Vandenberg LN et al. (2011), Low frequency vibrations disrupt left-right pat...

XB-ART-43685

PLoS One 2011 Jan 01;68:e23306. doi: 10.1371/journal.pone.0023306.

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Low frequency vibrations disrupt left-right patterning in the Xenopus embryo.

Vandenberg LN , Pennarola BW , Levin M .

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The development of consistent left-right (LR) asymmetry across phyla is a fascinating question in biology. While many pharmacological and molecular approaches have been used to explore molecular mechanisms, it has proven difficult to exert precise temporal control over functional perturbations. Here, we took advantage of acoustical vibration to disrupt LR patterning in Xenopus embryos during tightly-circumscribed periods of development. Exposure to several low frequencies induced specific randomization of three internal organs (heterotaxia). Investigating one frequency (7 Hz), we found two discrete periods of sensitivity to vibration; during the first period, vibration affected the same LR pathway as nocodazole, while during the second period, vibration affected the integrity of the epithelial barrier; both are required for normal LR patterning. Our results indicate that low frequency vibrations disrupt two steps in the early LR pathway: the orientation of the LR axis with the other two axes, and the amplification/restriction of downstream LR signals to asymmetric organs.

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Species referenced: Xenopus
Genes referenced: nodal1 nudt6

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	Figure 1. Heterotaxia is induced by various low frequency vibrations. A) A simple experimental device was produced from a Gwinstek GFG-8216A function generator (right) attached to a 4-inch Sony speaker (left). A petri dish, with the appropriate solution containing embryos, was placed on top of the speaker at various stages. B) Ventral views (V) showing organ position and dorsal views (D) showing normal dorsoanterior development in tadpoles with wildtype situs, heterotaxia, and situs inversus. In all panels of ventral views, the red arrowhead indicates the apex of the heart, yellow indicates the stomach, and green indicates the gall bladder. For wildtype organ situs, the apex of the heart loop is located on the animal's right side, the stomach coils to the animal's left, and the gall bladder is positioned on the animal's right. The heterotaxic tadpole shown here has an inverted heart, but normal position of the stomach and gall bladder. There are seven combinations of position of the three scored organs that are each examples of heterotaxia, including situs inversus, where all three organs are inverted. The right-most panel also shows an animal with heterotaxia, with an inverted stomach and gut, but normal position of the heart. Note that the coiling of the stomach is also abnormal, a phenotype observed in a minority of vibrated embryos (<5% at 7 Hz). C) Quantification of heterotaxia in embryos vibrated at different frequencies from 1 cell through st. 19 of development. 7 Hz and 15 Hz were the most effective at randomizing the LR axis, but 15 Hz vibration also produced other developmental abnormalities in a large subset of embryos (not shown); therefore, 7 Hz was selected for additional study. Numbers on bar graphs indicate sample sizes from a combination of two or more replicate studies. * p<0.01, but not biologically relevant because heterotaxia rates were below 10%, the minimal meaningful difference. ** p<0.001, Ï‡2 test.
	Figure 4. Localization of the asymmetric gene Xnr-1 is affected by vibration during early, but not late, cleavage stages. A) Control and vibrated embryos were examined at approximately st. 22 for the localization of Xnr-1 mRNA. All embryos were classified as having correct, left-sided expression (i), or incorrect right-sided (ii), bilateral (iii) or absent (iv) expression. Green arrows indicate correct expression, red indicate incorrect expression, and white indicate absent expression. All embryos are positioned with the anterior upward and the left (L) and right (R) sides are indicated. B) Quantification of incorrect Xnr-1 localization in control and vibrated embryos. When vibrations started at 1 cell, 2 cell, 4 cell or st. 6, there was a significant increase in incorrect Xnr-1 expression compared to controls (Ï‡2 test, *p<<0.001). Vibrations starting at st. 8 were not statistically different from untreated controls.
	Figure 5. Ternary plots reveal unique signatures of inverted organs depending on the timing of vibration treatment. All heterotaxic embryos were compiled by treatment and graphed on a ternary plot where one axis represents the % of embryos with single organ inversions, the second axis represents the % of embryos with two (double) organ inversions, and the third axis represent the % of embryos with situs inversus (all three organs inverted.) Data is graphed so a single point represents each treatment, surrounded by an oval that illustrates the 95% confidence interval (CI); when the ovals do not overlap, the groups are significantly different from each other [67]. A) Unique signatures of embryos vibrated from 1 cell to st. 19 (blue), 2 cell to st. 19 (red), 4 cell to st. 19 (green) or st. 6 to st. 19 (violet) demonstrate that the period of vibration significantly affects the organ inversions observed. Most striking are the differences in the incidence of situs inversus, which is highest in embryos vibrated from 1 cell to st. 19 and lowest in embryos vibrated from st. 6 to st. 19. B) Signatures of affected organs are very similar in heterotaxic embryos from groups treated with chemicals and molecular constructs targeting H+ pumps (yellow), K+ channels (blue), gap junctional communication (GJC, green), the cytoskeleton (red), and the LR signaling molecule serotonin (5HT, purple). For a complete list of the reagents used, see Supplemental Table 2. C) The ion flux data shown in panel B was combined and statistically collapsed to produce a datapoint that represents all ion flux-related treatments (black dots, inside the violet circle). This was compared to the signatures obtained from the vibration protocols (panel A). The ion flux data thus overlaps with the signatures obtained from vibrations occurring from st. 6 through neurulation (violet).
