Embryonic handedness choice in C. elegans involves the G protein GPA-16

DEV00839 Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Determination of the gpa-16(it143) temperature-sensitive period in temperature-shift experiments. Percent of embryonic viability (light blue) or dextral handedness of surviving animals (dark blue) is shown on the y-axis; stages in oogenesis and early embryogenesis are shown along the x-axis. Percentages on the left were determined in shift-down experiments; those on the right in shift-up experiments (see Materials and methods for details of procedure). The TSP for viability begins during oogenesis and is over by the eight-cell stage, while the TSP for handedness reversal is more restricted to the period of the first two AB cleavages, beginning at the two-cell stage and finishing by the six-cell stage.

• Supplemental Figure 2 -

  Fig. S2. gpa-16(pk481) homozygotes carry wild-type gpa-16 sequence. Arrows on the left show the positions of the ~900 bp wild-type product obtained with primers 1F and 6R (Fig. 4) and the ~700-bp deletion product obtained with 1F and 5R in PCR assays of single worms that were presumed homozygous or heterozygous for pk481 or were wild type (N2). Although these assays were not designed to assess quantitative differences, the apparent ratio of wild-type to deletion product in presumed homozygotes was consistently lower than the ratio seen in presumed heterozygotes, as is evident in the figure. Neither band was seen in a control to which no DNA was added (not shown). Primer sequences are available on request.

• Movie 1 -

  Movie 1. Spindle skewing during ABa and ABp cleavage. A four-cell embryo of genotype ruIs32[pAZ132: pie-1/histoneH2B::GFP] III ;  ojIs1[b-tub::GFP] (Praitis et al., 2001; Strome et al., 2001) (provided by C. Malone) was used to allow viewing of spindle and chromosome movements during cleavage. It was mounted ventral side upwards so that both ABa and ABp spindles were simultaneously in focus and was imaged during cleavage of these cells at 4 second intervals to detect both transmitted light and fluorescence, using a multi-photon microscope at the University of Wisconsin (Madison) (Wokosin et al., 2003), with help from C. Malone.

  References

  Praitis, V., Casey, E., Collar, D. and Austin, J. (2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217-1226.

  Strome, S., Powers, J., Dunn, M., Reese, K., Malone, C. J., White, J., Seydoux, G. and Saxton, W. (2001). Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos. Mol. Biol. Cell 12, 1751-1764.

  Wokosin, D. L., Squirrell, J. M., Eliceiri, K. W. and White, J. G. (2003). An optical workstation with concurrent, independent  multiphoton imaging and experimental laser microbeam capabilities. Rev Sci Instrum 74.

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