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Piezo1-mediated spontaneous calcium transients in satellite glia impact dorsal root ganglia development [1]

['Jacob P. Brandt', 'Department Of Biological Sciences', 'University Of Notre Dame', 'Notre Dame', 'Indiana', 'United States Of America', 'The Center For Stem Cells', 'Regenerative Medicine', 'Cody J. Smith']

Date: 2023-10

Spontaneous Ca 2+ transients of neural cells is a hallmark of the developing nervous system. It is widely accepted that chemical signals, like neurotransmitters, contribute to spontaneous Ca 2+ transients in the nervous system. Here, we reveal an additional mechanism of spontaneous Ca 2+ transients that is mechanosensitive in the peripheral nervous system (PNS) using intravital imaging of growing dorsal root ganglia (DRG) in zebrafish embryos. GCaMP6s imaging shows that developing DRG satellite glia contain distinct spontaneous Ca 2+ transients, classified into simultaneous, isolated, and microdomains. Longitudinal analysis over days in development demonstrates that as DRG satellite glia become more synchronized, isolated Ca 2+ transients remain constant. Using a chemical screen, we identify that Ca 2+ transients in DRG glia are dependent on mechanical properties, which we confirmed using an experimental application of mechanical force. We find that isolated spontaneous Ca 2+ transients of the glia during development is altered by manipulation of mechanosensitive protein Piezo1, which is expressed in the developing ganglia. In contrast, simultaneous Ca 2+ transients of DRG satellite glia is not Piezo1-mediated, thus demonstrating that distinct mechanisms mediate subtypes of spontaneous Ca 2+ transients. Activating Piezo1 eventually impacts the cell abundance of DRG cells and behaviors that are driven by DRG neurons. Together, our results reveal mechanistically distinct subtypes of Ca 2+ transients in satellite glia and introduce mechanobiology as a critical component of spontaneous Ca 2+ transients in the developing PNS.

Here, we use imaging of GCaMP6s in satellite glia of the dorsal root ganglia (DRG) in zebrafish as a model to investigate the role of glial activity in the developing PNS. The DRG is required for somatosensory stimuli in the PNS and contains somatosensory neurons and satellite glia that ensheath those neurons. We identify that satellite glia display at least 3 types (microdomain, isolated, and simultaneous) of spontaneous Ca 2+ transients in early phases of development. By mapping the GCaMP6s events, we identify that the DRG transitioned to synchronized Ca 2+ transients early in development, demonstrating the formation of glial networks within the first 3 days of DRG construction. In a pilot screen and follow-up experimental manipulations, we identify mechanosensitive ion channel Piezo1 as a modulator of the isolated Ca 2+ transients of satellite glia in development and identify that these satellite glia are mechanosensitive. Perturbation of Piezo1 causes not only changes in isolated Ca 2+ transients of DRG satellite glia but also in their expansion and function, demonstrating a potential consequence to altering isolated glial Ca 2+ transients during development. Together, we introduce the role of mechanosensitive ion channels in the spontaneous Ca 2+ transients of the developing PNS.

What we do know is that spontaneous Ca 2+ transients in the nervous system have largely been characterized as dependent on chemical signals. In neurons, Ca 2+ spontaneous activity is promoted by neurotransmitters and their receptors [ 17 , 18 ]. Similarly, glutamate and NMDA drive spontaneous Ca 2+ transients of glial cells like oligodendrocytes and astrocytes [ 19 – 22 ]. We also know chemical signals like ATP can induce purinergic receptors to drive Ca 2+ changes in glia, akin to activity of the glia [ 23 – 25 ]. Each of these chemical signals causes changes to ion channels that drive spontaneous Ca 2+ transients. However, in addition to ion channels that are induced by chemical signals, mechanosensitive ion channels are also present in the nervous system [ 26 , 27 ]. For example, Piezo proteins are mechanosensitive channels that are expressed in the nervous system [ 26 , 28 , 29 ]. These mechanosensitive channels are essential for evoking a subset of peripheral sensory neurons in response to mechanical stimulation [ 30 , 31 ]. Peripheral mechanosensitive glia are also present at the skin to ensure response to mechanical stimuli [ 32 ]. However, the role of mechanosensitive properties in the development of glia is less understood, especially in peripheral glia. This is despite knowledge that mechanical components can have profound effects on cell differentiation and tissue organization and that Trp channels, some of which are at least partially mechanosensitive, are important for Ca 2+ transients in glia-like astrocytes [ 4 , 8 , 29 , 33 – 36 ].

It is widely accepted that spontaneous activity is a critical feature of the developing nervous system [ 1 – 3 ]. This activity has been visualized by measuring Ca 2+ transients. For years, such spontaneous Ca 2+ transients have been investigated in neurons and are identified as neuronal firing or activity, but recent studies have also revealed an important role for spontaneous Ca 2+ transients in glia. These glial Ca 2+ transients can be in response to neuronal activity or independent of neuronal activity and can be characterized into distinct subtypes [ 4 – 7 ]. For example, glial cells exhibit whole cell and microdomain Ca 2+ transients, which are mechanistically and functionally distinct [ 8 – 10 ]. Glial cells can also exhibit synchronous Ca 2+ transients in physically connected networks [ 11 , 12 ]. Regardless of Ca 2+ transient subtype and unique from neurons, immature glia and their progenitors also proliferate throughout life [ 13 ]. How glial Ca 2+ transients, proliferation, and physically connected networks are related or regulated, remains largely unexplored. The importance of these concepts is underscored by the prevalence of such processes during normal brain development and in gliomas [ 4 , 5 , 14 – 16 ]. If glial Ca 2+ transients are critical for nervous system function, we need more investigation into how distinct Ca 2+ transients change over development, whether different molecular components control distinct transient subtypes, and if distinct Ca 2+ transients are linked to proliferation and/or network formation. Lastly, these concepts need to be explored in both the central nervous system and peripheral nervous system (PNS).

