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First published online 16 September 2003
doi: 10.1242/dev.00735

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C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation

Jeremy Nance1, Edwin M. Munro2 and James R. Priess1,*,{dagger}

1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
2 Center for Cell Dynamics and Friday Harbor Labs, Friday Harbor, WA 98250, USA



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  		Fig. 1. Localization and degradation of PAR proteins. (A) Schematic diagram of early stages of embryogenesis; sister cells are linked by short bars. Somatic precursors are indicated by bold outlines, germline precursors are indicated by a yellow asterisk. Germline precursors divide asymmetrically into a somatic precursor and a new germline precursor; the germline daughter has a high level of germline proteins, such as PIE-1, (dark red cells) and the somatic daughter contains a low level of PIE-1 (pink cells) that is degraded within one or two additional cell cycles (white cells). (B) PAR expression in early embryos. Embryos are oriented as in panel A; yellow asterisks mark germline precursors. (a,b) 1-cell embryos expressing either PAR-6GFP (a) or PAR-6ZF1-GFP (b); arrows point to the anterior cortex. (c,d) 4-cell embryos stained for endogenous PAR-3 (c) or PAR-3ZF1-GFP (d); large arrow in c points to the apical cortex of ABp. (d) Large arrow in d indicates the apical cortex of ABp, arrowhead indicates the apical cortex of EMS, and small arrow points to the cortex of the germline precursor. (e,f) 8-cell embryos expressing either PAR-6GFP (e) or PAR-6ZF1-GFP (f); note that PAR-6ZF1-GFP has disappeared from the oldest somatic cells (arrow) but is still detectable in the younger somatic cells (arrowheads). (g,h) 24-cell embryos showing PAR-3 (g) and PAR-3ZF1-GFP (h). (i,j) PAR-3 expression in epithelia of an organogenesis-stage wild-type embryo (i) and a par-3(ZF1) embryo (j); arrowheads point to the apical surfaces of cells forming the digestive tract. (k) Chimeric embryo formed by combining a wild-type embryo with a par-3 mutant embryo; arrowheads indicate former apical surfaces of the wild-type cells that now contact the par-3 mutant cells (p). (l) PAR-3 expression in the chimeric embryo in k; note localization of PAR-3 to the contact-free surface of the wild-type cell (arrows). In all panels, exposures were adjusted to visualize the background fluorescence of cells; the level of fluorescence in the ABa and ABp cells in d was similar to that in par-3 mutant embryos lacking the transgene. Transgene expression was identical in wild-type and par-3 mutant backgrounds; embryos shown are wild-type in a-c,e-g,i and par-3 in d,h,j. Embryos in this and subsequent figures are ~50 µm in length. Scale bar: 10 µm.

 


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  		Fig. 2. Anterior-posterior asymmetry. (A-D) Wild-type, 2-cell embryo in the light microscope (A) after staining for P granules (B, green), and PIE-1 (C, red). DNA is shown in blue. (D) A 24-cell, wild-type embryo with an end-1::gfp transgene; the GFP fluorescence image (green) was superimposed on the light microscope image. The middle and bottom rows show par-mutant embryos (middle) and par(ZF1) embryos (bottom) prepared as above. Embryo genotypes are as follows: par-3(it71) (E,F,H); par-6(zu222) (G); par-3(ZF1) (I,J,L); par-6(ZF1) (K).

 


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  		Fig. 3. Localization of PAR-2 and HMR-1/E-cadherin. (A-D) Each row shows a single 8-cell embryo, either wild-type or par-3(ZF1), immunostained for both PAR-3 (A,C) and PAR-2 (B,D). (B',D') higher magnifications of the boxed regions in B,D, respectively. The arrow in C points to PAR-3 that remains in a young somatic cell. Note presence of apical PAR-2 in the par-3(ZF1) embryo (arrow in D'). (E) Two cells in an 8-cell par-3(ZF1) embryo immunostained for PAR-3 (red) and HMR-1 (green). PAR-3 is present at the apical cortex (arrow) of one cell only. Note that HMR-1 is present at basolateral surfaces but is not detected at apical surfaces (arrowhead). DNA is stained blue in all panels. Yellow asterisks mark germline precursors. Scale bar: 5 µm.

 


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  		Fig. 4. Localization of apical PAR proteins. Each row depicts a single embryo immunostained for the protein at the bottom right of each panel. Embryo genotypes are shown above each row. Where visible, the germline precursor is indicated with a yellow asterisk and the youngest somatic precursors are indicated by arrows. Embryos are shown at representative stages; similar results were obtained for other stages. (A-B) An 8-cell embryo. (C-D) A 12-cell embryo. (E-F) A 14-cell embryo. (G-H) A 26-cell embryo. At this stage, PAR-3ZF1-GFP is present only in the three youngest somatic cells (arrows), whereas apical PAR-3 is present in several older somatic cells (arrowheads in H).

 


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  		Fig. 5. Lateral cell adhesion. (A,B) Surface (A) and internal (B) images of a wild-type embryo viewed by light microscopy. (C,D) Surface (C) and internal (D) views of a par-6(ZF1) embryo. (E) Transmission electron micrograph of a par-3(ZF1) embryo. The arrow indicates abnormal intercellular separations. Opposing arrowheads in E indicate contacts between lateral membranes. n, nucleus. Scale bars: 5 µm in D; 1 µm in E.

 


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  		Fig. 6. Ingression of the endodermal precursors. (A) Images from video recordings of the ingression of the E daughters, the endodermal precursors. The focal plane is through the center of the embryo. (a) 24-cell stage. The E daughters (nuclei indicated by single asterisks) are present on the surface of the embryo. (b) 28-cell stage. The E daughters have moved toward the interior of the embryo (top). (c) 46-cell stage. The E daughters have entered the body cavity and divided into the E granddaughters (double asterisks indicate the two visible granddaughters). Neighboring cells (triangle) cover the site of ingression. (d-f) Comparable stages of par-3(ZF1) embryos, labeled as above. Note that the E granddaughters remain on the surface of the embryo in f. (B) Confocal images of NMY-2GFP in living embryos during the early stages of ingression, labels as above. The arrowheads in b,e indicate levels of NMY-2GFP in the midbodies of dividing cells; arrows in c and f show the apical surfaces of the E daughters. Time points in minutes are indicated in the lower right-hand corner of each panel; t=0 was the 26-cell stage just after the MS(2) to MS(4) division. Scale bars: 5 µm.

 

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© The Company of Biologists Ltd 2003