Pai VP et al. (2012), Neurally Derived Tissues in Xenopus laevis Embr...

XB-ART-46589

Stem Cells Int 2012 Jan 01;2012:353491. doi: 10.1155/2012/353491.

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

Pai VP , Vandenberg LN , Blackiston D , Levin M .

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Consistent left-right asymmetry in organ morphogenesis is a fascinating aspect of bilaterian development. Although embryonic patterning of asymmetric viscera, heart, and brain is beginning to be understood, less is known about possible subtle asymmetries present in anatomically identical paired structures. We investigated two important developmental events: physiological controls of eye development and specification of neural crest derivatives, in Xenopus laevis embryos. We found that the striking hyperpolarization of transmembrane potential (V(mem)) demarcating eye induction usually occurs in the right eye field first. This asymmetry is randomized by perturbing visceral left-right patterning, suggesting that eye asymmetry is linked to mechanisms establishing primary laterality. Bilateral misexpression of a depolarizing channel mRNA affects primarily the right eye, revealing an additional functional asymmetry in the control of eye patterning by V(mem). The ATP-sensitive K(+) channel subunit transcript, SUR1, is asymmetrically expressed in the eye primordia, thus being a good candidate for the observed physiological asymmetries. Such subtle asymmetries are not only seen in the eye: consistent asymmetry was also observed in the migration of differentiated melanocytes on the left and right sides. These data suggest that even anatomically symmetrical structures may possess subtle but consistent laterality and interact with other developmental left-right patterning pathways.

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Species referenced: Xenopus laevis
Genes referenced: abcc8 abcc9 lefty1 nodal nodal1 pax6 pitx2 ranbp2 shh

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	Figure 1. Rightward bias in induction of polarization signal regulating eye development. (a) Incubation in voltage-sensitive CC2-DMPE dye of multiple Xenopus embryos tracked from stage 18 to stage 20 shows the representative temporal progression of hyperpolarization signal (red arrowheads) during development (ii)â€“(v). White arrowheads indicate the lack of a coherent (contiguous) spot of signal. (vi) Color bar representing the scale of relative depolarization and hyperpolarization as seen with the CC2-DMPE dye. (b) Bar graph comparing a group of Xenopus embryos (n = 44) that were tracked individually and analyzed for the first detectable hyperpolarization signal using CC2-DMPE; a significant bias is observed favoring the right side. A pairwise comparison and Chi-squared test analysis were done between the groups. (c) In situ hybridization analysis of Sonic Hedgehog (Shh) signal in stage 18 Xenopus embryos either untreated (control) ((i) white arrowhead) or treated with 1.5â€‰Î¼M retinoic acid receptor inhibitor Ro-41-5253 ((ii) blue arrowhead) from midgastrula stage. Ro-41-5253 treatment significantly enhances the Shh expression signal in 92% of treated embryos (n = 29). (d) Categorical data analysis using a ternary plot shows that the treatment with Ro-41-5253 (1.5â€‰Î¼M) that inhibits retinoic acid receptor signal resulted in no significant change in the rightward bias of polarization signal (as observed via CC2-DMPE staining) involved in Xenopus eye development. In control (untreated) embryos the polarization was 51.5% right first, 26.5% left first, and 22% simultaneous (n = 72). In the Ro-41-5253-treated embryos the polarization was 48% right first, 25% left first, and 27% simultaneous (n = 126). 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 only when there is no overlap of the confidence intervals.
	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).
	Figure 3. KATP channels are expressed in the putative eye regions and in the eye tissue in a left-right asymmetric manner. (a) Xenopus embryos at stage 18 (i)â€“(iv) and stage 30 (v)â€“(viii) were analyzed by in situ hybridization for KATP channel subunits showing their presence in the putative eye region ((i)â€“(iv) blue arrowheads) as well as in differentiated eye tissue ((v)â€“(viii) blue arrowheads). All four subunits (Kir6.1, Kir6.2, SUR1, and SUR2) were found to be present in the putative and developed eye tissue. In addition to the eye tissue, the KATP channel subunits were also present in the general head region and the dorsal region of the trunk. Illustration shows a stage 18 embryo with the dorsal-ventral and the left-right axes. (b) Transverse JB4 sections of in situ hybridized Xenopus embryos at stage 30 (i)â€“(iv) showing left-right distribution of the KATP channel subunits. Illustration shows the plane of sectioning of the stage 30 embryo. Kir6.1 expression is found in the inner retinal part of the eye vesicle ((i) blue arrowheads) and in the brain especially in the cells lining the ventricle and in some cells surrounding the eye tissue. Kir6.2, SUR1, and SUR2 expression is found in the inner layer of the 2-layer epidermis with intense staining at the lens placode ((ii)â€“(iv) blue arrowheads). Kir6.1, Kir6.2, and SUR2 expression is symmetric ((i), (ii), and (iv) blue arrowheads). SUR1 shows asymmetric distribution ((iii) red and blue arrowheads) where red arrowheads indicate the side with lessened expression. Scale bars = 100â€‰Î¼m. Schematic shows a transverse section of a stage 30 embryo with the dorsal-ventral and the left-right axes indicated. (c) Sense probes showed no signal detected at neurula (i) or tailbud (ii) stages.
	Figure 4. Biased migration of melanocytes along the left-right axis. (a) Xenopus embryos at stage 30 analyzed by in situ hybridization for melanocyte marker trp2 show a higher number of melanocytes on the left side (i) as compared to the right side (ii) of the embryo. Red arrowheads indicate the melanocytes being counted. (b) Quantification of a number of stage 30 embryos showing biased trp2 spots indicates that 78.6% of embryos show a leftward bias of trp2 staining. 21.4% of embryos showed higher trp2 staining on the right side. The data were analyzed using a two-tailed Binomial calculation; n = 28. (See Table 1 for details). (c) Quantification of Xenopus embryos at stage 26 analyzed by in situ hybridization for trp2 shows no asymmetry in melanocyte number prior to their migration. Due to dense staining at this early stage individual stained cells could not be resolved and counted, hence the stained region was marked, and the area was quantified. Quantification of the trp2 stain on the left and right sides of embryos (n = 10) shows no significant difference in the staining. The areas of the signal on right and left sides were compared using a t-test. (d) Transverse agarose sections of in situ hybridized Xenopus embryo at stage 26 showing left-right distribution of the melanocyte marker trp2 as measured. Illustration shows the plane of sectioning of the stage 26 embryo. Symmetric trp2 expression (red arrowheads) is found in the area around the neural tube. Scale bar = 200âÎ¼.
	Figure 5. A model of physiological asymmetries within the overall scheme of the left-right patterning pathway. In Xenopus, bilateral symmetry is first broken, and the left-right axis is consistently oriented with respect to the dorsoventral and anterior-posterior axes, during early cleavage stages [8, 81]. The intracellular chirality is amplified onto multicellular cell fields during cleavage and blastula stages by the voltage-dependent movement of small molecule determinants through gap junctions [82â84]. The transduction of voltage gradient differences by epigenetic mechanisms [85] and other biophysical events such as ciliary movement [86] initiates at least two asymmetric transcriptional cascades. The first is the well-known Nodal, Lefty, and Pitx2 cassette that drives asymmetric organogenesis of the visceral organs. The other is the asymmetry of KATP channel subunits expressed in neural tissues, which results in asymmetric gradients of resting potential that directs development of the eye [87]. Future work will determine the functional linkage of the reported asymmetry in neural crest cell movement.

References [+] :

Adams, Measuring resting membrane potential using the fluorescent voltage reporters DiBAC4(3) and CC2-DMPE. 2012, Pubmed, Xenbase