Results

DRG satellite glia exhibit distinct Ca2+ transients To understand if DRG satellite glia display spontaneous Ca2+ transients, we first explored the Ca2+ transients of DRG cells in intact ganglia using intravital imaging in zebrafish. To do this, we imaged transgenic animals expressing GCaMP6s in distinct DRG cell populations. It is known that the DRG has both a population of neurons and satellite glia. To image both of these populations, we used animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP). Cells expressing RFP were identified as neurons, while the glial population expressed GCaMP6s with the absence of RFP (Fig 1A). To further confirm that these sox10+ neurod- cells were satellite glia, we investigated the morphology of these cells (S1A and S1B Fig). Using these triple transgenics and identifying morphological features of DRG cells, we found that both neurons and glia that ensheathed those neurons were present in the DRG during the developmental window examined. We define satellite glia in this report as sox10+ neurod- cells located in the DRG with ensheathing phenotypes [37,38]. In addition to these transgenics, we also imaged neurons in the DRG using Tg(neurod:gal4+myl7); Tg(uas:GCaMP6s), which uses regulatory sequences of neurod that are expressed in DRG neurons. This allowed for another approach in which we only investigate the neuronal population. We imaged 3 dpf (days post fertilization) animals expressing GCaMP6s at a 15-s interval for 1 h, which allowed us to capture a 3D view of 3–4 DRG. To define a Ca2+ transient event in a cell, we calculated the z score of the integrated density of fluorescence of each individual cell during that 1-h time period. Time points with a z score greater than 2.58 (represents 99% confidence interval) were considered Ca2+ transient event-containing time points. If a Ca2+ transient event lasted for consecutive time points, it was still considered 1 Ca2+ transient event. Scoring of the z score of GCaMP6s integral density of fluorescence over the 1-h period revealed that all DRG displayed cells with spontaneous Ca2+ transients, with remarkable activity in satellite glia (Fig 1B and 1C). Within individual DRG, satellite glia displayed on average 3.600 ± 1.78 Ca2+ transient events in a 1-h period (n = 25 cells, 14 DRG, 5 animals) (S2A Fig). We also measured 2.364 ± 1.12 Ca2+ transient events in the neuronal population in a 1-h period (n = 11 cells, 9 DRG, 4 animals) (S2A Fig), indicating that both neurons and glia display Ca2+ transients during DRG construction. Ca2+ transients can be quick in cells, so it is possible that a 15-s imaging interval underrepresented the number of Ca2+ transients. To address this, we imaged smaller z-stacks but with short time intervals of 5 s. These results revealed that Ca2+ transients in sox10+ cells lasted on average 2.21 time points using 5-s imaging intervals and therefore were generally captured with 15-s intervals (n = 412 Ca2+ transient events, 97 cells, 20 DRG, 5 animals) (S2B Fig). We therefore utilized 15-s imaging intervals throughout this manuscript, allowing us to image z-stacks that covered the entire DRG at each time point. With the lack of knowledge about glial activity in the DRG, we further investigated the developmental, molecular, and functional features of Ca2+ transients in sox10+ satellite glia. PPT PowerPoint slide

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TIFF original image Download: Fig 1. DRG exhibit distinct calcium activity and increase synchronous activity. (A) Confocal z-projections of DRG in a 3 dpf animal expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP). Arrow notes satellite glia and asterisk notes neuron. (B) Confocal z-projections of DRG in a 3 dpf animal expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s). Images presented as heated scale (reds-more activity, blues-less activity). (C) Line graphs depicting z scores of integrated density of fluorescence for individual cells expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) over a 1-h period. A z score greater than 2.58 indicates a Ca2+ transient event. Red scale bar is a z score of 2.58. (D) Confocal z-projection of DRG in 3 dpf animal expressing Tg(sox10:gal4+myl7); uas:GCaMP6s-caax. Red colors indicate a higher intensity of fluorescence and blue colors indicate a lower intensity of fluorescence. Arrows indicate active Ca2+ microdomains. (E) Line graphs depicting z scores of integrated density of fluorescence for Ca2+ microdomains in 3 dpf animals expressing Tg(sox10:gal4+myl7); uas:GcaMP6s-caax over a 10-min period. A z score greater than 2.58 indicates an active Ca2+ event. Red scale bar is a z score of 2.58. (F) Confocal z-projection of DRG in a 3 dpf animal expressing Tg(sox10:gal4+myl7); Tg(uas:GcaMP6s). Left image depicts an isolated Ca2+ event and the right image depicts a simultaneous Ca2+ event. Arrows indicate active