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• W4385150413 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking. Editor's evaluation Sampaio and colleagues manipulate fluid dynamics in zebrafish Kupffer's vesicle to ask if fluid movement or something in the fluid governs the break in symmetry. These results support a role for fluid movement and detection as important in breaking symmetry in a ciliated left-right organizer and help set a time window when fluid flow is critical for this process. https://doi.org/10.7554/eLife.83861.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Vertebrate organisms break lateral symmetry during embryonic development and define left and right. The left-right axis is the third and final body axis to be established in the embryo and encompasses a biophysical process yet to be fully elucidated. In mouse, frogs, and fish it involves fluid movement in a specialized organ called the left right organizer (LRO). In the mouse embryo, cilia driven fluid flow inside the LRO has been proposed to activate either a mechanosensory mechanism in the epithelial cells that constitute the LRO, or to transport morphogens (as dissolved molecules or carried inside extracellular vesicles) towards the left side of the LRO (Nonaka et al., 1998; McGrath et al., 2003; Tanaka et al., 2005). While the mechanism is unknown, it culminates in asymmetric left-sided calcium signalling at the node cells and a subsequent conserved Nodal pathway on the left side of the embryo (Tanaka et al., 2005; Takao et al., 2013; Mizuno et al., 2020). The endpoint of this developmental process is the asymmetric localization of internal visceral organs, such as the heart and liver. Analysis of cilia-driven LRO flow is therefore crucial to understand this initial process of symmetry breaking. The transparent zebrafish embryo is a particularly suitable model system enabling flow visualization and subsequent scoring of organ asymmetry in the same individual. We and others have shown that wildtype (WT) zebrafish embryos have a stereotyped flow pattern in the LRO (known as Kupffer’s vesicle or KV), exhibiting higher dorsal anterior speed (Essner et al., 2005; Kramer-Zucker et al., 2005; Supatto et al., 2008; Sampaio et al., 2014; Smith et al., 2014; Ferreira et al., 2017). Our group has demonstrated that deviations from this flow pattern due to less cilia or shorter motile cilia, predictably generate larvae with wrong laterality outcomes, indicating a biological relevance to flow (Sampaio et al., 2014). Thus, flow dynamics and the mechanical-chemical readout are central to solving the problem of how left-right asymmetry is first established. Although the LRO is an organ with a limited lifetime, from ~2 ss to 14 ss (Oteíza et al., 2008), seminal work unveiled that the zebrafish Kupffer’s vesicle is dispensable for left-right establishment after 10 somite stages (ss) (Essner et al., 2005). Guided by this finding, most subsequent studies have been performed at 8–10 ss, when an asymmetry in dand5 gene expression is first detected (Lopes et al., 2010; Sampaio et al., 2014; Ferreira et al., 2017; Ferreira et al., 2019). The decrease of dand5 gene expression on the left side of the LRO is the first asymmetric molecular signal in left-right development, downstream of fluid flow, an event conserved across species (Marques et al., 2004; Hojo et al., 2007; Schweickert et al., 2010). Yuan and colleagues have shown that, as soon as zebrafish LRO cilia are formed and some start beating, at around 1–4 ss, intraciliary calcium oscillations (ICOs) can be registered in LRO cells (Yuan et al., 2015). These signals were reported to precede cytosolic calcium elevations that extend mostly to the left mesendodermal regions and were shown to be essential to trigger correct left-right patterning. Therefore, it is conceivable that an asymmetry in dand5 mRNA expression, first detectable at 8 ss in zebrafish (Lopes et al., 2010), results from molecular events that significantly precede that time point. Although it is evident from pioneer studies using mouse mutants without cilia or with immotile cilia that fluid flow has a critical role in conveying a LR biased signal to the nearby cells (Nonaka et al., 1998; Supp et al., 1999), both the nature of the signal and its detection mechanism remain elusive. The mechanosensory hypothesis postulates that fluid flow can be mechanically sensed by cilia, through a Polycystin protein complex involving PKD1L1 and PKD2 cation channel (Field et al., 2011; Kamura et al., 2011; Yuan et al., 2015). Conversely, the chemosensory hypothesis posits that vesicular parcels or extracellular vesicles containing an unknown chemical determinant