Complementation tests with ku234

From a cross between unc-62(e644)/+ males and ku234 dpy-11 hermaphrodites, three out of eight non-Dpy hermaphrodite progeny exhibited the egg-laying-defective (Egl) phenotype characteristic of ku234. From three separate crosses of individual dpy-18/+; unc-62(s472) unc-46/+ males with ku234 dpy-11 hermaphrodites, 31/81 non-Dpy hermaphrodite progeny were sickly, Unc and Egl. In contrast to these results showing noncomplementation, crosses between ku234/+ males and ct344 dpy-11 or t2012 dpy-11 hermaphrodites yielded no abnormal progeny. Half of a set of normal non-Dpy progeny from each cross produced ku234 Egl F2 progeny (n=20), demonstrating that the ku234 +/ct344 dpy-11 and ku234 +/t2012 dpy-11 heterozygotes are viable and phenotypically wild type, and that ku234 complements both these alleles. In tests with e917, all the F₁ progeny from a cross between e917/+ males and ku234 hermaphrodites were phenotypically wild-type, and five of ten F₁ progeny tested produced some F₃ progeny with the typical e917 Egl phenotype, indicating a viable, non-Egl phenotype for ku234/e917 and thus complementation.

Additional characterization of the LGV inversion e917

Near the left break point, the inverted segment includes a duplication of 94 bp from a region about 700 bp to the left of the break point, and additional complexities in the inverted region are not ruled out. Thus, e917, which was originally isolated after ³²P-decay mutagenesis (Babu, 1974), is a complex rearrangement (see Fig. S1). Preliminary tests for recombination suppression (unpublished) suggest that e917 could be a useful balancer for genes on LGV between -5 and +5 map units on the genetic map.

Fig. S1. Structure of the e917 rearrangement on LGV. The – strand of the wild-type chromosome is highlighted: dark gray for the left arm and light gray for the right arm. Vertical arrows indicate where breaks apparently occurred. Note that the two strands did not break at the same base pair at the ends of the inverted segment. The 94 bp duplication that is inserted at the left break point and its original location are boxed. Because sequences in bold face adjacent to the right breakpoints of the duplication and the inversion are identical on the + strand of the left arm and the – strand of the right arm, their strand of origin is uncertain.

Fig. S2. Enclosure defects in unc-62(s472) embryos compared with wild type. Each panel shows the s472 embryo on the left and the wild-type embryo, 10 minutes younger, on the right. Both embryos are oriented with anterior towards the left and ventral side up. (A,B) Ventral cleft closure at 3 hours 30 minutes after first cleavage at 25°C(A), and 6 minutes later (B). Arrowheads indicate the edges of the ventral cleft, which is only a remnant in the wild-type embryo but still open in the mutant embryo. (C) Dorsal focal plane at 5 hours 20 minutes from first cleavage, showing the elongating hypodermal cells as characteristic ridges in wild type (arrowheads) and lack of cellular elongation in the mutant. (D) Ventral focal plane at 5 hours 14 minutes after first cleavage, showing the leading cells (stars) moving towards the ventral midline. They are slower to move in the mutant embryo. (E) Ventral view at 5 hours 22 minutes as the leader cells meet in the wild-type embryo, but have not yet enclosed the mutant embryo. Several cells were subsequently extruded through the gap in the mutant embryo. (F) Morphology at 6 hours 10 minutes

Fig. S3. (A) Alignment of unc-62 splice variant 1a-7b predicted translation products from C. elegans and C. briggsae. The predicted protein-coding regions of exon 1b from both C. elegans and C. briggsae encode the same four amino acid sequence MAQR. Broken underline indicates the MEIS domain; solid underline indicates the homeodomain; ^ indicates exon boundaries between codons. Note the high degree of identity throughout the protein. (B) Spliced sequence of the unc-62 gene from C. briggsae (Genome Sequencing Center, personal communication). The sequence was determined by comparison of genomic sequences of C. briggsae and C. elegans and then confirmed by RT-PCR of C. briggsae RNA and direct sequencing. The 5¢ end of exon 1a is predicted to start just after a splice acceptor and is expected to be spliced to SL1 as is exon 1a in C. elegans. The 5¢UTR of both exons 1a and 1b is designated by normal typeface; coding region is designated by bold typeface. Exon boundaries are indicated by shading.

Fig. S4. Effects of unc-62 loss of function on nuclear localization of a CEH-20::GFP translational reporter in early and late embryos. All strains shown carried the reporter construct pHK110 (from H. Kagoshima and T. Burglin) and a rol-6dm marker construct as an integrated array. Embryos were photographed using Nomarski optics (left columns) and then with epi-illumination to visualize the GFP signal. (A-F) N2 wild type. (G-N) unc-62(ct344). (O-T) unc-62(e644). Mutant embryos exhibited a range of nuclear localization from apparently none to some, correlating with severity of the allele. In mutant embryos prior to morphogenesis, localization was always less than in N2 embryos. The mutant images shown include the extremes observed. Variable results were also obtained with unc-62(RNAi) embryos (not shown). Attempts to test the effect of unc-62(s472) on this localization failed because, interestingly, the required s472/+ starting strain carrying the reporter construct was inviable, suggesting that an increased dose of CEH-20 substantially enhances the lethality of the s472/+ genotype. Presence of the reporter also increased the lethality resulting from e644 by 27% and caused additional morphogenetic defects and severe Egl defects in ct344 mutants.

Fig. S5. Disorganization of hypodermal seam cells in nob-1(ct223) embryos stained with MH27 antibody. Late embryos corresponding in age to the threefold stage in the wild-type N2 are shown in dorsolateral view. Seam cells are indicated by arrowheads. Double-headed arrows indicate the width of the hypodermal cells along the AP axis. In nob-1 embryos, the seam cells fail to elongate properly along the AP axis and are sometimes stacked rather than aligned end-to-end as in N2. However, these embryos have enclosed and are less disorganized than the late unc-62 mutant embryos shown in Fig. 4 of the paper or pal-1 mutant embryos of the same stage (Edgar et al., 2001). Reproduced, with permission, from Van Auken (Van Auken, 1998