cells. (G) Average number of isolated Ca2+ transient events per sox10+ cell at 2, 3, and 4 dpf in animals expressing Tg(sox10:gal4+myl7); Tg(uas:GcaMP6s) (2 dpf: n = 6 animals, 10 DRG, 46 cells, 3 dpf: n = 4 animals, 6 DRG, 27 cells, 4 dpf: n = 4 animals, 7 DRG, 34 cells). (H) Average number of simultaneous Ca2+ transient events per sox10+ cell at 2, 3, and 4 dpf in animals expressing Tg(sox10:gal4+myl7); Tg(uas:GcaMP6s) (2 dpf: n = 6 animals, 10 DRG, 46 cells, 3 dpf: n = 4 animals, 6 DRG, 27 cells, 4 dpf: n = 4 animals, 7 DRG, 34 cells). (I) Average number of seconds for Ca2+ microdomains duration in 3 dpf animals (n = 7 animals, 8 DRG, 15 microdomains). (J) Average volume (μm3) of Ca2+ microdomains in 3 dpf animals (n = 7 animals, 8 DRG, 15 microdomains). (K–M) Three (K) and 4 (L) dpf DRG in an animal expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s). ROIs are traced for individual cells. Line graphs of the z score of the integrated density of fluorescence correspond to the individual ROIs. (M) A corresponding network map for 3 and 4 dpf DRG (K, L) indicates both the number of isolated Ca2+ transient events and the high correlation coefficients present in the DRG. (N) Percent of high correlation coefficient per sox10+ cell in animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) at 2, 3, 4 dpf (2 dpf: n = 6 animals, 10 DRG, 50 cells 3 dpf: n = 4 animals, 6 DRG, 27 cells 4 dpf: n = 4 animals, 7 DRG, 34 cells). (O) Immunohistochemistry for Cxn43 in DRG of animals expressing Tg(sox10:meGFP) at 2, 3, 4 dpf. Magenta indicates Tg(sox10:meGFP) and cyan indicates Cxn43. (P) Quantification of the average number of Cxn43 puncta present in DRG at 2, 3, and 4 dpf (2 dpf: n = 6 animals, 18 DRG 3 dpf: n = 10 animals, 29 DRG 4 dpf: n = 8 animals, 24 DRG). (Q) Quantification of the average percent of high correlation coefficients per sox10+ cell following treatment with DMSO or CBX (DMSO: n = 4 animals, 7 DRG, 26 cells CBX: n = 3 animals, 5 DRG, 34 cells). (R) Depiction of targeted cellular processes for molecular screen. (S) Quantification of the average number of Ca2+ events per DRG following pharmacological screen (DMSO: n = 19 animals, 64 DRG, HMR1556: n = 5 animals, 15 DRG Apyrase: n = 5 animals, 15 DRG Thaps: n = 4 animals, 13 DRG, GsMTx4: n = 5 animals, 13 DRG CBX: n = 5 animals, 14 DRG). Scale bar is 10 μm (A, B, D, F, K, L, O). Ca2+ transient events are time points containing a z score of the integrated density of fluorescence greater than 2.58 (C, E, G, H, I, J, K, L). Statistical tests: one-way ANOVA followed and represented by post hoc Tukey test: (I, J, N, P), unpaired Student t tests: (Q), one-way Brown–Forsythe ANOVA followed and represented by post hoc Dunnett test: (S), correlation coefficient test: (M, N, Q). The data underlying this figure can be found in S1 Data. dpf, days post fertilization; DRG, dorsal root ganglia; ROI, regions of interest. https://doi.org/10.1371/journal.pbio.3002319.g001 Neural cells can exhibit distinct spontaneous Ca2+ transient events. To explore if DRG satellite glia exhibit distinct subtypes of Ca2+ transients, we created activity profiles for each cell in a given DRG from z-score calculations in 1 h movies of Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP) 3 dpf animals. Using this data, we could then compare when each individual cell in a DRG was active compared to the other cells in the DRG. We found that individual sox10+ cells displayed Ca2+ transients simultaneously with other sox10+ cells in the DRG (Fig 1F), consistent with previous descriptions of simultaneous Ca2+ transients in glial networks [11,39,40]. However, we also identified a subset of Ca2+ transients that occurred in cells when no neighboring cell is active (Fig 1F). We define these Ca2+ transients in this report as isolated Ca2+ transients. Calcium microdomains are also known to be present in several glial types [10,34,41]. Therefore, we tested if Ca2+ microdomains are also present in the DRG during development. To do this, we imaged animals expressing a membrane localized GCaMP6s by injecting Tg(sox10:gal4+myl7) embryos with uas:GCaMP6s-caax and imaging at 3 dpf. In order to initially capture and identify these quick dynamic events, we imaged animals for a 10-min period with 5-s intervals capturing the entire DRG. We defined Ca2+ microdomains as small regions with significant changes in integrated density of fluorescence of GCaMP6s-caax (Fig 1D and 1E). We quantified the duration of these microdomains and found that they lasted on average for 11.81+/−9.914 s (n = 15 cells, 8 DRG, 7 animals) (Fig 1I). We also quantified the average volume of these microdomains and found that they were on average 30.10+/−18.51 μm3 (n = 15 cells, 8 DRG, 7 animals) (Fig 1J). Together, these results indicate DRG satellite glia exhibit at least 3 distinct Ca2+ transient events during development: isolated, simultaneous, and microdomains.

