Endothelin-3 and T-type Ca^2+ channels drive enteric neural crest cell calcium activity, contractility and migration

Domain: neuroscience
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
Enteric neural crest cells (ENCCs) colonize the gut during embryogenesis and migration defects give rise to Hirschsprung disease (HD). Mutations in GDNF/RET and EDN3/EDNRB are known to be causal in HD. Here, we show that migrating ENCCs in mice exhibit endogenous EDN3/EDNRB-gated calcium activity, mediated by chloride channels, T-type Ca2+ channels and inositol trisphosphate-sensitive intracellular-store release. We find that inhibiting Ca2+ activity results in ENCC migration defects, while exciting it promotes migration by increasing ENCC contractility and traction force to the extracellular matrix. Our study demonstrates that embryonic endothelin-mediated neural crest migration and adult endothelin-mediated vasoconstriction is one and the same phenomenon, taking place in different cell types. Our results suggest a functional link between rare mutations of CACNA1H (the gene encoding CaV3.2) and HD, and pave the way for understanding neurocristopathies in terms of neural crest cell bioelectric activity deficits.

Introduction
Neural crest cells (NCC) are a population of multipotent, highly migratory cells common to all vertebrates. They colonize developing organs during embryonic development to give rise to a variety of structures including cranio-facial bone, chromaffin cells of the adrenal medulla, Schwann cells of the peripheral nervous system and the neurons and glia of the enteric nervous system (ENS)1. NCCs have attracted much scientific attention because of their paramount contributions to vertebrate physiological and pathological2,3 development, to domestication-induced phenotypes4,5 and because aberrantly expressed NCC developmental programs can lead to cancer tumor invasiveness6,7. Vagal-derived8 enteric neural crest cells (ENCCs) invade the gut rostro-caudally between 9.5 and 13.5 days of development in mice to form the ENS. ENCC migration defects result in colonic aganglionosis at birth, a syndrome known as Hirschsprung disease (HD). HD is one of the most frequent neurocristopathies, affecting ~1:5000 births9. The mutations hitherto identified, mostly in the GDNF/RET10–12 and EDN3/EDNRB13,14 signaling cascades, are of incomplete penetrance and account for only 50% of the familial and 15-20% of the sporadic cases15: the cause of most HD cases is today not understood. Bioelectric activity16–18, as measured by Ca2+ imaging or membrane potential changes, is increasingly recognized as a key player of stem cell16–18 and NCC19–22 behavior. Spontaneous propagating Ca2+ waves in ENCCs23 were found to depend on purinergic signaling, but their implication for migration and HD remained uncertain. Here, we show that most of the Ca2+ activity in ENCCs occurs as unsynchronized single-cell events and not as waves, that ENCC Ca2+ activity is driven by EDN3/EDNRB, via the opening of Cl- and T-type Ca2+ channels, and that Ca2+ activity is intimately correlated with ENCC migration potential through a simple mechanism: elevated intracellular Ca2+ leads to increased ENCC contractility and traction force to the extracellular matrix that allows them to migrate down the gut. This mechanism renews our understanding of how EDN3/EDNRB affects ENCC migration. Disruption of any components of this mechanism can result in a NCC migration defect, opening-up new perspectives for neurocristopathy and collective cell migration research.

Results
Enteric neural crest cells display spontaneous Ca2+ activity during gut invasion

We monitored Ca2+ activity (CA) on ex-vivo mouse embryonic guts expressing the intracellular Ca2+ reporter GCaMP6f specifically in NCCs (Fig. 1a, Supplementary Movie 1), and measured its spatial distribution and characteristics with an automated analysis pipeline (Fig. S1). CA in migrating ENCCs was spontaneous and occurred mostly as asynchronous, non-propagating, single-cell events. Ca2+ transients could sometimes propagate radially to neighboring cells, but such wave-like events23 occurred, at E11.5, at an activity of CA = 7.5 × 10-4 ± 2 × 10-4 wave events/min/100µm² (±SEM, n=15), i.e. ~100-1000 times less frequently than single-cell events (Fig. 1b). Endogenous CA at the ENCC migration front decreased from E10.5 to E12.5 (Fig. 1b, Fig. S2, Supplementary Movie 1). At E11.5, it was significantly higher at the ENCC wavefront located at the ileo-cecal junction (ilcc) than in trailing ENCCs (Fig. 1c), concentrating at the cecum and anti-mesenteric ileum border (Figs. 1a and S3). CA was also present in transmesenteric ENCCs (Fig. S3). CA was insensitive to tetrodotoxin (Figs. 1d and S4), and therefore did not stem from neural activity; the latter could however be elicited by veratridine in proximal gut segments (Figs. 1e and S4). Endogenous CA co-localized with the ENCC-specific marker Sox10, but only very partially with the neuronal marker Tuj1 (Supplementary Movie 2). These results indicate that spontaneous CA originated in ENCCs and was most intense during early gut colonization stages, decreasing as ENCCs gradually differentiated to neurons and glia.