	Figure 6. Epistasis and dye movement experiments indicate that low frequency vibrations alter nocodazole-sensitive pathways and intracellular movements. For epistasis experiments, embryos were vibrated, treated with a drug, or given a combination of these two treatments from 1 cell to st. 12, and organ laterality was assessed at st. 45. If the combination produced an additive effect, we concluded that these treatments were likely affecting different aspects of the left-right (LR) pathway; if the combination was not additive, we concluded that the treatments were likely targeting the same aspects of the LR pathway. A) Embryos were vibrated, treated with the microtubule disruptor nocodazole, or a combination of the two. No additive effects on heterotaxia rates were observed, therefore it is likely that vibrations target nocodazole-sensitive LR pathways. B) In contrast, embryos treated with lindane, a gap junction blocker, and vibration showed additive effects over each treatment alone. These results show that additive effects can be obtained, and indicate that vibrations are not likely to be targeting gap junctions. C) 1 cell embryos were injected in the central area of the animal pole with a mixture of two dyes, then vibrated or left untreated and examined 10 minutes later. In both treatments, the low molecular weight dye (10 kDa) spread throughout most of the embryo with the strongest concentration in the vegetal hemisphere. In controls, the high molecular weight dye (70 kDa) typically spread throughout the top third of the embryo, whereas the same dye remained localized much more strongly to a single point in the upper quadrant of animal pole in the vibrated embryo. Orientation of the embryos is indicated in the merged panels with a (animal) and v (vegetal). D) Sections were obtained from 1 cell embryos after 10 or 20 minutes of vibration, and time-matched unvibrated controls. The percentage of A/V sections containing TMR dye were calculated, giving a quantitative measure of how far the dye had spead laterally through the embryo. The data indicate that over time, the high molecular weight dye spread out laterally in control embryos, but in vibrated embryos, the dye spread to a lesser degree.
	Figure 7. Epithelial integrity is disrupted in vibrated embryos. A) Using a biotin permeability assay, we determined that epithelial integrity was maintained in control embryos such that the biotin signal was normally observed at the outer edges of the embryo, less than 1 cell depth (arrowheads). B) In vibrated embryos, the biotin signal was observed much farther into the embryo, with signal several cell layers deep (arrows).
	Figure 2. Vibration period influences the frequency of laterality defects.When embryos were vibrated at 7 Hz during different periods of development, striking differences in the rate of heterotaxia were observed. Most effective were vibrations that started at 1 cell and went through mid-gastrula (st. 10.5) or neurula stages (st. 19, see black arrow). Somewhat less effective were vibrations starting just a short time later, at the 2 cell stage (blue arrow). Finally, vibrations that included a second period of time, around st. 6, were able to disrupt left-right patterning, but to a lesser degree (see yellow arrow, for example). For all groups, a red line indicates significant differences from non-vibrated controls (Ï2 test, p<0.01) and heterotaxia rates above the minimal meaningful difference of 10%. A green line indicates that the treatment either was not significantly different from controls or was below the minimal meaningful difference of 10% heterotaxia. All lines are the summed data from two or more replicate studies.
	Figure 3. Vibration from 1 cell to 2 cell, paired with various later vibration periods, increases the rate of heterotaxia.One set of embryos was vibrated for a select period of time later in development (white bars). A second set was vibrated from 1 cell to 2 cell, allowed to rest in a vibration-free environment, and then re-vibrated for the same select period of time later in development (black bars). In all cases, adding vibrations from 1 cell to 2 cell increased the incidence of heterotaxia observed at st. 45. Vibrations from 1 cell to 2 cell without a later period of vibration were not significantly different from untreated controls (not shown here, see Figure 2). For statistical comparison, the actual numbers of embryos from two or more replicate studies with wildtype versus heterotaxic situs were compared for single late vibrations and the two paired vibrations (Ï2 test, p<0.05, *p<0.001).
	Figure 8. Vibrations affect patterning of the left-right (LR) axis in two distinct ways, producing different phenotypes depending on the timing of treatment.Early events involved in the patterning of the LR axis have been split into three basic steps [1], [42], [94]: breaking of symmetry, orientation of the axes, and amplification/restriction of LR signals. Targeting each of these steps is expected to produce different phenotypes: altered symmetry breaking leads to mirror-image left and right halves, i.e. isomerisms, a LR defect rarely if ever seen in Xenopus but often observed in mutant mice; altered orientation of the axes will produce a mixed population of wildtype and mirror-image individuals (with situs inversus) because the LR axis is randomly oriented with respect to the anterior-posterior and dorsal-ventral axes; altered amplification/restriction of LR signals will cause each organ to make independent decisions, thus producing a population of heterotaxic individuals. Here, we show that early vibrations produce large amounts of situs inversus, therefore we propose that the effects of early vibrations on the cytoskeleton are responsible for the second step, proper orientation of the three axes. The effects of vibrations during the second period of sensitivity largely produce heterotaxia, thus we propose that the effects of vibration on tight junction integrity, part of the ion flux pathway, alters the third step in the pathway, the amplification/restriction of LR signals.

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