Satellite glia cell networks are established during early DRG construction To understand how these types of activity may change over development, we quantified the average amount of isolated and simultaneous Ca2+ transients events in the same animal at 2, 3, and 4 dpf. While we did not see a significant change in isolated Ca2+ transients over this developmental period (2 dpf: n = 46 cells, 10 DRG, 6 animals, 3 dpf: n = 27 cells, 6 DRG, 4 animals, 4 dpf: n = 34 cells, 7 DRG, 4 animals) (Fig 1G), there was a noted significant increase in the number of simultaneous Ca2+ transient events after 2 dpf (2 dpf versus 3 dpf: p = 0.0019, 2 dpf versus 4 dpf: p = 0.0449 post hoc Tukey test) (2 dpf: n = 46 cells, 10 DRG, 6 animals, 3 dpf: n = 27 cells, 6 DRG, 4 animals, 4 dpf: n = 34 cells, 7 DRG, 4 animals) (Fig 1H). This work indicates distinct developmental properties between isolated and simultaneous subtypes. Current research proposes that DRG satellite glia form networks in vitro [42–44]. To further test if this occurs in vivo and to determine when in development it arises, we measured synchronized networks in sox10+ cells. To identify a synchronized network of cells, we compared the Ca2+ transient profiles of individual cells by computing the correlation between 2 Ca2+ transient profiles. To determine how this changed in development, we quantified the percent of high correlation coefficients (>0.5) per cell in each DRG at 2, 3, and 4 dpf. By creating network maps of individual DRG that show how the activity of each cell is related (Fig 1K–1M), we found that sox10+ cells at 2 dpf had an average of 37.48% ± 32.08% high correlation coefficients (n = 50 cells, 10 DRG, 6 animals). At 3 dpf, we measured that sox10+ cells had an average of 39.33% ± 30.61% high correlation coefficients (n = 27 cells, 6 DRG, 4 animals) and by 4 dpf, sox10+ cells had a significant increase in the percent of high correlation coefficients, with an average of 58.26% ± 32.13% high correlation coefficients (n = 34 cells, 7 DRG, 4 animals) (2 dpf versus 4 dpf: p = 0.0044 post hoc Tukey test, 2 dpf n = 46 cells, 3 dpf n = 27 cells, 4 dpf n = 34 cells) (Fig 1N). Additionally, we observed that the percent of cells displaying Ca2+ transients together increased by 4 dpf (S3A–S3C Fig). These data are consistent with the hypothesis that DRG satellite glial networks are present in vivo and form by at least the third day of DRG construction in zebrafish. If glial networks are forming, we hypothesized that gap junctions may also increase during the time when synchronized Ca2+ transients are present. Cxn43 is known to be present in satellite glia and contribute to gap junctions in synchronized neural networks [45–47]. Therefore, we stained for Cxn43 at 2, 3, and 4 dpf in animals expressing Tg(sox10:meGFP), which labels satellite glia in the DRG with membrane-localized GFP. The 2 dpf DRG had an average of 0.500 ± 0.707 Cxn43 puncta (n = 18 DRG, 6 animals). This increased to an average of 1.000 ± 0.845 Cxn43 puncta per DRG at 3 dpf (n = 29 DRG, 10 animals) and by 4 dpf, there was a significant increase in the number of Cxn43 puncta present in the DRG with an average of 2.208 ± 1.062 Cxn43 puncta per DRG (n = 24 DRG, 8 animals) (2 dpf versus 4 dpf: p < 0.0001, 3 dpf versus 4 dpf: p < 0.0001 post hoc Tukey test) (Fig 1O and 1P). These results support the hypothesis that DRG cells begin forming glial connections during its earliest construction. To determine if there are functional gap junction connections, we treated animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP) with either carbenoxolone (CBX), a gap junction inhibitor, or a control treatment of DMSO. Animals treated with CBX at 3 dpf demonstrated a significant decrease in the percent of high correlation coefficients with an average percent of 22.00% ± 22.34% (n = 34 cells, 5 DRG, 3 animals) compared to an average percent of high correlation coefficients of 36.58% ± 31.22% when treated with a DMSO control (n = 26 cells, 7 DRG, 4 animals) (DMSO versus CBX: p = 0.0391 unpaired t test) (Fig 1Q). These results strongly support the idea that functional gap junctions are present in glial networks in DRG during its early construction.

Satellite glia Ca2+ transients are impacted by altering mechanobiology Our measurements indicated that DRG satellite glia cells demonstrate distinct Ca2+ transients. To identify potential molecular components involved in these Ca2+ transients, we performed a chemical screen targeting various chemical signals shown to affect Ca2+ transients using transgenic animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP) (Fig 1R and 1S). Additionally, we included a broad-mechanosensitive ion channel antagonist, GsMTx4, because of the underappreciated role that mechanobiology has during neurodevelopment. We hypothesized that GsMTx4 would reduce the amount of observed Ca2+ transients if mechanobiology had an important role during early development. Each animal was exposed to the pharmacological agent 30 min prior and during the imaging window and then GCaMP6s intensity was measured for 1 h with a 15-s imaging interval. We reasoned that an overall change in the abundance of Ca2+ transients could help us identify molecules that are important for either isolated or simultaneous spontaneous Ca2+ transients. We found that GsMTx4 significantly reduced the amount of Ca2+ transients observed compared to DMSO (Fig 1S) (DMSO versus GsMTx4: p < 0.0001 post hoc Dunnett test). We also measured a significant change following treatment with Thapsigargin (Thaps) (Fig 1S) (DMSO versus Thaps: p = 0.0010 post hoc Dunnett test). While chemical signaling has been widely described in spontaneous Ca2+ transients, the role of mechanobiology in the process is less known, which