Neurally Derived Tissues in Xenopus laevis Embryos Exhibit a Consistent Bioelectrical Left-Right Asymmetry
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) () in comparison to untreated controls (). 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 () 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 (). 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 ().