are transported by the directional fluid flow towards the left side of the mouse node (Cartwright et al., 2020). However, the existence of such signaling morphogen has not yet been confirmed or any evidence for extra-cellular vesicle endocytosis or content release has ever been revealed with the exception of the work from Hirokawa’s lab (Tanaka et al., 2005). The difficulty in testing which hypothesis is correct relies on the fact that it is virtually impossible to perform genetic manipulations of the LRO cell secretion pathway without impacting cilia length or motility. This is not surprising if we think that any perturbation in lipid or protein trafficking affects motile cilia function, which reflects an experimental impossibility, because to test chemosensation we need to keep fluid mechanics constant. To address these 20 year-long-standing questions, we decided to take a step back and unequivocally determine the developmental period for left-right initiation. We envisaged that by determining the critical time-window within the 7 hr lifetime of the zebrafish LRO, we could then characterize its fluid flow and, thus, narrow down the variables that contribute to left-right establishment. Ultimately, we aimed at obtaining evidence for one or the other model for LR initiation as current scenarios fail to fully uncouple the LRO hydrodynamics from the potential role of fluid flow in transporting vesicles or molecules with signalling properties (Cartwright et al., 2020). The LRO is filled with fluid, but the nature of the fluid content is unknown because, so far, its minute volume, around one hundred picolitres (Roxo-Rosa et al., 2015), prevented a detailed biochemical analysis. However, the swelling mechanism of the LRO lumen is known to be driven by the chloride channel known as cystic fibrosis transmembrane conductance regulator (CFTR) that, when impaired, predictably leads to empty LROs and left-right defects (Navis et al., 2013). Thus, it is possible to block the swelling process by using pharmacology, anti-sense technology or mutants for CFTR, without affecting cilia number or motility. However, manipulation with precision and higher resolution in time was lacking. To determine the sensitive time-window for LR initiation, we devised a novel assay that allows for highly controlled mechanical manipulation of the LRO fluid. We performed total fluid extractions from the LRO at different developmental time points only 30 min apart. After confirming that the KV was still present and able to refill, we followed the development of each embryo until organ laterality could be determined. This approach led us to narrow the time-window for symmetry breaking to 1-hr, from 4 to 6 ss, corresponding to the cell rearrangement period mechanistically explained by Wang et al., 2011 and initially noted by Kreiling et al., 2007 and Okabe et al., 2008. During this time interval, we found fluid flow to be mild but already directional. Extraction of LRO fluid at 5 ss led to 35% of WT embryos with both heart and liver incorrect localization. Embryos did not develop LR defects when the original KV fluid was replaced with Danieau’s buffer, but LR axis was perturbed when viscosity of the same buffer was increased. Altogether, our experiments suggest that fluid flow mechanics, contrary to the nature of fluid content, has a major role in LR initial establishment. Results Fluid extraction affects LR during 1-hr time-window KV fluid flow was disrupted by performing fluid extraction at each LRO developmental stage using a microscope-based micromanipulation setup connected to microfluidic actuators (Figure 1A). We envisaged that if we found the sensitive time-period, we could then investigate with more precision the properties that were necessary and sufficient to break LR. For this purpose, we challenged WT embryos by extracting the liquid from their LRO lumen from 3 to 12 ss (see extraction Figure 1—video 1 at 8 ss and Figure 1—video 2 at 5 ss). Manipulated embryos that recovered the LRO liquid, were later characterized according to heart and liver laterality. This procedure intendedly depleted the LRO fluid transiently, disrupting the flow generated by motile cilia, while at the same time it potentially extracted any molecular signals that are present in the fluid. Figure 1 with 6 supplements see all Download asset Open asset Fluid extractions uncover a sensitive one-hour interval. (A) Schematic representation on the micromanipulation setup developed for the zebrafish LRO fluid extraction throughout development. (B) Evaluation of organ patterning after 48 hr. Heart and liver position were scored for each embryo manipulated in the respective developing stage