led us to investigate the potential role of mechanobiology in spontaneous Ca2+ transients in the DRG. To first explore the possibility that mechanical features impact spontaneous Ca2+ transients in the DRG, we tested if the cells in the developing DRG are sensitive to mechanical perturbation. To do this, we imaged the DRG of transgenic zebrafish expressing GCaMP6s in satellite glial Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) cells during tissue compression (Fig 2A). Tissue compression was administered by bending the animal with a microneedle as they were imaged on the confocal microscope (Fig 2B). At 2 dpf, 57% of DRG expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) responded to tissue compression (n = 7 DRG, 7 animals) (Fig 2C and 2D). In order to better understand how much compression was needed, we measured the distance that the animal was compressed. This compression impacted not just the DRG itself, but also all surrounding tissue. We found that the average amount of compression needed to elicit a response in the DRG was 207.8 μm (n = 16 DRG, 16 fish) (Fig 2H). There was notable variability in this compression assay. This was due to variability in the amount of force, size of the tip of the needle, positioning of the animal, and angle that the microneedle was positioned. It is possible that this response of sox10+ cells was secondary to neuronal firing. We, therefore, tested if neurons fired in response to compression at 2 dpf in Tg(neurod:gal4+myl7); Tg(uas:GCaMP6s) animals but could not detect Ca2+ transients in neurons after compression (n = 5 DRG, 5 animals) (Fig 2C). Examining DRG axonal projections in Tg(ngn1:GFP) animals also showed that neurons at 2 dpf did not have peripheral axons at their final targets in the periphery (Fig 2G). It therefore seems unlikely that such Ca2+ transients in sox10+ satellite glia after tissue compression are secondary to neuronal activity. To understand if sox10+ satellite glia continued to be sensitive to mechanical compression, we repeated this assay at 3 dpf. By 3 dpf, 82% (n = 11 DRG, 11 animals) of DRG expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) responded to tissue compression (Fig 2C). At 3 dpf, 100% (n = 5 DRG, 5 animals) of DRG expressing Tg(neurod:gal4+myl7); Tg(uas:GCaMP6s) also demonstrated Ca2+ transients after tissue compression (Fig 2C). While the neuronal population of the DRG does respond to tissue compression at a later age, our data suggests satellite glia respond to the mechanical tissue compression at early ages without neuronal activation. PPT PowerPoint slide

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TIFF original image Download: Fig 2. DRG are mechanosensitive and express piezo1. (A) LEFT Depiction of mechanical compression assay where animal is mounted dorsally on inverted spinning disk confocal with a dextran loaded microneedle mounted above the animal. RIGHT image of mechanical compression assay apparatus. (B) Depiction of mechanical compression assay with needle placing force on DRG. (C) Quantification of the percent of DRG responding to mechanical force in animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) (labeled sox10) or expressing Tg(neurod:gal4+myl7); Tg(uas:GcaMP6s) (labeled neurod) and treated with either DMSO or GsMTx4 at both 2 and 3 dpf (sox10 2 dpf DMSO: n = 7 animals, 7 DRG, sox10 2 dpf GsMTx4: n = 5 animals, 5 DRG neurod 2 dpf DMSO: n = 5 animals 5 DRG neurod 2 dpf GsMTx4: n = 4 animals, 4 DRG sox10 3 dpf DMSO: n = 11 animals, 11 DRG sox10 3 dpf GsMTx4: n = 4 animals, 4 DRG neurod 3 dpf DMSO: n = 9 animals, 9 DRG neurod 3 dpf GsMTx4: n = 4 animals, 4 DRG). (D) Confocal image taken of the mechanical compression assay in 2 dpf animal expressing Tg(sox10:gal4+myl7); Tg(uas:GcaMP6s). Images show an inactive time point, a time point with tissue compression, and an active time point in response to tissue compression. Inactive and active DRG marked with an arrow. (E) Quantification of the change in integrated density of fluorescence during each phase of mechanical compression assay of a DRG in a 3 dpf animal treated with either DMSO or GsMTx4. Change in integrated density of fluorescence is scored as time point subtracting the initial time point divided by time point (Δf/f). (F) Quantification of the average change in fluorescence during the phases of mechanical compression following treatment with either 2% DMSO or 1 μm GsMTx4 for 30 min (Resting, Compression, and Decompression DMSO: n = 9 DRG, 9 animals, Resting, Compression, and Decompression GsMTx4: n = 4 DRG, 4 animals). (G) Confocal z-projection of peripheral DRG axon in an animal expressing Tg(ngn1:GFP) at 2 and 3 dpf. Arrow notes the end processes of the peripheral axon. Arrowhead denotes peripheral axons from Rohon beard neurons. (H) Average distance (μm) of DRG displacement needed to elicit a response (n = 16 animals, 16 DRG). (I) Confocal images of RNAscope-piezo1 and Immunohistochemistry-GFP in Tg(sox10:meGFP) animals. GFP is shown in magenta and piezo1 is shown in cyan. Arrowheads indicate piezo1 puncta. Arrows indicate autofluorescence. (J) Quantification of DRG at 3 dpf with piezo1 puncta and without piezo1 puncta (n = 8 animals, 24 DRG). (K) Confocal images of 3 dpf animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s). Red colors indicate a higher intensity of fluorescence and blue colors indicate a lower intensity of fluorescence. Images depicted are of animals either treated with 2% DMSO, 40 μm Jedi2, or 1 μm GsMTx4. Arrows note active cells. (L) Line graphs of z score of integrated density of fluorescence for a 1-h time period in 3 dpf animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) that were treated with either 2% DMSO, 40 μm Jedi2, or 1 μm GsMTx4. A z