Tung A et al. (2024), Embryos assist morphogenesis of others through ...

XB-ART-60527

Nat Commun 2024 Jan 17;151:535. doi: 10.1038/s41467-023-44522-2.

Show Gene links Show Anatomy links

Embryos assist morphogenesis of others through calcium and ATP signaling mechanisms in collective teratogen resistance.

Tung A , Sperry MM , Clawson W , Pavuluri A , Bulatao S , Yue M , Flores RM , Pai VP , McMillen P , Kuchling F , Levin M .

???displayArticle.abstract???
Information for organismal patterning can come from a variety of sources. We investigate the possibility that instructive influences for normal embryonic development are provided not only at the level of cells within the embryo, but also via interactions between embryos. To explore this, we challenge groups of embryos with disruptors of normal development while varying group size. Here, we show that Xenopus laevis embryos are much more sensitive to a diverse set of chemical and molecular-biological perturbations when allowed to develop alone or in small groups, than in large groups. Keeping per-embryo exposure constant, we find that increasing the number of exposed embryos in a cohort increases the rate of survival while incidence of defects decreases. This inter-embryo assistance effect is mediated by short-range diffusible signals and involves the P2 ATP receptor. Our data and computational model emphasize that morphogenesis is a collective phenomenon not only at the level of cells, but also of whole bodies, and that cohort size is a crucial variable in studies of ecotoxicology, teratogenesis, and developmental plasticity.

???displayArticle.pubmedLink??? 38233424
???displayArticle.pmcLink??? PMC10794468
???displayArticle.link??? Nat Commun
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: mrc1
GO keywords: embryo development [+]

???attribute.lit??? ???displayArticles.show???