Endogenous Ca2+ activity is induced by EDN3/EDNRB

We next investigated whether the ligand/receptor pair EDN3/EDNRB involved in ENCC development was related to ENCC electric activity. Application of 1nM EDN3 at E11.5 led to a threefold CA increase (Supplementary Movie 3, Figs. 2a and S5), while further addition of EDN3 had more variable effects, presumably caused by the exhaustion of Ca2+ reservoirs upon repeated stimulation with EDN3. In stage E12.5 ileum, CA also increased threefold at 1nM EDN3 (Fig. S5), and the application of 10nM EDN3 induced an immediate increase of intracellular Ca2+ across all ENCCs (Fig. 2b, Supplementary Movie 3, Fig. S5). Unlike EDN3, GDNF, the other major ligand involved in ENCC development, did not modify CA at the 10ng/mL concentration known to drive ENCC chemotaxis24 (Figs. 2c and S6). EDN3 is endogenously expressed by the gut mesenchyme, concentrating at the cecum and at the anti-mesenteric ileal border25,26. This pattern mirrored the CA heatmaps we recorded (Figs. 1a and S3). We therefore blocked endogenous EDN3/EDNRB signaling with the EDNRB antagonist BQ788: it almost extinguished CA (−77 ± 14%, n=14) (Figs. 2d and S5, Supplementary Movie 4) for at least 24h (Fig. 2e), while the drug vehicle, DMSO, did not affect CA (Fig. S7). In addition, BQ788 systematically led to a rounding of ENCCs and a retraction of their cell processes (Fig. 2f, Supplementary Movie 4). We finally tested whether CA differed in an EDN3 missense mutation model that develops Hirschsprung disease, the ls/ls mouse. Because this mouse didn’t express GCaMP, we resorted to 1 day on-substrate-culture of intestinal explants in medium with GDNF and loaded the preparation with Fluo4-AM. The average frequency of ENCC Ca2+ transients was lower by ~25% in the ls/ls homozygote than in controls (Fig. 2g-i).

T-type Ca2+ channels and Cl- channels mediate Ca2+ oscillations

We next investigated the ionic transport mechanisms leading to Ca2+ oscillations downstream of EDNRB. Removing extracellular Ca2+ with EDTA led to a complete cessation of CA (Fig. 3a, Supplementary Movie 5). Consistent with the requirement for extracellular Ca2+, GdCl3, a cation channel blocker, significantly reduced CA (Figs. 3a and S8). Interestingly, transients could still be elicited by acute administration of EDN3 10nM in the presence of EDTA (Figs. 3e and S9, Supplementary Movie 5), indicating a contribution of intracellular Ca2+ stores. 2-APB halted activity (Figs. 3a and S8) while ryanodine did not affect CA (Figs. 3a and S8). These findings show that both extracellular Ca2+ and intracellular IP3-, but not ryanodine-sensitive Ca2+ stores contribute to CA.