score greater than 2.58 indicates an active Ca2+ event. Red scale bar shows a z score of 2.58. (M) Heatmaps of the z score of individual sox10+ cells from animals in G and H during a 1-h period of Ca2+ imaging. Yellow notes a high z score (2.58 or greater) (DMSO: n = 8 animals, 17 DRG, 52 cells, Jedi2: n = 7 animals, 18 DRG, 79 cells, GsMTx4: n = 6 animals, 16 DRG, 44 cells). (N) Quantification of the average number of Ca2+ events per sox10+ cell in animals treated with either 2% DMSO, 1 μm GsMTx4, 100 μm Yoda1, or 40 μm Jedi2 (DMSO: n = 8 animals, 17 DRG, 52 cells, Jedi2: n = 7 animals, 18 DRG, 79 cells, GsMTx4: n = 6 animals, 16 DRG, 44 cells, Yoda1: n = 4 animals, 9 DRG, 35 cells). (O) Quantification of the average number of Ca2+ events per neurod+ cell in 3 dpf animals expressing Tg(neurod:gal4+myl7); Tg(uas:GCaMP6s) that were treated with either 2% DMSO, 1 μm GsMTx4, 40 μm Jedi2, or 100 μm Yoda1 (DMSO: n = 10 animals, 24 DRG, 33 cells, Jedi2: n = 5 animals, 18 DRG, 35 cells, GsMTx4: n = 4 animals, 8 DRG, 8 cells, Yoda1: 4 animals, 7 DRG, 8 cells). (P) Quantification of the average number of Ca2+ events per sox10+ cell following genetic manipulation via injection of uas:cas9mkate-u6:piezo1gRNA or uas:cas9mkate-u6:emptygRNA into animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) at 3 dpf. Additionally, a group of Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) injected with uas:cas9mkate-u6:piezo1gRNA and treated with 40 μm Jedi2 treatment was also included in the experiment (u6:emptygRNA: n = 6 animals, 16 DRG, 40 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells, u6:piezo1gRNA+Jedi2: n = 4 animals, 4 DRG, 7 cells). Scale bar is 10 μm (D, G, I, K). Statistical tests: unpaired t test (H, N, O), Fisher’s exact (C), multiple unpaired t tests (F). The data underlying this figure can be found in S1 Data. dpf, days post fertilization; DRG, dorsal root ganglia. https://doi.org/10.1371/journal.pbio.3002319.g002 If this response to mechanical force is mediated by mechanosensitive ion channels, we would hypothesize that it would be reduced upon treatment of GsMTx4, which broadly blocks mechanosensitive ion channels. To test this hypothesis, we imaged animals expressing either Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP), or Tg(neurod:gal4+myl7); Tg(uas:GCaMP6s) that were treated with GsMTx4. We found that treatment with GsMTx4 reduced the response to mechanical stimuli to 20% of animals (n = 5 DRG, 5 animals) expressing sox10+ GCaMP6s at 2 dpf (Fig 2C and 2E). At 3 dpf when treated with GsMTx4, there was a significant reduction in response with 0% of animals (n = 4 DRG, 4 animals) expressing sox10+ GCaMP6s responded to mechanical force and 25% of animals (n = 4 DRG, 4 animals) expressing neuronal GCaMP6s responded to mechanical force (sox10 3 dpf DMSO versus GsMTx4: p = 0.0110, neurod 3 dpf DMSO versus GsMTx4: p = 0.0476 Fisher’s exact test) (Fig 2C). Additionally, we investigated the effect of GsMTx4 treatment on 3 phases of this assay. We assessed the change in fluorescence in DRG expressing Tg(sox10:gal4); Tg(uas:GCaMP6s) during the resting state, compression state, and decompression state (Fig 2E). We define these phases as follows: resting phase is the initial fluorescence before compression with the needle, compression phase is when the needle is actively putting force on the animal, and decompression phase is when the needle has been released. We compared the average change in fluorescence during these phases between DMSO and GsMTx4-treated animals at 3 dpf expressing Tg(sox10:gal4); Tg(uas:GCaMP6s). We found that animals treated with DMSO had an average change in fluorescence of 0.003+/−0.006 during resting phase, an average change in fluorescence of 0.010+/−0.010 during compression, and an average change in fluorescence of 0.029+/−0.019 during decompression (n = 9 DRG, 9 animals). We found that animals treated with GsMTx4 had an average change in fluorescence of 0.006+/−0.006 during resting phase, an average change in fluorescence of −0.030+/−0.021 during compression, and an average change in fluorescence of −0.027+/−0.034 during decompression (n = 4 DRG, 4 animals) (Fig 2F). When comparing these phases between DMSO treated and GsMTx4-treated animals, we found a significant difference in the change in fluorescence during the compression and decompression phases (Compression DMSO versus Compression GsMTx4: p = 0.0005, Decompression DMSO versus Decompression GsMTx4: p = 0.0025, multiple unpaired t tests) (Fig 2F). These data support the hypothesis that DRG are responsive to mechanical forces and identify that sox10+ cells are mechanosensitive, at least partially independent of neuronal activity.

Satellite glia Ca2+ transients can be altered by manipulating Piezo1 We next explored the potential molecular determinant of this mechanical component. The mature DRG is known to express mechanosensitive channels Piezo1 and Piezo2; however, Piezo2 is restricted to neurons while Piezo1 is expressed in neurons and satellite glia in mice [31]. To investigate this in zebrafish, we utilized RNAscope to determine spatiotemporal distribution of piezo1 RNA in animals expressing Tg(sox10:meGFP). We found that 79% of DRG (n = 24 DRG, 8 animals) at 3 dpf contained piezo1 RNAscope puncta within sox10+ satellite glia (Fig 2I and 2J). Additionally, we utilized Whole-mount HCR-FISH targeting piezo1 RNA 3 dpf animals expressing Tg(sox10:meGFP) and found similar expression of piezo1 (S4 Fig). To test if DRG contain functional Piezo1, we treated animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s); Tg(neurod:tagRFP) with Yoda1 and Jedi2, known Piezo1 specific agonists (Fig 2K–2M) [48,49]. We found the average amount of Ca2+ transients per sox10+ cell in a 1-h