Morphological Characterization of small, dumpy, and long Phenotypes in Caenorhabditis elegans

Abstract

The determinant factors of an organism's size during animal development have been explored from various angles but remain partially understood. In Caenorhabditis elegans, many genes affecting cuticle structure, cell growth, and proliferation have been identified to regulate the worm's overall morphology, including body size. While various mutations in those genes directly result in changes in the morphological phenotypes, there is still a need for established, clear, and distinct standards to determine the apparent abnormality in a worm's size and shape. In this study, we measured the body length, body width, terminal bulb length, and head size of mutant worms with reported Dumpy (Dpy), Small (Sma) or Long (Lon) phenotypes by plotting and comparing their respective ratios of various parameters. These results show that the Sma phenotypes are proportionally smaller overall with mild stoutness, and Dpy phenotypes are significantly stouter and have disproportionally small head size. This study provides a standard platform for determining morphological phenotypes designating and annotating mutants that exhibit body shape variations, defining the morphological phenotype of previously unexamined mutants.

Keywords: Caenorhabditis elegans; K-means clustering; dumpy; long; small.

Fig. 1. Measurement of the body size.

Body length (BL) was measured from the nose to the tail tip, following the worm’s centerline (blue). Body width (BW) was measured at the position of the vulva, perpendicular to the line at the vulva (orange). Head size (HS) was measured as the direct distance from the nose to the terminal bulb’s posterior end (red). Pharynx length (PL) was measured by tracing the centerline from the buccal cavity to the terminal bulb’s posterior end (yellow). BL/BW and HS/PL differences (BL/BW D and HS/PL D) were calculated as percentages of BL/BW and HS/PL of wild type N2. Each phenotype of dumpy, small, and long is characterized according to the values of BL/BW D and HS/PL D determined in this study (see below and Fig. 5). Dumpy is described as ‘shorter and thicker’, small is ‘shorter’, and long, ‘longer and thicker’.

Fig. 2. The body length and body width of the N2 wild type and mutants with reported Dpy, Sma, or Lon phenotypes.

All measurements were taken at the 1-day adult stage. (A) Nomarski differential interference contrast (DIC) images of the dpy-4(e1166), sma-4(e729), and lon-1(e185) mutants and the wild type (N2). (B) The average body length of N2 compared to that in the mutants with reported Dpy, Sma, and Lon phenotypes. (C) The average body width of N2 compared to that in the mutants with reported Dpy, Sma, or Lon phenotypes. The number of animals measured was 36 for N2, or 21 to 37 for each mutant type. n.s., no significance. *P < 0.05, **P < 0.01 (ANOVA with Bonferroni test).

Fig. 3. The pharynx length and head size of N2 wild type and mutants with reported Dpy, Sma, or Lon phenotypes.

All measurements were taken at the 1-day adult stage. (A) Nomarski differential interference contrast (DIC) images of the pharynx and head region of the dpy-4(e1166), sma-4(e729), and lon-1(e185) mutants and the wild type (N2). (B) The average pharynx length of N2 compared to that in the mutants with reported Dpy, Sma, or Lon phenotypes. (C) The average head size of the wild type compared to that in the mutants with reported Dpy, Sma, or Lon phenotypes. The number of animals measured was 36 for N2, or 21 to 37 for each mutant type. n.s., no significance. **P < 0.01 (ANOVA with Bonferroni test).

Fig. 4. Body sizes of individual mutants with reported dpy, sma, and lon phenotypes.

All measurements were taken 24 h after L4 pick. #, mutants reported as having both dpy and sma phenotypes. (A) Body length. (B) Body width. (C) Pharynx length. (D) Head size. The number of animals measured was 36 for N2, or 21 to 37 for each mutant type. *P < 0.05 compared to the wild-type (Student t-test).

Fig. 5. The illustration of the mutant phenotypes relative to the wild-type phenotype.

The differences (%) in the ratio of body length to body width (BL/BW D, x-axis) and the ratio of head size to pharynx length (HS/PL D, y-axis) were plotted relative to the wild type located at (0,0). K-clustering generates four clusters of dumpy (dpy; orange), small (sma; blue), long (lon; magenta), and wild type (WT; black), and mild phenotype (shaded in semi-circles) is also indicated. Dumpy is characterized in case BL/BW D is less than –25% and HS/PL D is less than –2%, small is in case BL/BW D is less than –4% and HS/PL D is more than –2%, and long, BL/BW D is more than 9% and HS/PL D is more than 0% (also see Fig. 1).