Neurally Derived Tissues in Xenopus laevis Embryos Exhibit a Consistent Bioelectrical Left-Right Asymmetry

A similar asymmetry was observed in neural crest-derived pigment cells, melanocytes. Melanocytes follow specific migration paths along the side of the Xenopus embryo after differentiation from neural crest. Interestingly, a consistent left-right asymmetry in the number of melanocytes was found upon careful analysis. Our analysis of Trp2-positive cells prior to onset of migration (stage 26) revealed that there is no apparent asymmetry in the extent of melanocyte differentiation from the neural crest cells, suggesting that the observed asymmetry is due to asymmetric migration patterns. The source of asymmetric migratory cues or mechanisms ensuring left-right asymmetrically biased response of cells to migration cues (as has been observed in mammalian cells [132, 133]) remains to be analyzed in future work. The evolutionary or developmental role of such asymmetric melanocyte distributions is not yet known; indeed, it is possible that careful quantification of cell number or other properties in other developmental events may reveal other heretofore-unrecognized consistent asymmetries in systems that are currently considered to be symmetrical.

The identification of subtle physiological and cell positioning asymmetries in Xenopus development raises a number of open questions. It is possible that consistent asymmetries in bioelectric state, cell number, and sensitivity to specific perturbations remain to be discovered in many different paired (symmetric) organs. The anatomical and genetic profiling studies carried out to date may have only scratched the surface of the developmental information embedded in various somatic tissues at the level of mRNA and protein profiles and would likely not reveal physiology differences. The identification of such asymmetries, as well as their molecular origins, and the characterization of their interactions with the major left-right axial patterning cascade are likely to reveal fascinating aspects of developmental biology with significant evolutionary implications. Moreover, a mechanistic understanding of these subtle asymmetries will provide insight into the form, function, and robustness of the nervous system and may help address the lateralized diseases of both nervous and nonnervous organs.

Xenopus laevis embryos were collected and fertilized in vitro according to standard protocols [134], in 0.1X Modified Marc's Ringers (MMR; pH 7.8). Xenopus embryos were housed at 14–18°C and staged according to Nieuwkoop and Faber [135]. All experiments were approved by the Tufts University Animal Research Committee in accordance with the Guide for Care and Use of Laboratory Animals, protocol number: M2011-70.

CC2-DMPE (molecular probes) stocks (stock: 1 mg/mL in DMSO) were diluted 1 : 1000 in 0.1X MMR for a final concentration of 0.2 μM. Stage 15–16 embryos were soaked in dye for 1.5 h. Embryos in solution were imaged using the CC2 cube set on an Olympus BX61 microscope with an ORCA digital CCD camera (Hamamatsu) with Metamorph software. See [66, 67] for additional details. For determining the bias in eye CC2-DMPE signal, embryos were imaged at regular intervals. The side of the embryo that first showed the eye CC2-DMPE signal was noted, and the embryos were kept under observation until signal was seen on both sides. Time difference between the right and left eye CC2-DMPE signals was measured by taking images at regular intervals of 10 minutes. While CC2-DMPE used alone cannot precisely quantify V mem levels, the strong fluorescence from this positively charged dye has been shown previously to reliably identify hyperpolarized regions as confirmed by electrophysiological impalement [65–67, 136] and to identify the same locations of hyperpolarized cells in the early face as did prior studies using ratiometric imaging [69].

Capped, synthetic mRNAs were generated using the Ambion mMessage mMachine kit, resuspended in water, and injected (2.7 nL per blastomere) into embryos in 3% Ficoll. Results of injections are reported as percentage of injected embryos showing eye phenotypes, sample size (n) and P values comparing treated groups to controls. For lineage tracing, 4-cell embryos were injected with mRNA encoding β-galactosidase. At stage 45, fixed embryos were stained by incubation with X-gal substrate.

In situ hybridization was performed according to standard protocols [137]. Accession numbers for the sequences used as probes were Kir6.1 NM_008428.4, Kir6.2 NM_001204411.1, SUR1 NM_011510.3, and SUR2 NM_001044720.1. Xenopus embryos were collected and fixed in MEMFA. Prior to in situ hybridization, embryos were washed in PBS + 0.1% Tween-20 and then transferred to ethanol through a 25%/50%/75% series. In situ probes were generated in vitro from linearized templates using DIG labeling mix (Invitrogen, Carlsbad, CA, USA). Chromogenic reaction times were optimized to maximize signal and minimize background. Histological sections were obtained by embedding embryos after in situ hybridization in JB4 according to manufacturer's instructions (Polysciences). Prior to sectioning, one corner of the block was physically marked to allow unambiguous orientation of section images with respect to the left-right axis (for sections in Figure 3(b)).

Trp2 in situ hybridized embryos were embedded in 4% low melting point agarose as previously described and sectioned at 100 μm [138]. The sections were imaged using a Nikon SMZ1500 microscope with a Q-imaging Retiga 2000R camera. Using NIH's ImageJ software the regions exhibiting purple signal on the left and right side of the sections were marked using the freehand selection tool, and the area of the Trp2 stain was automatically quantified. The 3rd section from the last section containing eye was selected from each of the 10 different embryos and used for quantification.

Embryos were kept in 0.1X MMR (pH 4.0–4.05) from fertilization until stage 12-13 and then moved to 0.1x MMR (pH 7.8) for the remainder of the experiment. At stage 45, the situs of the internal organs was scored by visual inspection of the heart, gut, and gallbladder as previously reported [17]. Categorical data analysis of the CC2-DMPE staining following drug treatment was done using ternary plots [75].

Embryos were kept in 0.1X MMR from fertilization. At ~ stage 11 Ro-41-5253 was added into 0.1X MMR with a final concentration of 1.5 μM. At stage 18, a portion of the embryos were fixed and used for in situ hybridization. Categorical data analysis of the CC2-DMPE staining following drug treatment was done on the remaining live embryos using ternary plots [75].

All Statistical analyses were performed using Graphpad Prism (GraphPad Software, La Jolla, CA, USA), except in case of the categorical data analyses where ternary plots algorithms were used (https://webscript.princeton.edu/~rburdine/stat/three_categories). For data pooled from various iterations, Chi-square test or two-tailed Binomial calculation was used. Nonpooled data was analyzed by t-test (for 2 groups) or ANOVA (for more than two groups).

V. P. Pai and M. Levin designed research, V. P. Pai, M. Levin, L. N. Vandenberg, and D. Blackiston performed research, and M. Levin and V. P. Pai wrote the paper.

The authors thank Punita Koustubhan, Amber Currier, Joan Lemire, and Claire Stevenson for Xenopus husbandry and general lab assistance and Daryl Davies and Miriam Fine for the GlyCl expression construct. They also especially thank Dany S. Adams and Brook Chernet for microscopy assistance and Sherry Aw for many helpful ideas and discussions on this project. This work was supported by NIH Grants EY018168, MH081842, and GM077425 to M. Levin. The authors are also grateful for the support of the G. Harold and Leila Y. Mathers Charitable Foundation.