time-lapse in DMSO controls was 2.69 ± 1.55 Ca2+ transient events, which was significantly less than the average 4.48 ± 1.58 Ca2+ transient events or 4.58 ± 2.54 Ca2+ transient events observed when treated with Yoda1 or Jedi2, respectively (DMSO n = 52 cells, 17 DRG, 8 animals, Yoda1 n = 33 cells, 9 DRG, 4 animals, Jedi2 n = 79 cells, 18 DRG, 7 animals) (DMSO versus Yoda1: p < 0.0001 unpaired t test, DMSO versus Jedi2: p < 0.0001 unpaired t test) (Fig 2N). Furthermore, we repeated this assay treating with GsMTx4 to investigate whether inhibiting mechanosensitive ion channels reduces spontaneous Ca2+ transients. This treatment significantly reduced the average amount of Ca2+ transients per sox10+ cell to an average of 2.05 ± 1.36 Ca2+ transient events (n = 44 cells, 16 DRG, 6 animals) (DMSO versus GsMTx4: p = 0.0342 unpaired t test) (Fig 2K–2N). One possible explanation for an increase in Ca2+ transients in sox10+ satellite glia is that the sox10+ satellite glia are active in response to neuronal activity. To investigate whether the observed change in Ca2+ transients in sox10+ satellite glia was a consequence of altered neuronal activity, we treated animals expressing Tg(neurod:gal4+myl7); Tg(uas:GCaMP6s) with Piezo1 agonists and quantified the amount of Ca2+ transients per DRG neuron. We found following DMSO treatment that DRG neurons exhibited an average of 2.42 ± 1.71 Ca2+ transient events per hour. When animals were treated with either Yoda1 or Jedi2, an average of 3.50 ± 2.39 or 2.57 ± 2.00 Ca2+ transient events per hour, respectively, could be detected. When animals were treated with GsMTx4, there was an observed 3.75 ± 1.67 average number of Ca2+ transient events (DMSO n = 33 neurons, 24 DRG, 10 animals, Yoda1 n = 8 neurons, 7 DRG, 4 animals, Jedi2 n = 35 neurons, 18 DRG, 5 animals, GsMTx4 n = 8 neurons, 8 DRG, 4 animals) (Fig 2O). Overall, we found that Piezo1 agonists did not contribute to an increase in Ca2+ transients in the neurod+ population. These data are most consistent with the hypothesis that sox10+ satellite glia display Ca2+ transients in response to Piezo1 agonists independent of an increase in neuronal activity. In addition to these pharmacological treatments, we also sought to do a genetic manipulation to identify if the endogenous Piezo1-mediated Ca2+ transient was present in satellite glia. We utilized a uas:cas9mkate-u6:piezo1gRNA construct designed with a verified piezo1 gRNA that produced genetic indels in 86% of animals that were injected and sequenced (S5 Fig) to knockout piezo1 in satellite glia [50]. This construct was injected into animals expressing Tg(sox10:gal4); Tg(uas:GCaMP6s). We then imaged animals expressing Tg(sox10:gal4); Tg(uas:GCaMP6s); uas:cas9mkate-u6:piezo1gRNA and quantified the average amount of Ca2+ transients in comparison with animals injected with uas:cas9mkate-u6:emptygRNA, a construct containing an empty gRNA cassette. We found in animals injected with uas:cas9mkate-u6:piezo1gRNA, there was an average of 1.579+/−1.117 Ca2+ transients (n = 57 cells, 13 DRG, 4 animals). This was significantly lower than the average amount of Ca2+ transients observed in uas:cas9mkate-u6:emptygRNA injected animals where there was an average of 2.500+/−1.536 Ca2+ transient events (n = 40 cells, 16 DRG, 6 animals) (p = 0.0028, post hoc Tukey test) (Fig 2P). Additionally, we injected animals expressing Tg(sox10:gal4); Tg(uas:GCaMP6s) with uas:cas9mkate-u6:piezo1gRNA and treated the animals Jedi2. These animals had on average 1.857+/−1.464 Ca2+ transients (n = 7 cells, 4 DRG, 4 animals) that was not significantly different from untreated uas:cas9mkate-u6:piezo1gRNA injected animals (p = 0.8578, post hoc Tukey test) (Fig 2P), supporting that this manipulation is specific to Piezo1. Interestingly, when comparing uas:cas9mkate-u6:emptygRNA injected animals with animals injected with uas:cas9mkate-u6:piezo1gRNA/ treated with Jedi2, we did not see a significant difference (p = 0.4602, post hoc Tukey test). This could be result of multiple different factors including the penetrance of our gRNA. Together, these findings support the idea that piezo1 contributes to spontaneous Ca2+ transients that are observed. We demonstrated that DRG satellite glial cells have distinct Ca2+ transients (Fig 1F–1H) but the underlying mechanism of those transients is unknown. We therefore tested if subtypes of Ca2+ transients were differentially modulated by Piezo1. To investigate the role of Piezo1 in isolated and simultaneous Ca2+ transient events, we quantified the effect of Yoda1, Jedi2, GsMTx4, and DMSO treatment on these Ca2+ transient events. In DMSO-treated animals, sox10+ cells displayed an average 0.46 ± 0.78 isolated Ca2+ transient events. When treated with Yoda1 or Jedi2, sox10+ cells displayed an average of 2.28 ± 1.72 or an average of 1.31 ± 1.22 isolated Ca2+ transient events, respectively. We found that when treated with GsMTx4, sox10+ cells displayed an average of 0.91 ± 1.2 isolated Ca2+ transient events (DMSO n = 24 cells, 5 DRG, 4 animals, Yoda1 n = 28 cells, 6 DRG, 3 animals, Jedi2 n = 59 cells, 18 DRG, 7 animals, GsMTx4 n = 23 cells, 5 DRG, 3 animals) (Fig 3A–3C). These results show that Piezo1 agonists significantly increased the amount of isolated Ca2+ transients (DMSO versus Yoda1: p < 0.0001, DMSO versus Jedi2: p = 0.0024 unpaired t tests), while mechanosensitive antagonists did not significantly decrease isolated Ca2+ transients (DMSO versus GsMTx4: p = 0.1294 unpaired t test) (Fig 3C). In contrast, simultaneous Ca2+ transient events were not significantly different in Yoda1 or Jedi2-treated animals compared to controls (DMSO n = 24 cells, 5 DRG, 4 animals, Yoda1 n = 28 cells, 6 DRG, 3 animals, Jedi2 n = 59 cells, 18 DRG, 7 animals, GsMTx4 n = 23 cells, 5 DRG, 3 animals) (Fig 3D). In the case of mechanosensitive antagonists, animals displayed a significant decrease in the number of simultaneous Ca2+ transient events compared to DMSO control when treated with GsMTx4 (DMSO versus GsMTx4: p = 0.0003 unpaired t test). