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doi: 10.1242/10.1242/dev.00561

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lin-35/Rb and ubc-18, an E2 ubiquitin-conjugating enzyme, function redundantly to control pharyngeal morphogenesis in C. elegans

¹ Department of Molecular Biology, University of Wyoming, PO Box 3944, Laramie, WY 82071-3944, USA
² Howard Hughes Medical Institute and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA

View larger version (118K): [in a new window]   Fig. 1. Synergism between mutations in lin-35 and ubc-18. DIC (A) and corresponding GFP fluorescence (B) images. The large adult with GFP fluorescence is of genotype lin-35; ubc-18; kuEx119. The white arrows indicate the position of an arrested lin-35; ubc-18 L1 larva that failed to inherit the array; the white arrowheads indicate a sterile lin-35; ubc-18 young adult. Inset in A shows an arrested lin-35; ubc-18; KuEx119 larval-stage animal treated with lin-35(RNAi) (same animal is shown in the inset in B). (C,D) L1 animals of genotypes lin-35; ubc-18 and wild type, respectively. White and black arrowheads indicate anterior and posterior pharyngeal boundaries, respectively. Note the failure of the mutant pharynx to extend to the anterior end of the animal. (Inset in D) myo-2::GFP fluorescence in the wild-type background, highlighting the bi-lobed shape of the pharynx. Scale bars: in A, 100 µm for A,B; in C, 10 µm for C; in D, 10 µm for D.

View larger version (41K): [in a new window]   Fig. 2. Alignment of UBC-18 with UBCH7 and Ubc5. Clustal analysis was used to align C. elegans UBC-18 with UBCH7 from humans and Ubc5 from S. cerevisiae. Black boxes indicate identity; gray boxes indicate similarity. The location of the mutation in ku354 mutant animals is indicated by an asterisk.

View larger version (94K): [in a new window]   Fig. 3. pha-4::GFP expression in wild-type and mutant animals. DIC (A,C,E,G) and corresponding pha-4::GFP fluorescence (B,D,F,H) images. (A-D) 1.5-fold-stage wild-type (A,B) and ubc-18; lin-35(RNAi) mutant (C,D) embryos. Anterior is towards the left and ventral is downwards. Arrows indicate the dorsoventral pharyngeal boundaries. Note gross differences in pharyngeal shape and borders in double mutants versus wild-type embryos. In addition, note the overall similarity in the numbers of GFP-expressing cells in wild-type and double-mutant embryos. (The mutant and wild-type embryos contained 102 and 101 GFP+ nuclei, respectively.) The white bracket (D) demarcates the presence of several GFP-expressing arcade cells that failed to integrate with the rest of the pharynx. Intestinal cell nuclei, which are large, round and reside to the posterior, can readily be distinguished from pharyngeal nuclei at this stage. (E-H) pha-4::GFP expression in wild-type (E,F) and ubc-18; lin-35(RNAi) mutant L1 larvae (G,H). White and black arrowheads indicate anterior and posterior pharyngeal boundaries, respectively. As above, note the overall similarity in the number of GFP-expressing cells in contrast to the gross differences in pharyngeal shape between wild-type and double-mutant animals. The bracket (H) indicates the non-integrated arcade cells. Scale bars: in A, 10 µm for A-D; in E, 10 µm for E-H.

View larger version (115K): [in a new window]   Fig. 5. Pharyngeal morphogenesis in wild-type and mutant animals. DIC (A,C,E,G,I,K) and corresponding GFP fluorescence (B,D,F,H,J,L) images showing the outline of pharyngeal (and intestinal) cells in wild-type (A-D) and ubc-18; lin-35(RNAi) mutant (E-L) embryos. Anterior is towards the left and ventral is downwards. Black arrows demarcate the anterior pharyngeal boundary. (M) A schematic for pharyngeal extension involving three discrete steps (also see text). (A,B) An early comma-stage embryo, before overt signs of morphogenesis have begun. Note the teardrop shape of the leading-edge epithelial cells (white arrowheads, B). (C,D) 1.5-fold-stage embryo with pharynx now extended to the anterior. Note the transformation in the shape of the leading-edge epithelial cells (white arrows, D). (E,F) 1.5-fold and (G,H) 2-fold-stage mutant embryos where leading edge epithelial cells have failed to reorient (white arrowheads in F,H). (I-L) 1.5-fold-stage mutant embryos in which dorsal epithelial cells have failed to reorient their axes (white arrowheads) whereas ventral cells in the same embryo appear to have undergone the proper transformation (white arrows). (M) Pharyngeal extension with three distinct steps (Portereiko and Mango, 2001) (see text). Scale bars: in A, 10 µm for A,C,E,G,I,K; in B, 10 µm for B,D,F,H,J,L.

View larger version (125K): [in a new window]   Fig. 4. Expression of differentiation markers in mutant animals. DIC (A,C,E) and corresponding GFP fluorescence (B,D,F) images of ubc-18; lin-35(RNAi) mutant animals. White and black arrowheads indicate anterior and posterior pharyngeal boundaries, respectively. (A,B) Expression of the differentiated muscle cell marker, myo-2::GFP. (C,D) Expression of the adherens junction component and marker for epithelial cell differentiation, ajm-1::GFP. Bracket indicates the region of ajm-1 expression in the non-extended pharynx. (E,F) Neuronal marker (unc-119::GFP) expression in a pharynx that underwent partial extension. White arrows (F) indicate the positions of several GFP+ nuclei that lie within the pharyngeal borders. Scale bar: 10 µm.