from 3 to 12 ss, situs inversus in zebrafish means both organs are localized on the right side and heterotaxia means that at least one organ is wrongly localized. (C) dand5 expression pattern at 8 ss after single intervention at 5 ss and double intervention at 5 and 7 ss for LRO liquid extraction. LRO. left-right organizer, ss. somite stage. Figure 1—source data 1 Characterization of heart and gut situs in zebrafish larvae. https://cdn.elifesciences.org/articles/83861/elife-83861-fig1-data1-v1.xlsx Download elife-83861-fig1-data1-v1.xlsx Controls for these experiments included confirmation that cilia number, length and anterior-posterior (A/P) distribution were not perturbed after fluid extraction at 5 ss, in fixed samples at 8 ss (Figure 1—figure supplement 1) and that number of motile / immotile cilia and their A/P distribution was not changed when evaluated by live imaging at 6 ss (Figure 1—figure supplement 2 and Supplementary file 1). Concomitantly, we analysed cilia beat frequency (CBF) of motile cilia, by high-speed video microscopy. Sham controls, where the LRO was perforated with the extraction needle without applying suction, were included. At 6 ss, no significant difference was found between Sham controls with an average of 38.93 Hz (n=56 cilia; 6 embryos) and fluid extracted embryos with 37.85 Hz (n=60 cilia; 6 embryos); (Figure 1—figure supplement 2). To determine if after extraction at 5 ss there were significant anterior-posterior cilia distribution differences between ‘Sham’ and manipulated experimental groups the spatial distribution of cilium types was compared by live imaging as well as the ratio of motile to immotile cilia (Figure 1—video 3). Consistent with previous studies (Tavares et al., 2017), cilia from control ‘Sham’ embryos at 6 ss (n=7) were predominantly motile (82±4 SEM %) and localized preferentially to the anterior half of the LRO (Fisher test, P-value <0.05, Figure 1—figure supplement 2). The total number of motile cilia for each analysed ‘Sham’ embryo revealed some intrinsic embryo variability even among the control group as reported before (Sampaio et al., 2014). Similarly, in the manipulated group (n=6 embryos), motile cilia were more abundant (78 ± 10 %) and were distributed more anteriorly (Fisher test p-value <0.05; Figure 1—figure supplement 2). Therefore, no significant differences were found for the ratio of motile / immotile cilia between manipulated group compared to ‘Sham’ controls (Figure 1—figure supplement 2). Although most embryos recovered the fluid extraction procedure without LR defects (Figure 1B), some presented both heart and liver misplacements, starting with intervention at 3 ss (2/13 embryos, 15%, p-value = 0.235), becoming more pronounced for intervention at 4 ss (7/26 embryos, 27%, Fisher test, p-value = 0.002) and peaking with intervention at 5 ss (14/40 embryos, 35%; Fisher test, p-value = 6.44e-7). LR organ misplacements then became progressively non-significant from 6 ss onwards (4/27 embryos, 19%; Fisher test, p-value = 0.865; Figure 1B). At this point, as a response to the fluid extraction challenge, we had identified the most sensitive time-window for LR establishment. Next, we wanted to investigate in detail what were the factors that did not allow some of the challenged embryos to recover the correct laterality. To elucidate how fluid depletion affected LR signalling, we examined the expression pattern of dand5. It is well established that in WT embryos, zebrafish dand5 is mainly expressed on the right side of the LRO at 8 ss (Lopes et al., 2010) due to left-sided decrease of dand5 mRNA. Thus, we first evaluated dand5 expression pattern at 8 ss in embryos that were manipulated at 5 ss, which was the developmental stage with highest incidence of LR defects upon liquid extraction. We observed that dand5 expression also became abnormally bilateral in 35% of the cases (6/17 manipulated embryos compared to 0/26 controls, Fisher test, p-value = 0.002; Figure 1C) suggesting that either lack of fluid flow or depletion of a molecular signal prevented left-sided degradation of dand5 mRNA. Next, to further challenge the system we performed an additional fluid extraction 1 hr after the first intervention when enough liquid was replenished, at 7 ss. Results showed a similar proportion of bilateral dand5 expression (6/16, 37.5%) to that observed at 5 ss (comparing both plots in Figure 1C, Fisher test p-value = 0.728). This experiment confirmed that fluid extractions at 7 ss no longer affect LR development significantly, as already implied in Figure 1B. To continue the investigation on a potential deficiency in flow recovery in the ‘LR Defects’ group after fluid extraction at 