We further questioned the entry pathway of extracellular Ca2+, first considering voltage-gated Ca2+ channels (VGCCs). The L-type Ca2+ channel (CaV1.2) blocker nifedipine inhibits activity in fetal and adult ENS neurons27 but did not significantly alter CA in E11.5 guts (Figs. 3b and S10). Ruthenium red, a non-selective blocker of P/Q-type (CaV2.1) and N-type (CaV2.2) channels as well as of cationic TRPV channels, did not affect CA either (Figs. 3b and S10). Blockade of all 3 T-type Ca2+ channels (CaV3.1, 2, 3) with Z944 induced a dose- and time-dependent decrease of CA (Figs. 3b, S10, and S12, and Supplementary Movie 6). CaV3.2 (encoded by Cacna1h) is strongly expressed in E11.5 ENCCs28; we found that specific blockade of CaV3.2 with ascorbic acid (AA) 1mM29 also induced a dose- and time-dependent decrease of CA (Figs. 3b, S10, and S12, and Supplementary Movie 6). The only commercially available T-type Ca2+ channel agonist, SAK3, is specific for CaV3.1 and CaV3.330. This molecule induced a significant CA increase at 10µM (Figs. 3b and S10, and Supplementary Movie 6), suggesting that all three T-type Ca2+ channels are involved in ENCC CA. IHC for CaV3.1, CaV3.2 and CaV3.3 (Fig. 3d-f) showed that all three channel types were indeed expressed by ENCCs (Sox10+ cells), but not by the surrounding mesenchyme. SAK3 also led to a significant CA increase post-EDNRB blockade with BQ788, but CA remained well below physiological levels, even after 1 day culture (Fig. S14).

We next targeted Cl- channels by applying three different classes of inhibitors: 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid (NFA) and 4,4’-Diisothiocyanato-2,2’-stilbenedisulfonic acid (DIDS). These compounds led to a drastic CA decrease (Figs. 3c and S11, and Supplementary Movie 7) and a retraction of cell processes (Supplementary Movie 7). Transient CA could also be elicited by EDN3 10µM after 15min incubation in 100µM NPPB, but the transients had low amplitude (Fig. S9). NPPB and DIDS systematically induced ENCC death after 24h (Fig. S15). NFA 50µM inhibition kinetics were similar to Z944 50µM (Fig. S12): CA had recovered after 24h, ENS morphology was normal, and the fraction of dead ENCCs similar to control samples (Fig. S16). Cl- channels likely contribute to Ca2+ transients by depolarizing the cell membrane through Cl- efflux, thereby opening VGCCs31,32. This idea is in line with the fact that the membrane depolarizer 4-AP markedly increased CA (Fig. S13). 4-AP could not however re-stimulate CA post-EDNRB block (Fig. S14c), and also induced cell death after 24h (Fig. S15).

The purinergic pathway is an important actor of ENCC multicellular Ca2+ waves23. We found that the ATP enzymatic degradation inhibitor ARL 67156 and exogenous ATP could transiently increase wave activity after EDNRB blockade (Fig. S14d,e). Higher ATP concentrations could even result in a network-spanning intracellular Ca2+ rise (Fig. S14d and Supplementary Movie 8). These effects were however not sustained in time (Fig. S14e), indicating that the purinergic pathway could not bypass the main regulator EDN3/EDNRB.

Ca2+ activity correlates with ENCC migration speed

We next investigated how pharmacological upregulation of CA by EDN3 or SAK3 affected ENCC migration from E11.5 midgut explants embedded in a 3D collagen gel. We found that both EDN3 1nM and GDNF 10ng/mL increased migration distances from the explant compared to control conditions, and that they acted synergistically when combined (Fig. 4). Strikingly, we found that the CA activator SAK3 was able to mimic the effect of EDN3, i.e. migration distances were similarly high in GDNF + SAK3 10µM and GDNF + EDN3 1nM conditions (Fig. 4).

We next assessed the effect of CA inhibitors on ENCC migration in the colon in full E11.5 guts cultured for 1 day in agarose (a gel not permissive to migration). We found that the four conditions that significantly and long-lastingly (Figs. S12 and 2e) lowered CA - Z944 50µM, NFA 50µM, AA 1mM and BQ788 10µM - all decreased ENCC migration distances in the colon (Fig. 5a, b). These effects could not be attributed to toxicity, as cell death in most (n=49/54) samples was on the same level as control samples (Fig. S16). Addition of SAK3 to BQ788 did not improve ENCC migration down the colon, consistent with the fact that CA was still very low in these conditions (Fig. S14).

These results show that, in addition to the well-known inhibitory effect of BQ788 on ENCC migration33,34, Cl- channel and T-type VGCC inhibition also result in a migration defect leading to colonic aganglionosis.

