Figure 2.

Right-ward bias in eye development is linked to the body left-right axis. (a) (i) Brightfield images of tadpoles at stage 45 showing normal positioning of the organs (situs solitus); rightward looping heart (red arrow), leftward coiling of gut (yellow arrow), and right side placement of gallbladder (green arrow) along the left-right axis in untreated controls. (ii)-(iii) Brightfield images of tadpoles at stage 45 after incubation in pH 4.00 0.1XMMR. (ii) Showing heterotaxic positioning of organs; rightward looping heart (red arrow) rightward coiling of gut (yellow arrow), and left side placement of gallbladder (green arrow). (iii) Showing inverse positioning of the organs (situs inversus); leftward looping heart (red arrow), rightward coiling of gut (yellow arrow), and left side placement of gallbladder (green arrow) along the left-right axis in tadpoles. (b) Bar graph showing percentage of embryos with heterotaxia upon incubation in 0.1XMMR (pH 4) (n = 481) in comparison to untreated controls (n = 419). The controls and treated groups were analyzed using Chi-squared test. (c) Categorical data analysis using a ternary plot shows that treatment with pH = 4 0.1XMMR that induced left-right body axis randomization also resulted in randomization and loss of the rightward bias of the polarization signal (observed via CC2-DMPE staining) involved in Xenopus eye development. In control embryos the polarization bias was 51% right first, 23% left first, and 26% simultaneous. The pH = 4 0.1XMMR-incubated embryos showed randomization of polarization signal 31% right first, 39% left first, and 30% simultaneous. The circles in the plot represent 95% confidence intervals. Using the calculations provided by a ternary plot algorithm (https://webscript.princeton.edu/~rburdine/stat/three_categories), the results are statistically significant (P < 0.05) when there is no overlap of the confidence intervals. (d) Bar graph showing right-ward bias in malformed eye upon perturbation of polarization signal. Embryos were injected with GlyR in the dorsal two cells (eye precursor cells) at the 4-cell stage and treated with IVM to induce depolarization in injected cells. Percentages of phenotypic embryos with a single malformed eye are depicted (n = 763). Data was analyzed using a Chi-square test comparing the right and left groups. (e) Bar graph showing no left-right bias in malformed eye upon perturbation of Pax6. Embryos were injected with DNPax6 in the dorsal two cells (eye precursor cells) at 4-cell stage. Percentages of phenotypic embryos with a single malformed eye are depicted. Data was analyzed using a Chi-squared test (n = 294).