5 ss, we next assessed how fluid flow dynamics varied with time by retrospectively comparing embryos with and without LR defects. LRO recovers lumen area after fluid extraction The lumen of manipulated LROs started to expand soon after fluid extraction (Figure 1—figure supplement 3), indicating that the fluid secretion machinery mediated by CFTR (Navis et al., 2013; Roxo-Rosa et al., 2015) was not collaterally affected by the manipulation procedure and was able to drive the swelling of the LRO lumen. Our observations contrasted with previous reports (Essner et al., 2005; Hojo et al., 2007) of irreversibly emptied LROs after manipulations, highlighting that the technical approach developed in this work confers minimal damage to the LRO core structure. Gokey et al., 2016 showed that below 1300 μm2 of KV cross-sectional area at 8 ss, LR patterning could be compromised. Therefore, a scenario where some LROs replenish better than others could explain a differential recovery, enabling the correct organ patterning in most, but not all embryos. Thus, we first investigated if there were significant differences in fluid recovery between manipulated embryos that developed LR defects from those that did not. We performed LRO fluid extraction at 5 ss and followed the recovery of the LRO cross-sectional area and fluid dynamics along development (at 6 ss, 7 ss and 8 ss, Figure 1—figure supplement 3A). As expected, the LROs from the control group (‘Sham’) were larger at each sampled time point (t-test, p-value <0.05) compared to the fluid extracted groups Further, our results showed that the LRO area was similar as well as the area change per somite (t-test, pvalue >0.05) in manipulated embryos that developed a normal organ patterning (‘No Defects’ group) and those that developed incorrect LR axis establishment ‘Defects’ group, (Figure 1—figure supplement 3B, C). Fluid flow angular velocity is reduced in manipulated embryos showing LR developmental defects Flow can be determined by following native particles in KV as a proxy for tracking fluid directly. The general pattern of fluid flow in the LRO is roughly circular with local variations (Sampaio et al., 2014; see Figure 2—video 1 for an embryo that developed normal LR development and Figure 2—video 2 for an embryo that showed abnormal LR development). Local flow speed is a useful measure for identifying regional flow patterns, but it does not fully describe the directional material transport of the LRO fluid (Ferreira et al., 2017; Juan et al., 2018). Thus, we determined the angular velocity at discrete locations as a proxy for the circular flow speed (Figure 2A–C) and show angular velocity components: tangential and radial velocities in Figure 2—figure supplements 1 and 2, respectively. Considering the two sensing hypotheses, we predicted that any type of detectable LR signal, either as flow-induced shear force or as a signalling molecule, must happen in the vicinity of the cell membrane. Taking this into consideration, we analysed the fluid dynamics within the outer half of the radius of the LRO lumen. Figure 2 with 4 supplements see all Download asset Open asset Angular velocity during LRO fluid flow recovery. Angular velocity polar plots for 6 ss, 7 ss, and 8 ss for the three different groups (A) 'Sham’ control embryos colour coded in green, (B) ‘No LR Defects’ group of embryos colour coded in blue and (C) 'LR Defects’ group of embryos colour coded in red. LR defects refer to misplacement of heart or liver encountered at the larval stage after fluid extraction was performed at 5 ss; number of tracks refers to the number of particle trajectories identified for the quantifications and respective angular velocity plots. Colour code on polar plots refers to the median angular velocity for pooled embryos. (D–E) Violin plots showing quantifications of angular velocities found for the tracks analysed (D) anterior-posterior and (E) left and right. Dots contained in the violin plots correspond to median values per embryo. A statistical linear mixed effects regression was applied (see results in Table 1). Figure 2—source data 1 Angular velocity quantifications at 6, 7, and 8 somite stages after fluid extraction at 5 somite stage. https://cdn.elifesciences.org/articles/83861/elife-83861-fig2-data1-v1.zip Download elife-83861-fig2-data1-v1.zip Angular velocity data from the many tracks obtained from each of the sham and extraction experiments were analysed at 6, 7, and 8 ss, first dividing the LRO in 8 radial sections and plotting the median angular velocity per section (Figure 2A–C). To account for the hierarchical structure of the data with both within- and between-embryo variability in angular velocity