Ca2+ activity drives cell contractility and traction force to the extracellular matrix

ENCCs exert a traction force on the tissue as they migrate24, visible in-situ as a backflow of mesenchyme (Supplementary Movie 9). Several observations indicated that Ca2+ oscillations could modulate this traction force by influencing cell contractility, as in smooth muscle: 1°) CA blockers led to process retraction and rounding (Fig. 2f, Supplementary Movie 4, 7), indicating a loss of ENCC-extracellular matrix (ECM) traction; 2°) In a 2D migration assay at x60 magnification, we noticed that Ca2+ transient could induce abrupt lamellipodium detachment from the substrate (n=4 such events observed, Fig. S17a, Supplementary Movie 10). This could be driven by actomyosin or by calpain activity35; 3°) Most Ca2+ transients (79/102) in this assay were correlated with an abrupt change of nucleus speed, as measured by deep-learning assisted tracking and by kymographs (Fig. S17b-c, Supplementary Movie 11).

To quantify the force exerted by ENCCs on the ECM, we let ENCCs migrate from E11.5 midgut explants in a collagen gel seeded with fluorescent beads that served as fiducial markers of gel deformation (Fig. 6a inset). The traction force on the collagen scaffold induced bead displacements towards the migration front (Fig. 6, Supplementary Movie 12). Immediately after induction of CA by addition of EDN3 1nM, the bead speed abruptly increased (Fig. 6, Supplementary Movie 12), and the tip of the migration “fingers” accelerated (Supplementary Movie 12). When EDNRB was blocked by BQ788, the beads suddenly relaxed, indicating a sudden loss of ENCC-ECM traction force (Fig. 6a, b, d, Supplementary Movie 12), and the migration fingers retracted (Supplementary Movie 12). The effect of the T-type Ca2+ channel blocker Z944 was similar to that of BQ788, although the decrease in traction force was less pronounced (Fig. 6c, e). This experiment shows that, similarly to endothelin-induced smooth muscle contraction, EDN3 could stimulate Ca2+-dependent actomyosin, increasing the ENCC-ECM traction force necessary for migration, whereas blocking EDNRB or T-type Ca2+ channels relaxed it.

Methods
Ethics

Mice were hosted at the Institut Jacques Monod (GCaMP) and at the Institut Imagine LEAT (ls/+ heterozygotes) animal husbandries. They had access to housing, food and water ad-libitum. Temperature in the husbandry was 20–24 °C and humidity in the range 45–55%. The mice were exposed to light from 7 am to 7 pm. Females were kept in common cages with a maximum of 5 females per cage. For mating, the male is introduced in the evening and removed in the morning after detection of plugs in the morning. Pregnant mice were killed by cervical dislocation to retrieve embryos age E10.5 to E12.5. The embryos were separated and immediately beheahed with surgical scissors. The methods used to kill the mice conform to the guidelines of CNRS and INSERM animal welfare committees. Killing of mice for retrieval of embryosis a terminal procedure for which neither CNRS or INSERM assign ethics approval codes hence none are given here.

Mouse gut samples

The Cre reporter mice C57BL/6N-Gt(ROSA)26Sortm1(CAG-GCaMP6f) Khakh/J mice is referred to as Gcamp6fl/fl. A transgenic mouse line in which the transgene is under the control of the 3-kb fragment of the human tissue plasminogen activator (Ht-PA) promoter Tg(PLATcre) 116Sdu16 is referred to as Ht-PA::Cre. Gcamp6fl/fl males were crossed with Ht-PA::Cre females to generate embryos carrying the calcium fluorescent reporter in neural crest cells and their derivatives27. The gut of each embryo was dissected in PBS with 1mM Ca2+, 0.5mM Mg2+ and 1% penicillin-streptomycin, from stomach (cut at the oesophagus-stomach junction) to colon (cut at the colon-anus junction). The mesentery of E11.5 guts was kept intact. 57% (118/207 for which the full count was performed) of the embryos expressed GCaMP specifically in NCCs, while 43% expressed GCaMP in all cells, with the mesenchymal signal being dominant67; these phenotypes could be easily recognized during confocal imaging. We do not know the reason of this partial specificity of GCaMP. Only embryos expressing GCaMP in NCCs were used for Ca2+ imaging. Ubiquitously GCaMP expressing embryos, which were otherwise morphologically normal, were used for experiments where Ca2+ imaging was not required.

