Figure 1
The twisted pharynx phenotype in mnm-4;etIs2 worms of different stages, and in adult mnm-4;unc-61. (A) An example of a twisted pharynx and the three measurements that can be obtained by DIC microscopy to estimate the actual degree of twist within the isthmus using the formula shown on the right side (D is diameter; L is isthmus length; and θ is the angle between the torsion lines and the pharyngeal axis). (B) Analysis of etIs2 [pRF4 pRIC-19::GFP] transgenic worms [12]: DIC images (left column), M2 neurons of the same worms visualized via their GFP expression (middle column), and the average degree of twist within the isthmus for at least three similar worms scored using confocal microscopy (pie charts). Genotypes and stages are as indicated in the DIC images. See Table 1 for actual numerical data and list of alleles. For the confocal microscopy analysis, worms were mounted on dried agarose pads (2% in dH2O), paralyzed with a small drop of 100 mM levamisole and covered with a coverslip. The worms were examined using a Zeiss LSM 510 META system connected to an inverted Zeiss Axiovert 200 microscope. The z-stacks were projected in 360° using 32 or 64 steps and then exported as full resolution images in avi or mov format using the in-microscope software LSM 510 ConfoCor2 Combination, version 3.2. These movies were then used to determine the degree of twisting in the isthmus using video editing software (Sorenson squeeze, trial version). "180° twist" means that the distal ends of the M2 neurons would have to be rotated by 180° in order to be parallel with the cell bodies from which they originate.