filaments appear first in the vegetal hemisphere and are initially thin. Subsequently, they form bundles that grow thicker through the period of first to second cleavage, at which point large cytokeratin filament bundles form a loose, fishnet-like system that encompasses the vegetal portion of each blastomere. In the animal region, cytokeratin filaments do not appear to form large fibre networks, but rather appear to be organized into a system of fine filaments. The animal-vegetal polarity in cytokeratin organization persists until early blastula (stage 5); in later-stage embryos, both animal and vegetal blastomeres possess qualitatively similar cytokeratin filament systems. The entire process of cytokeratin reorganization in the egg is initiated by prick activation. These observations indicate that the cortical cytoskeleton of Xenopus oocytes and early embryos is both dynamic and asymmetric.

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Species referenced: Xenopus laevis
Genes referenced: krt12.4 krt61 slc4a1 vim
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	Fig. 1. Analysis of Ih5 specificity in Xenopus A6 cells. (A) A micrograph, taken in fluorescein-optics of methanol-fixed, Ih5-labelled A6 cells demonstrates that Ih5 labels cytokeratin filaments (Bar, lOjjm). (B) Detergent-insoluble cell residue proteins of Xenopus A6 cells were separated by SDS-polyacrylamide gel electrophoresis and either stained with Coomassie brilliant blue (lane marked 'cell residue') or were electrophoretically transferred to nitrocellulose paper and probed with the monoclonal antibodies antilFA, Ih5, 14h7, AE1 or Id7. Relative molecular mass marker positions (xlO~3) are noted on the left hand side of the figure; the positions of major protein bands have been marked across the figure.
	Fig. 2. Cytokeratin organization in the mature oocyte. The labelling of cytokeratin filaments by Ih5 in the animal region of mature oocytes varied dramatically from barely detectable (A, specific staining marked by white arrows; grey regions are areas of yolk autofluorescence visible through pigment cap); through robust, but apparently disorganized (B); to clear systems of interconnected filaments (C). In the vegetal hemisphere, the organization of cytokeratin filaments is much more regular (D), consisting of thin fibres that interconnect distinct vertices. This type of cytokeratin organization is characteristic of the entire vegetal region of mature oocytes; in some regions it can approach a geometric precision (E). Arrows in part E mark vertices of the cytokeratin lattice. The bar in part E marks 10/im for all parts.
	Fig. 3. Cytokeratin organization in eggs and early postfertilization embryos. In some eggs, Ih5 labelling consisted of scattered filament fragments (A, negative image; white arrows point to Ih5-labelled fibres); more often (B, positive image) Ih5 labelled amorphous masses and irregular aggregates in the cortex. The following series of micrographs were taken from a batch of eggs in which the unfertilized eggs showed no punctate Ih5-labelling. By 15min after fertilization, Ih5-labelling appeared as a pattern of small spots (C, positive image) which grew larger and more irregular (D, positive image - 30min postfertilization) and were eventually transformed into a fibrous array (E, positive image - 60min postfertilization), with an apparent overall directionality (marked by white arrow). Bar in part E marks 10^m for all parts.
	Fig. 4. Cytokeratin organization in 2-, 4- and 8-cell embryos. By the time of first cleavage (A) the cytokeratin filament system of the vegetal hemisphere has beguh to coalesce into a system of anastomosing filament bundles; this reorganization of cytokeratin filaments continues through second (B) and third (D) cleavage to produce a highly regular cytokeratin filament network. The cytokeratin system does not appear to descend into the cleavage furrows (marked by large white arrows in part D). In a negative image of a sectioned, Ih5-labelled, 4-cell embryo (C) (Ih5 labelling is black), it is clear that the cytokeratin system is entirely cortical. Bar in part B marks 10/jm for all parts.
	Fig. 5. Animal-vegetal polarity in cytokeratin organization in early embryos. By the 4- to 8-cell stage, the cytokeratin system of the animal region (A) consists of scattered filament fragments; the vegetal cytokeratin network (B) (shown at the same magnification) is dramatically different. In some batches of embryos, the gradient in cortical cytokeratin organization (C) (negative image) from the animal (a) to the vegetal (v) pole included a region in which the networktype organization of cytokeratin filaments appears to break down into cytokeratin aggregates. By stage 6/7, the cytokeratin filaments of the animal hemisphere blastomeres (D) have begun to form integrated filament systems similar to those found in the vegetal blastomeres (E). Arrow in part E marks what appears to be a desmosomal junction. Bar in part D marks lOjnn for parts A,B,D,E; bar in part C marks 10/im.
	Fig. 6. Appearance of cytokeratin polarity in prick-activated eggs. Unfertilized eggs were activated by pricking with a clean glass needle. Cytokeratin labelling appeared first as a pattern of spots (A, 30min postactivation) which later was transformed in the vegetal hemisphere into an anastomosing filament bundle system (B,C, 60min postactivation). The animal-vegetal gradient in cytokeratin organization is clearly visible in parts B and C. By 120 min postactivation (D,E), the cytokeratin system'appeared to be mature and similar to that seen in fertilized eggs. In part D, the cortex has ripped and folded over itself, revealing that the bulk of the cytokeratin system is located in the cortex (white arrows mark limit of folded over region). By 180 min after activation (F), the cytokeratin system appeared to be breaking down into a more amorphous type of organization. Bar in part A marks 100nm for parts A,C,E,F; bar in part D marks 100^m for parts B,D.
	Fig. 7. Schematic of changing cytokeratin organization in oocytes, eggs and embryos. The changing organization from mature oocytes (left) through to the 2-cell embryo (right) is diagrammed here. Cytokeratin organization, as visualized by Ih5-labelling, is shown in red. Various benchmarks in the period from fertilization (0) to first cleavage (100) have been indicated on the time line at the bottom of the figure. In this cartoon, a continuous gradient between animal and vegetal cytokeratin systems in the 2-cell embryo has been depicted. A, anterior; V, ventral; GV, germinal vesicle; GVBD, germinal vesicle breakdown; 3O'pf, 30min postfertilization; 60'pf, 60min postfertilization.