	Fig. 2: Conspecific effect beyond thioridazine exposure. a Images of control (left) and forskolin-treated animals (right), demonstrating hyperpigmentation following forskolin exposure (purple arrows). b The graph shows the total frequency of hyperpigmentation in embryos exposed to forskolin in small (n = 25), medium (n = 50), and large (n = 300) groups. **p < 0.001.. c Control animal raised in 0.1x MMR with normal brain structures (left). p <0.0001. Representative animals from cohorts subjected to nicotine treatment were then raised at a group size of 75 animals (right). Purple arrows highlight misshapen eyes, malformed forebrain, and midbrain. d Percent total of embryos with brain defects in groups treated with nicotine at densities of n = 10, 25, 50, 75, and 125. p < 0.0001, p = 0.0008, p = 0.0197. e Effect of group size on incidence of defects in dominant-negative Kir6.1-injected embryos. Images of control (left) and injected (right) embryos at stage 45. Blue arrows indicate normal eyes and head shape while purple arrows point out misshapen heads and eyes. f Quantification of malformed and normal individuals at stage 45. Group size of n = 10, 25, 50, and 100. Both cells at the 2-cell stage were injected and embryos were split into different size groups post injection. p = 0.0111, *p < 0.0001, p = 0.0004, and *p = 0.0028. All animal images are dorsal views and oriented so anterior is facing up and dorsal is down. For all data: values are plotted as mean ± SD, one-way ANOVA, and Tukey tests were conducted. Each dot on the graph is a separate replicate. All treatments are normalized to the mean of non-treated controls’ spontaneous defects. Scale bars represent 2 mm. Source data are provided as a Source Data file.
	Fig. 3: Genetically diverse populations can also benefit from CEMA but unperturbed cohort members do not aid in stabilization. a Examples of wildtype and albino animals that were untreated (controls) or treated with thioridazine (treated). Blue arrows highlight normal head shape and pigmentation. Purple arrows indicate square heads and hyperpigmentation. b Percent total of embryos with square heads in two strains of Xenopus laevis, wildtype (either n = 150 or n = 300, respectively labeled on the graph) and albino (n = 150 or n = 300, respectively labeled on the graph). **p < 0.0001. c Averages of the frequency of square head defects in a mixed group of n = 150 thioridazine-treated wildtype embryos + 150 untreated albinos, n = 300 treated wild-type embryos, and n = 150 treated wild-type embryos. p = 0.0094 and p = 0.0133. d Average of the frequency of brain defects and square heads in nicotine, thioridazine, and mixed treatments (n = 150 in each group). p = 0.0019 and p = 0.0291 for brain defects and ****p < 0.0001 for square head. All animal images are dorsal views with the anterior end at the top of the image. For all data: values are plotted as mean ± SD, one-way ANOVA, and Tukey test were conducted. All treatments are normalized to the mean of non-treated controls’ spontaneous defects. Scale bars represent 2 mm. Source data are provided as a Source Data file.
	Fig. 4: Computational agent-based model of CEMA. a Cartoon example of healthy and teratogen-influenced development. Left, an ECA, a 1D array of 0's and 1's, has an initial configuration of 40% 0's (white) and 60% 1's (black). ECA follows the GKL update rule, solving the majority problem by converging to all 1's. Right, an ECA that has noise induced by teratogens, which does not successfully solve the majority problem. b A cartoon example of inter-embryonic signaling from time t to time t + 1. Embryos signal their current health (red) at time t and supportive signals (black) at the next timestep to their neighbors and nearest neighbors. Embryos that have not been exposed to the teratogen cannot participate. c Data from the simulation shows that inter-embryonic signaling, CEMA, aids in development in the presence of noise (blue line). Without this communication, development fails in the presence of noise (red line). When half of a cohort is comprised of untreated embryos, they do not participate, and therefore the effect is lower (orange line). Each experiment included the number of embryos equal to the total embryos indicated on the x-axis and each experiment was repeated 50 times. The bars on the graph are 95% confidence intervals and the center represents the mean. Source data are provided as a Source Data file.
	Fig. 5: Increasing cohort size induced changes to the transcription of different small sets of genes in control and thioridazine-treated embryos. Volcano plots of differential expression data for large (300 embryos) compared to small (100 embryos) groups of a control and b thioridazine-treated embryos. Genes undergoing significant changes in expression (FDR < 0.05) are highlighted in red (increased expression) and blue (decreased expression). Pathway analysis for comparison of large vs. small numbers of conspecifics in the c control and d thioridazine treatment conditions. Pathway analysis was performed by over-representation testing of the differentially expressed genes (FDR < 0.1) in the gene ontology (GO) terms/pathways and over-represented pathways were identified at a significance threshold of FDR < 0.1. The size of the dots reflects the gene ratios (number of significant genes associated with the GO term/total number of significant genes associated with any GO term), and the adjusted p-value (FDR) reflects the significance. Source data are provided as a Source Data file.
	Fig. 6: CEMA requires diffusion but not physical contact. a Images of embryos undergoing thioridazine treatment in a standard physical isolation device (left) and an image of a diffusion-enhanced physical isolation device (right). b Groups of n = 300 embryos were treated with thioridazine and were isolated using windowed wells, solid wells, or unseparated. **p < 0.0001 and p = 0.0010. c Groups of n = 300 embryos were either put into clear or opaque separation devices or left unseparated and treated with thioridazine. p = 0.0021 between -separated,+vision and +separated,+vision and *p = 0.0012 between -separated,+vision and +separated,-vision. b, c were both analyzed using one-way ANOVA and Tukey tests. Values are means ± SD. All treatments are normalized to the mean of non-treated controls’ spontaneous defects. Source data are provided as a Source Data file