tracks, linear mixed effects regression was employed to characterise how angular velocity varied across the LRO and over time (Figure 2D–E), incorporating somite stage, normalized LR/ AP axis position and group (Sham/ Defects/ No Defects) as independent variables. Coefficient values can be interpreted as showing relative effect sizes and direction (see Table 1). Table 1 Linear mixed-effects regression: Results for fluid extraction experiment. Fixed effects coefficient estimates are given. Number of observations: 15775; embryos n=23; Fixed effects coefficients: 12; Random effects coefficients 69; Covariance parameters: 7. Regression formula: AAV ~1 + Group*LR +Group*PA +Group*Time + (1+Group | EmbryoID). Results relate to Figure 2D–E; *** indicates p<0.001; ** indicates p<0.01; * indicates p<0.05. NameEstimateLower CIUpper CIp-value(Regression line intercept)0.25150.207620.295443.8408E-29 ***No defects group–0.032775–0.123210.057660.47748Defects group–0.12728–0.18434–0.0702151.2396E-05 ***Left-right axis0.0042934–0.00926860.0193630.57655Posterior-anterior axis0.161310.146360.176266.285E-98 ***Somite stage0.021450.0120910.0308097.0847E-06 ***Interaction of no defects group and left-right axis0.0266530.0075770.045730.006176 **Interaction of defects group and left-right axis–0.0026553–0.0227530.0174430.79567Interaction of no defects group and posterior-anterior axis0.0336450.0121180.0551710.0021912 **Interaction of defects group and posterior-anterior axis–0.041406–0.06219–0.0206229.461E-05 ***Interaction of no defects group and somite stage0.0266350.0143170.0389532.2637E-05 ***Interaction of defects group and somite stage0.0134970.000566210.0264270.040778 * The linear mixed regression for experiments with fluid extraction and ‘Sham’ intervention at 5 ss identified the following significant fixed (embryo-independent) effects: (i) a significant reduction of angular velocity in the ‘Defects group’ (p-value = 1.2e-5, coefficient –0.13 rad/s), (ii) greater angular velocity in the anterior compared with posterior region of the LRO (p-value = 6.3e-98, coefficient 0.16 rad/s), (iii) increased flow velocity over time (p-value = 7.1e-6, coefficient 0.021 rad/s/stage), (iv) enhanced left-right difference in the ‘No Defects group’ (p-value = 0.006, coefficient 0.027 rad/s), (v) reduced posterior-anterior difference in the ‘Defects group’ (p-value = 9.5e-5, coefficient –0.041 rad/s), (vi) enhanced posterior-anterior difference in the ‘No Defects group’ (p-value = 0.002, coefficient 0.034 rad/s), (vii) enhanced degree of velocity increase over time in the ’No Defects’ group (p-value = 2.3e-5, coefficient 0.027 rad/s/stage) and (viii) (at marginal significance level) some degree of velocity increase over time in the 'Defects group’ (p-value = 0.041, coefficient 0.013 rad/s/stage). It is important to note that the increase in anterior angular velocity (Figure 2D) in the 'No defects' group compared with the 'Sham group' can be explained by the change in size of the Kupffer’s vesicle after extraction (Figure 1—figure supplement 3B): assuming that cilia beating and, therefore, material transport at the periphery is the same in ‘Sham’ group and ‘No-defect’ group, the fluid flow absolute velocity is also unchanged, but in a smaller sphere (R = ~25 µm versus R = ~30 µm) it leads to a proportionally higher angular velocity. In the ‘Defects’ group, despite this geometric effect, the angular velocity suggests that flow velocity was decreased. Next, to clarify for further differences we showed that radial velocity was modestly stronger and positive on the left-anterior sections of the LRO (Figure 2—figure supplement 2) denoting directionality towards the membrane was enhanced in the ‘No defects’ group. Further, between the ‘Defects’ group and the other groups we investigated the directionality of the individual particles as it could provide more cues for localized differences that we observed denoted by negative values for angular velocity indicating the existence of some particles with clockwise flow. We divided the LROs into sections of 30 degrees each and by looking at the direction of each tracked particle in detail (Figure 3A and Figure 3—figure supplements 1–6), we only found significant differences in directionality when comparing the group ‘Defects’ with the ‘Sham’ control group in anterior sectors of the LRO at 6 ss (Figure 3B and C). In this region the ‘Defects’ group presented disoriented particle trajectories compared to both the ‘Sham’ control group and the ‘No Defects’ group at 6 ss. This suggests that the manipulation might have regionally perturbed the fluid so that at 6 ss, the earlier time point after extraction, the effect of the transient