Heterozygous ls/+ animals were crossed and embryos retrieved at E11.5. DNA extraction and subsequent genotyping were performed from head biopsies of each embryo, using the direct PCR lysis reagent (Viagen). Genotyping was performed using primers (Eurogentec, Belgium) m-Edn3lsF GACTGTGCCCTATGGACTGT and m-Edn3lsR GGTGACATCTCTGGTGCGTG68,69.

Organ culture and Ca2+ imaging

After dissection, each gut was placed on a pre-solidified 0.5mL layer of 1% low-melting point agarose (Condalab 8050.11, dissolved in PBS with Ca2+ 1mM and Mg2+ 0.5mM), in a 35mm diameter Petri dish (Greiner). This gel layer prevents adhesion of the gut and subsequent ENCC migration on the dish bottom, confining it to the organ. The gut was then immobilized by pouring additional 0.5mL of liquid agarose on top and letting it gel at 4°C for 10 minutes. Capillary forces of the shallow liquid agarose layer gently press down the gut, most of the time positioning the stomach, midgut and colon in a horizontal plane that was optimal for microscopy. Immobilization is crucial both for Ca2+ imaging and to prevent shrinking/retraction of the sample during culture. The 1mL gut & gel were covered by 2mL of complemented DMEM:F12 Glutamax (Gibco 31331-028). Final concentrations after diffusion and dilution of the medium in the PBS-based agarose were Ca2+ 1mM, Mg2+ 0.5mM, penicillin-streptomycin 1%, Fetal Veal Serum 6.6%, while all other molecules that are present in DMEM:F12 but not in PBS (e.g. glucose, amino acids etc.) had their concentration divided by a factor 2/3 compared to DMEM:F12 alone. Samples were incubated at 37°C in a 5% CO 95% air atmosphere for at least 45min before imaging.

Calcium imaging was performed on an inverted spinning-disk microscope (Olympus IX-81, Yokogawa CSU-X1) equipped with an ILE laser-base (Andor) and a Zyla camera (Andor, resolution 1392×1040 pixel). The GCaMP signal was excited at 488nm and the emission filtered at 497-527nm. Time-lapse videos were recorded at x10 magnification, 400ms exposure time, 1Hz acquisition rate, for at least 3min, and often longer depending on the type of experiment. We did not observe any illumination-induced bleaching or alteration of the sample. Sample basal (endogenous) Ca2+ activity was first recorded, and the position of the ENCC wavefront determined by a z-stack at the level of the ileo-caecal junction (E11.5). Drugs were then added from stock solutions directly in the Petri dish, far from the sample, without displacing it, and homogenized by up & down movements with a 1mL pipette. Products used in this report as well as solvent and stock concentration are listed in Fig. S18. In preliminary experiments, addition of the drug was performed during the time-lapse to capture potential immediate effects. For most drugs, CA was then recorded 10-15min post-administration. After imaging, samples were placed back in the incubator for further culture. Time-lapse and z-stack imaging was performed after 24h to assess ENCC wavefront position and CA. Some samples were imaged multiple times during the culture period to assess drug kinetics (Fig. S12). Some samples were fixed and processed for IHC as described below.

The experiment with ls/ls embryos required that the ENCCs migrate out of the intestine because Fluo4AM does not penetrate inside the tissue. We achieved this by placing E11.5 explants on 35mm Greiner Petri dishes and covering them with a thin meniscus of collagen gel (0.5mL, 1mg/mL, as described below) to hold them down against the Petri surface. After gelling, the preparation was topped with 2mL complemented medium with GDNF 10ng/mL, cultured for 1 day, loaded with 1.8µM Fluo4-AM for 5min, and 3min timelapse movies were recorded in 3 different locations per sample. The samples were immediately fixed after calcium imaging for IHC.