	Fig. 7: ATP/P2X receptors may mediate CEMA. a Suramin eliminates the protective effect of large group size against thioridazine-induced craniofacial defects and death. Tadpoles in groups of 300 were treated with 100 µM suramin, 90 µM thioridazine, 90 µM thioridazine and 100 µM suramin together, and compared to 90 µM thioridazine exposure as singletons. Blue arrows showing normal eyes, pigmentation, and head shape. Purple arrows show defects in those regions. b Percent defect of each group (n = 300 for each group) was then quantified. **p < 0.0001 and p = 0.0002. c Thioridazine concentration was constant at 90 µM across all treatments and calcium or P2 receptor blockers were added. Calcium was depleted using 5 µM BAPTA. P2 receptors were inhibited with 100 µM PPADS. Each treatment group had a total of n = 300 embryos. **p < 0.0001. d Quantification of extracellular ATP concentration. p = 0.0443. Each dot represents the average of a group of three replicates. All animal images are dorsal views, and are oriented with the anterior end at the top of the image. For all data: values are plotted as mean ± SD, one-way ANOVA, and Tukey tests were conducted, and *p ≤ 0.001, p ≤ 0.01, *p ≤ 0.05. All treatments are normalized to the mean of non-treated controls’ spontaneous defects. Scale bars represent 2 mm. Source data are provided as a Source Data file.
	Fig. 8: Mechanical damage in Xenopus induces calcium waves in distant, uninjured conspecifics. a Dimensions for holder and embryo arrangement for injury experiments. b Fluorescent images of GCAMP6S-expressing embryos at 0-, 10-, and 20-min post-injury for untreated stage 10–12 embryos. The injured embryo is on the left and the neighboring uninjured embryo is on the right. c The speeds of GCAMP6S signal propagation between and within embryos. Speeds were compared using the Kruskal–Wallis test, assessing differences in speed between embryos (n = 7) and within injured (n = 8) and receiver (n = 9) embryos; p < 0.05. d Kymographs of receiver embryo calcium dynamics before injury (spontaneous activity) and post-injury under control, suramin, and ATP conditions. Maximum GCAMP6S signal for e injured and f receiver embryos from the control (n = 10) and suramin-treated (n = 5) groups. Intensities are normalized to the mean of the pre-injury spontaneous activity. Groups were compared separately for injured and receiver embryos using the two-tailed Welch’s t-test; p = 0.009. g Maximum GCAMP6S signal for embryos exposed to a bolus of MMR media (control; n = 3) or ATP (n = 4). Groups were compared using the two-tailed Welch’s t-test; p = 0.01. All error bars represent the standard deviation. Source data are provided as a Source Data file.
	Fig. 9: Mechanistic model of CEMA effects. a Naive condition in which embryos are undisturbed. Damaged conditions in which embryos are subjected to injury and ATP expulsion increases. Damaged embryo + PPADS/Suramin showing the environment in which P-type receptors are blocked and inhibiting the binding of ATP. Damaged + BAPTA shows that internal calcium stores are depleted and there is a reduction of calcium response. b Enlarged view of internal events during injury. Top: The embryo on the left is injured and elicits a calcium response and increased ATP release. ATP binds to P2X receptors on neighboring, uninjured embryos and elicits its own calcium response. Middle: In the presence of PPADS/Suramin, the injured embryo has a calcium and ATP response but P2X receptor blockade prevents ATP binding and subsequent calcium wave in the uninjured embryo. Bottom: In the presence of BAPTA, the injured embryo no longer has a calcium response, but is still able to increase the release of ATP. ATP can still bind to P2X receptors in the neighboring embryo but there is no calcium wave, indicating that the calcium responses in both the injured and uninjured neighbor are dependent on calcium. Created with BioRender.com.
	Supplemental Figure 1. Additional images of thioridazine treated tadpoles and additional noted defects. (A) Whole body images of thioridazine treated animals. (B) Variations in square head phenotypes. (C) Quantification of all observed defects from thioridazine treatment in large and small groups with their respective controls.
	Supplemental Figure 2. Supplementary images of tadpoles treated with different teratogens. (A) Example control embryo. Example of forskolin (B), nicotine (C), and kir6.1 (D) treated embryos.
	Supplemental Figure 3. A visual example of how the control and ‘teratogenexposed’ ECAs update from one time step to another. (Left) Normally, cells follow the GKL rule 84, successfully solving the majority problem. There is an initial distribution of ones (black) and zeros (white) with more ones than zeros. The ECA evolves (top to bottom) and converges to all ones. (Right) In the presence of noise (red dashed line), the rules shift (and stay shifted) to random updating, and the ECA fails to solve the majority problem. There is the same majority in the right as in the left, but at the end of the simulation not all cells are one/black. These examples were generated as singletons, and no collective effects are shown here.
	Supplemental Figure 4. Two representative examples of health values over time. In each graph, the y-axis is embryos in the group and the x-axis is time. At each time point the health value of the embryo is reported as a color (yellow is more health and blue is more unhealthy). For low numbers of total embryos, health values quickly collapse to low values. This can be seen in the top graph as the colors slowly drift to a dark blue color. Conversely, under the same conditions, large numbers of embryos better resist noise, resulting in healthy, although not perfectly healthy, embryos. This can be visually seen in the lower graph, where the health values are more yellow than the upper graph, meaning higher health. While not a central area of investigation for this study, it is particularly interesting how health and noise balance one another.
	Supplemental Figure 5. (A) Left, a schematic of two tested conditions – first with only neighbor’s interactions involved in the simulations and second with neighbor’s neighbors’ involved. Right, a graph showing how these two conditions (orange and blue, respectively) effect how the cohort resists noise. Even when lowering noise (green), small, local-only interactions are not enough to resist noise. (B) Left, a schematic showing how health threshold values were tested. Right, a graph showing how these three conditions altered the simulations while keeping all other parameters set
	Supplemental Figure 6. Principal component analysis of RNA-sequencing data for Stage 25 and Stage 35 Xenopus housed in small (100-embryo) and large (300-embryo) group sizes in control or thioridazine-treated media.
	Supplemental Figure 7. Intensity vs. time plots for control and suramin-treated embryos. Baseline (0-10 minutes) and post-injury (10- 30 mins) traces are shown for the injured and receiver embryos. The time point of injury is denoted by the lightning bolt.

References [+] :

Abbracchio, International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. 2006, Pubmed