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First published online 19 May 2004
doi: 10.1242/dev.01146

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PAR-3 is required for epithelial cell polarity in the distal spermatheca of C. elegans

Shinya Aono^1,*, Renaud Legouis², Wendy A. Hoose¹ and Kenneth J. Kemphues^1,

¹ Department of Molecular Biology and Genetics, Cornell University, 107 Biotechnology Building, Ithaca, NY 14853, USA
² CNRS-CGM, Avenue de la Terasse, 91198 Gif-sur-Yvette, France

View larger version (99K): [in a new window]   Fig. 1. Post-embryonic phenotype of par-3(RNAi) worms. Worms carrying AJM-1::GFP were collected after 48 hours of growth on bacteria producing par-3 dsRNA (B,B') or control bacteria (A,A') and stained with DAPI (A,B). Spermathecae were visualized by AJM-1::GFP (arrows in A',B'). The small condensed DNA signals represent the sperm nuclei (arrows in A,B) that are present in the spermathecae of control worms (A) but are restricted to the proximal ovary of par-3(RNAi) worms (B). Endomitotic oocyte nuclei (B) and normal nuclei (A) are indicated by arrowheads in A, B. Scale bar: 10 µm.

View larger version (88K): [in a new window]   Fig. 2. Defective ovulation in par-3(RNAi) worms. Images from time-lapse Nomarski video microscopic observations of meiotic maturation and ovulation in the wild type (left) and a first ovulation in par-3(RNAi) (right). Real time (minutes:seconds) is indicated in the top left corner, with the zero time point corresponding to the time that nuclear envelope break down (NEBD) was observed (black arrows). An oocyte (o), the oocyte nuclear envelope (ne), the sheath (sh) and the spermatheca (sp) are indicated. The oocyte enters the spermatheca in the wild type at 0:45 and is transferred to the uterus at 1:10. However, in par-3(RNAi) worms, the oocyte remains in the proximal gonad even 4:28 after the NEBD. The contraction of sheath cells and distal extension of the spermatheca was observed in both worms (arrowheads). Scale bar: 10 µm.

View larger version (107K): [in a new window]   Fig. 3. Differentiation of gonad sheath cells is not affected by par-3(RNAi). (A,B) Expression of CEH-18 protein. Gonads were dissected 48 hours after growth with (right) or without (left) par-3 RNAi treatment, and stained with anti CEH-18 antibodies. Sheath nuclei are indicated by arrowheads. The size difference in the sheath nuclei in the gonads shown was not reproducible and might have been an artifact of fixation. (C,D) Distribution of myosin heavy chain A (MHCA) in gonad sheath cells of par-3(RNAi) worms. Worms were collected after 48 hours of growth with (right) or without (left) RNAi treatment and localization of MHCA was analyzed by epifluorescence microscopy. DNA was stained by DAPI. Sheath cells are numbered and sheath nuclei are indicated by numbered arrowheads; gray arrowheads indicate out of focus nuclei. Scale bar: 10 µm.

View larger version (103K): [in a new window]   Fig. 4. Specification of spermatheca is not affected by par-3 RNAi, but distal cell morphology and tissue organization are abnormal. let-502::gfp worms were collected after 48 hours of growth with (C,D) or without (A,B) RNAi treatment, and fixed and stained with rhodamine-phalloidin. The number, shape and distribution of cells with high levels of GFP in the distal end of spermatheca was compared. (A,C) Projections of 20 adjacent optical sections. Note the more spherical shape of cells in (C) and the gap between GFP-positive cells in (D') (arrow). Such gaps are not seen in controls. (E) Summary of the results of the tissue organization analysis. Scale bar: 5 µm.

View larger version (79K): [in a new window]   Fig. 5. Apical polarity of spermatheca cells is affected by par-3(RNAi). (A-D) Mislocalization of AJM-1::GFP in spermathecae of RNAi-treated worms. Worms carrying AJM-1::GFP were collected at 39 hours after growth with (right) or without (left) RNAi treatment and localization of AJM-1::GFP and anti-LET-413 was analyzed by confocal microscopy. The distal spermatheca is to the right in all panels; the distal-most cell pairs are indicated by brackets in (C) and (D). Panels (A) and (B) show projections of 36 and 26 optical sections through the entire spermatheca, respectively. (C,D) Single optical sections selected to show the middle focal plane through the distal spermatheca. (C',D') Summary of our interpretation of the observed defects in the distal spermatheca. In control worms, AJM-1::GFP is precisely localized to the apical junctions of the distal spermathecal tube (arrowheads). However, in par-3(RNAi) worms, AJM-1::GFP is widely dispersed in puncta that extend into the lateral, and in some worms into the basal, domains. Gaps of GFP signals (arrowheads) are often observed. (E,F) Mislocalization of microfilaments in PAR-3-depleted spermatheca. Worms carrying LET-413::GFP were collected after 48 hours of growth with (right) or without (left) RNAi treatment and stained with rhodamine-phalloidin (red in top panels) after dissection. Ovary (ov) and the spermatheca (sp) are indicated. In control worms, microfilaments are concentrated beneath the apical cell membrane in cells near the distal end of the spermathecal tube (left column, arrow). However, in par-3(RNAi) worms, microfilaments are lacking in this region (right column, arrow). Note that although cell shape is abnormal, LET-413::GFP remains restricted to the basolateral membrane domain (arrowheads). Scale bar: 8 µm (A-D); 5 µm (E-F'').

View larger version (120K): [in a new window]   Fig. 6. Time course of PAR-3 and AJM-1::GFP expression. Worms carrying AJM-1::GFP were collected 27 hours (A), 30 hours (B), 33 hours (C), or 36 hours (D) after incubation. Localization of PAR-3 was examined by immunohistochemistry. Multiple sections were stacked in each panel for analysis. PAR-3 was detected in vulval precursor cells (vulva), uterine precursor cells (ut), spermathecal precursor cells (sp) and sheath cells (arrow in B'). PAR-3 levels begin to decrease in 36-hour-incubated worms (arrow in D'). Scale bar: 5 µm.

View larger version (111K): [in a new window]   Fig. 7. Effect of par-3 RNAi on expression of PAR-3, PAR-6 and PKC-3. Worms were collected after 30 hours of growth with (right) or without (left) RNAi treatment. Localization of PAR-3 (A,B), PAR-6 (C,D) and PKC-3 (E,F) was examined by immunohistochemistry. Multiple optical sections were stacked in each panel for analysis. The vulval precursor cells (vulva), uterine precursor cells (ut), and spermathecal precursor cells (sp) are indicated. The amounts of all three proteins were significantly reduced in the distal part of the spermathecal primordium (arrows) where cells had not been polarized (arrowheads), as indicated by the absence of apical enrichment of HMP-1::GFP. (L). Expression of PAR-3 in par-3(it71) homozygous worms after 30 hours of growth on OP50. Scale bar: 5 µm.