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First published online 21 February 2007
doi: 10.1242/dev.02833

Development 134, 1259-1268 (2007)
Published by The Company of Biologists 2007
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PAR-6 is required for junction formation but not apicobasal polarization in C. elegans embryonic epithelial cells

Ronald Totong*, Annita Achilleos* and Jeremy Nance{dagger}

Skirball Institute of Biomolecular Medicine and NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA.


Figure 1
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  		Fig. 1. PAR-6 localization in epithelial cells. In this and all subsequent figures, embryos (~50 µm in length) or embryo insets are oriented anterior to the left. Nuclei are stained with DAPI (blue) and show cell-type differences in staining intensity. (A,B) Lateral views of embryos when epithelial cells are polarizing (A, bean stage) or after most epithelial cells have completed polarization (B, 1.5 fold stage). PAR-6 localizes to apical surfaces of cells in each of the major epithelia (labeled); staining in epidermal epithelial cells is weaker and more difficult to detect in older embryos (compare A and B). {alpha}-PAR-6 antibodies also recognize germline P granules (P). (A',B') Boxed regions magnified in insets below also show PAR-3 staining; inset contrast has been adjusted to indicate nuclei. (C-H) Dorsoventral views of polarizing intestinal cells showing PAR-6GFP and junction proteins. The intestinal midline is indicated with opposing arrowheads. (C-E) PAR-6GFP and HMP-1/{alpha}-catenin at initial stages of intestinal cell polarization (before apical migration of intestinal nuclei). (F-H) PAR-6GFP and DLG-1 in polarizing intestinal cells (after apical migration of intestinal nuclei). Scale bars: 2.5 µm.

 

Figure 2
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  		Fig. 2. PAR-6ZF1GFP localization and degradation. Panels show par-6(zu170) embryos expressing maternal PAR-6ZF1GFP stained with {alpha}-PAR-6 antibodies (green) and/or {alpha}-GFP antibodies (red). {alpha}-PAR-6 antibodies also react non-specifically with P granules (P). (A,B) One-cell embryo co-stained for PAR-6 and GFP; PAR-6ZF1GFP is at the anterior cortex (arrow). (C) A 26-cell embryo; PAR-6ZF1GFP has degraded from most cells except the germ-line precursor cell (asterisk) and the youngest somatic cells (arrows). (D) ~100-cell embryo; PAR-6ZF1GFP is detectable only in the two germ cells (asterisks). (E,F) 1.25 fold stage embryos; PAR-6 (arrow in E) but not PAR-6ZF1GFP (F) is detectable in epithelial cells. Scale bar: 2.5 µm.

 

Figure 3
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  		Fig. 3. The par-6(tm1425) deletion and PAR protein localization in par-6(M/Z) embryos. (A) The par-6 gene (top) and its predicted full-length product (bottom). Exons (rectangles), introns (lines), and the region deleted in tm1425 are indicated. The PB1, CRIB (C) and PDZ domains are shown in the predicted protein, and sequences encoding these domains are colored in par-6 exons. (B,C) PAR-6, PKC-3, and PAR-3 localization in wild-type (B) and par-6(M/Z) (C) embryos. (B',C') Magnifications of the boxed regions in B,C. PKC-3 is uniform in par-6(M/Z) embryos. Nonspecific staining of P granules by PAR-6 antibodies is indicated (P). Scale bars: 2.5 µm.

 

Figure 4
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  		Fig. 4. Cell adhesion in par-6(M/Z) embryos. Nomarski micrographs of wild-type and par-6(M/Z) embryos. The wild-type embryo (A) has elongated threefold; the par-6(M/Z) embryos (B,C) have arrested at the 1.5 fold stage and the embryo in C has developed epidermal lesions (arrowheads). Scale bar: 2.5 µm.

 

Figure 5
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  		Fig. 5. Epithelial polarity in par-6(M/Z) embryos. (A-B') Microtubules in wild-type (A,A') and par-6(M/Z) (B,B') embryos concentrate at apical regions of intestinal epithelial cells (arrows in A',B'); embryos are co-stained for {alpha}-tubulin and PAR-6. (C-E) IFB-2 localization in intestinal cells of wild-type and par-6(M/Z) embryos. IFB-2 localizes in a continuous apical band in wild type (C). In younger par-6(M/Z) embryos (D), IFB-2 is apical but not always continuous between cells (arrow); (E) apical IFB-2 patches separate and circularize (arrow) in older par-6(M/Z) embryos. Scale bars: 2.5 µm.

 

Figure 6
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  		Fig. 6. Apical junctions in par-6(M/Z) embryos. Wild-type or par-6(M/Z) embryos immunostained as indicated. Dashed boxes are magnified in the insets below; the contrast within insets has been increased to highlight intestinal epithelial cell staining. Nonspecific staining of P granules by PAR-6 antiserum is indicated (P). (A,B) PAR-6 and HMP-1. In wild-type (A), PAR-6 is apical and HMP-1 is present in more lateral junctions. HMP-1 is apical but fragmented in par-6(M/Z) embryos (B). (C,D) HMR-1 and DLG-1. In wild type (C), HMR-1 and DLG-1 are present together in apical junctions within adjacent domains. In par-6(M/Z) embryos (D), HMR-1 and HMP-1 are apical but fragmented and do not colocalize (arrow, D'). (E,F) LET-413 and PAR-3 in epidermal cells, lateral view in bean stage embryos. In both wild-type (E) and par-6(M/Z) (F) embryos, LET-413 is found at lateral surfaces (arrows) but not apical surfaces (arrowheads); PAR-3 is present only at the apicolateral interface. Scale bars: 2.5 µm.

 

Figure 7
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  		Fig. 7. DLG-1 localization during junction formation. (A,B) Fluorescence timelapse movie stills (taken from Movies 1, 2 in the supplementary material) of wild-type (A) and par-6(M/Z) (B) epidermal cells expressing DLG-1GFP. Times are indicated in minutes:seconds, with 00:00 representing the time DLG-1GFP is first expressed in epidermal cells. DLG-1GFP forms continuous junctions in wild-type but not par-6(M/Z) embryos. (C,D) DLG-1 and PKC-3 localization in intestinal epithelial cells of wild-type (C) and par-6(M/Z) (D) embryos. Embryos are at the 1.25 fold stage, just after DLG-1 organizes into continuous junctions in wild-type embryos. DLG-1 in par-6(M/Z) embryos is always fragmented. Scale bars: 2.5 µm.

 




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