manipulation was still detectable. Conversely, the ‘No Defects’ group never showed significant differences in directionality compared to the ‘Sham’ control group (Figure 3—figure supplement 1; see also Figure 3—figure supplements 2–6) for complete data on all sectors of the LRO for 7–8 ss. Figure 3 with 6 supplements see all Download asset Open asset Directionality of vector fields changes in embryos that develop LR defects. (A) Diagram representing particle directionality. LRO area sections were delimited based on intervals of 30 degrees. Highlighted are regions (B) from 30 to 60 degrees and (C) 60–90 degrees, that showed significant differences in particle movement between the groups ‘Sham’ control embryos and embryos with ‘LR Defects’. Each density plot represents the pooled tracked trajectory of all moving particles at any given point in time. Respective area region analysed is represented on the top right corner of each plot. To assess differences upon fluid manipulation ‘Sham’ and ‘Defect’ groups were plotted for the 6 ss. Kolmogrov-Smirnov test was used for comparing trajectory distribution between the two groups. Full data can be found in Figure 3—figure supplements 1–6. In summary, our data showed that after fluid extraction at 5 ss, the group of embryos that later developed LR defects showed a decreased angular velocity, preferentially at the anterior LRO region resulting in reduced AP difference and it also presented deviations from the counterclockwise flow direction anteriorly. The study of the angular velocity highlighted that a robust flow recovery mechanism is in place in most zebrafish WT embryos, ensuring that LR succeeds in most of the manipulated embryos. Both the groups of embryos that recovered or failed to recover LR development after the extraction manipulation, exposed new sensitive mechanical properties of the anterior region of the LRO that will be discussed below. A mechanosensory mechanism likely drives LR asymmetry Extraction of the LRO fluid annihilated both flow dynamics and fluid content, thus it did not allow to discern a potential mechanosensory mechanism from a chemosensory one. Benefitting from the micromanipulation setup we next designed an experiment to help us to uncouple these two sensory systems. We therefore diluted the fluid content in a physiological buffer (Danieau buffer, DB) without changing the flow dynamics for more than a few seconds, so that we could test whether the fluid content was crucial for LR establishment. To address how this dilution experiment affected flow dynamics, we monitored angular velocity over time as before, from 6 to 8 ss. As a positive control for flow dynamics perturbation, we used methylcellulose (MC, viscosity ~1500 cP; water, viscosity 1 cP) as previously used in Xenopus and mouse (Schweickert et al., 2007; Shinohara et al., 2012) to make the fluid more viscous and cause LR defects. First, we extracted the LRO fluid from 5 ss embryos and diluted it in approximately 1 µl volume of Danieau buffer (DB) previously loaded into the needle. Next, after a few seconds, we re-injected the resulting mixed liquid (Figure 4A). To test if dilution of the fluid content was indeed occurring, we performed the same experiment using a rhodamine tracer and confirmed that the LRO lumen became fluorescent red after the procedure (Figure 4B). The data for median angular velocities is presented in Figure 4—figure supplement 1 and the application of a linear mixed effects regression for this set of experiments with DB and MC dilution versus ‘Sham’ controls at 5 ss identified the following significant fixed effects (Figure 4C–D; Table 2): (i) as expected we saw a significant reduction of angular velocity following MC dilution (p-value = 4.7e-4, coefficient –0.12 rad/s), but not DB alone; (p-value = 0.17), (ii) as noted before controls showed greater velocity in the anterior compared to the posterior region of the LRO (p-value = 4.4e-142, coefficient 0.16 rad/s), (iii) controls also showed increased flow velocity over time (p-value = 5.6e-8, coefficient 0.021 rad/s/stage), (iv) an unpredicted faster flow on the right compared with the left following MC dilution was highlighted (p-value = 0.0039, coefficient 0.026 rad/s), but not by DB dilution alone (p-value = 0.11) (v) we noted a modest difference in flow between anterior and posterior following DB dilution (p-value = 0.0072, coeffic" @default.
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• W4385150413 title "Editor's evaluation: Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking" @default.
• W4385150413 doi "https://doi.org/10.7554/elife.83861.sa0" @default.
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