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Piezo1 overactivation increases isolated calcium activity in DRG. (A) Confocal z-projection of DRG in 3 dpf animal expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) and treated with either 2% DMSO or 40 μm Jedi2. Individual cells are traced for ROIs and labeled with a number. (B) Line graphs of the z score of the integrated density of fluorescence over a 1 h period. Each numbered line graph corresponds to a numbered ROI. Red scale bar represents a z score of 2.58. Blue arrowheads note simultaneously active time points. Red arrowheads note isolated active time points. (C) Quantification of the average number of isolated Ca2+ transient events per sox10+ cell in 3 dpf animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) that were treated with either 2% DMSO, 1 μm GsMTx4, 100 μm Yoda1, or 40 μm Jedi2 (DMSO: 4 animals, 5 DRG, 24 cells, GsMTx4: n = 3 animals, 5 DRG, 23 cells, Jedi2: n = 7 animals, 18 DRG, 59 cells, Yoda1: n = 3 animals, 6 DRG, 28 cells). (D) Quantification of the average number of simultaneous Ca2+ transient events per sox10+ cell in 3 dpf animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) that were treated with either 2% DMSO, 1 μm GsMTx4, 100 μm Yoda1, or 40 μm Jedi2 (DMSO: n = 4 animals, 5 DRG, 24 cells, GsMTx4: n = 3 animals, 5 DRG, 23 cells, Jedi2: n = 7 animals, 18 DRG, 59 cells, Yoda1: n = 3 animals, 6 DRG, 28 cells). (E) Quantification of the average number of isolated Ca2+ transient events following genetic manipulation via CRISPR/Cas9 targeting piezo1 or empty gRNA cassette in 3 dpf animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) uas:cas9mkate-u6:piezo1gRNA or uas:cas9mkate-u6:emptygRNA (u6:emptygRNA: n = 6 animals, 14 DRG, 38 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells). (F) Quantification of the average number of simultaneous Ca2+ transient events following genetic manipulation via CRISPR/Cas9 targeting piezo1 or empty gRNA cassette in 3 dpf animals expressing Tg(sox10:gal4+myl7); Tg(uas:GCaMP6s) uas:cas9mkate-u6:piezo1gRNA or uas:cas9mkate-u6:emptrygRNA (u6:emptygRNA: n = 6 animals, 14 DRG, 38 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells). (G) Quantification of the average number of microdomains per DRG in 3 dpf animals expressing Tg(sox10:gal4+myl7); uas:GCaMP6s-caax that were treated with either 2% DMSO, 1 μm GsMTx4, or 40 μm Jedi2 for 30 min prior to imaging (DMSO: n = 7 animals, 7 DRG, GsMTx4: n = 7 animals, 7 DRG, Jedi2: n = 6 animals, 8 DRG). (H) Quantification of the average duration of microdomains in 3 dpf animals expressing Tg(sox10:gal4+myl7); uas:GCaMP6s-caax that were treated with either 2% DMSO or 40 μm Jedi2 for 30 min prior to imaging (DMSO: n = 8 animals, 8 DRG, Jedi2: n = 5 animals, 7 DRG). Scale bar is 10 μm (A). Statistical tests: unpaired t test (C, D, E, F, H), one-way ANOVA followed and represented by post hoc Dunnett test (G). The data underlying this figure can be found in S1 Data. dpf, days post fertilization; DRG, dorsal root ganglia; ROI, regions of interest. https://doi.org/10.1371/journal.pbio.3002319.g003 To complement the pharmacological approach, we also quantified these distinct Ca2+ transient events in genetic manipulations following the injection of uas:cas9mkate-u6:piezo1gRNA and uas:cas9mkate-u6:emptygRNA. Animals injected with the empty-gRNA had an average of 1.132+/−1.474 isolated Ca2+ transient events and an average of 1.342+/−0.7453 simultaneous Ca2+ transient events at 3 dpf (n = 38 cells, 14 DRG, 6 animals) (Fig 3E and 3F). Animals injected with the piezo1-gRNA had an average of 0.4912+/−0.7820 isolated Ca2+ transient events and an average of 1.088+/−0.9688 simultaneous Ca2+ transient events at 3 dpf (n = 57 cells, 13 DRG, 4 animals) (Fig 3E and 3F). We found there was a significant reduction in the number of isolated Ca2+ transient events in animals injected with uas:cas9mkate-u6:piezo1gRNA compared to uas:cas9mkate-u6:emptygRNA (p = 0.0071, unpaired t test). The average number of simultaneous Ca2+ transient events were not significantly different following these manipulations (p = 0.1740, unpaired t test). Together, the pharmacological and genetic manipulations support the hypothesis that Piezo1 contributes specifically to isolated Ca2+ transients. We also quantified the number of Ca2+ microdomains following manipulations of Piezo1 in Tg(sox10:gal4+myl7) animals injected with uas:GCaMP6s-caax. In DMSO-treated animals, sox10+ cells displayed an average number of 0.14 ± 0.38 Ca2+ microdomains at 3 dpf. Animals treated with Jedi2 displayed an average number of 1.38 ± 0.92 Ca2+ microdomains at 3 dpf. When animals were treated with GsMTx4, we observed an average of 0.14 ± 0.38 Ca2+ microdomains at 3 dpf (DMSO n = 7 DRG, 7 animals, Jedi2 n = 8 DRG, 6 animals, GsMTx4 n = 7 DRG, 7 animals) (Fig 3G). Similar to isolated Ca2+ transients, we found a significant increase in the average number of Ca2+ microdomains per DRG when animals were treated with a Piezo1 agonist (DMSO versus Jedi2: p = 0.0025 post hoc Dunnett test) (Fig 3G). We also quantified the average duration of the identified Ca2+ microdomains and did not find a significant difference (Fig 3H). These additional findings suggest that Piezo1-mediated mechanical forces contribute to the number of observable Ca2+ microdomain events in addition to isolated Ca2+ transient events.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002319

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