Ca2+ activity analysis

Analysis of CA was performed using custom-written ImageJ 1.54r macro and Matlab R2018a scripts, and is synthesized in Fig. S1. Briefly, a fixed mesh comprised of 2745 square regions-of-interest (ROIs), each 250µm², was overlayed on the 8-bit video. A matrix of the average pixel intensity over time in each square was retrieved and input into Matlab for peak detection. The “findpeaks” function was applied, specifying, to filter out noise, a minimum peak prominence of 2 (8-bit pixel intensity unit), a minimum peak width of 3 sec, a minimum inter-peak time of 3 sec. Illumination and peak filtering settings were identical for all videos acquired, allowing quantitative comparison of GCaMP signal intensity across samples. We measured in each ROI the number of peaks, and, for ROIs in which there was at least one peak, the frequency (number/acquisition time), average duration (width at half-maxima) and average intensity ratio I/I0; the data of all ROIs was collected to generate heatmaps of these variables, assess their spatial distribution, and compute their spatial average. To reflect the general activity of the region imaged, we defined the Ca2+ activity CA = N peaks / (A cells * T), where N peaks is the total number of transients in a given video, A cells the total area of the GCaMP positive cells within the field of view and T the duration of the acquisition. A cells was determined by first time-projecting the average intensity to obtain a crisp image of the ENCC network, and by then applying iteratively stricter Bernsen local thresholds under ImageJ, stopping just before it diverged (Fig. S1e). A cells should remain constant for an experiment with a fixed z and field of view. Because our procedure yielded some variability depending on Ca2+ basal level and activity, we selected for a given experiment the biggest A cells and applied it to all other conditions of this experiment (e.g. before/after drug). CA was expressed in units of events/100µm²/min; because a cell has typically an area of 100µm², CA has values lying in the same range as frequencies expressed in cycles-per-minute (cpm).

For ls/ls samples, we followed the same procedure but used a finer mesh of 86µm² (9890 ROIs in total) to gain resolution to differentiate ENCC from mesenchymal CA. After registration of Sox10 & α-SMA IHCs with frequency heatmaps, we drew ROIs around Sox10 positive aggregates using the ImageJ ROI manager, and then measured the average pixel intensity of these ROIs on the greyscale frequency heatmap. The average frequency was obtained by weighing the measured frequency of each aggregate by its area (i.e. bigger aggregates contributed more to the average). Area-weighted frequency and CA are proportional; we present the former in Fig. 2i because the finer mesh size used results in a higher event count compared to the standard mesh used in the remainder of the study.

Collagen gel organ culture

Collagen gels (Figs. 4 and 6) were prepared from Cultrex Rat Collagen I (Bio-Techne) at 1mg/mL on ice, following the manufacturer’s guidelines. For 3D contractility experiments (Fig. 6), fluorescent deep-red 0.2µm diameter spheres (Thermofisher, F8807) were added to the mixture at 1:100000 dilution to serve as fiducial tracers. Midgut segments from E11.5 embryos were cut in 2-3 pieces with micro-scissors and each segment was embedded in 1mL of liquid collagen, that gelled after incubating at 37°C for 45min. 2mL of complemented medium with 10ng/mL GDNF were then added. Collagen gel migration was assessed after culture for 3 days at 37°C, in a 5% CO2 – 95% air humidified atmosphere. We measured the area S1 occupied by the gut explant and the ENCC halo, the area S2 occupied by the gut explant alone, and computed the average migration distance from the explant as S = sqrt(S1/π) - sqrt(S2/π). This approach yielded an average radius which took into account halo asymmetries.

Immunohistochemistry

For registration of CA with IHC (Supplementary Movie 2, Fig. 2g, h), guts were fixed in 4% PFA in PBS for 20min, washed 3 times, then blocked and permeated in 1% BSA and 0.1% triton in PBS for 1h, immersed in 1:200 mouse monoclonal Anti-SOX10 (Sigma, AMAB91297) for 24h, washed 3 times, immersed in 1:500 anti-mouse AF647 secondary antibody with 1:500 βIII-tubulin FITC conjugated antibody (Abcam 224978) or 1:500 α-SMA Cy3 conjugated antibody (Sigma C6198) for 24h, washed 3 times in PBS, and imaged in the same location as during calcium imaging. Slight translation and rotation registration corrections between the CA time-lapse movie and the IHC were performed manually using the GIMP software.

For IHC of T-type Ca2+ channels (Fig. 3d-f), we used explants from the 3D contractility assay (see below). Some of these explants touched the bottom of the Petri-dish and ENCCs can, in addition to the 3D halo, also migrate on the dish surface, as a 2D sheet, which facilitated imaging. After fixation, blocking and permeation, samples were incubated for 1 day in 1:200 mouse Anti-SOX10 and 1:100 rabbit anti CaV3.1 (Alomone Labs #ACC-021) or 1:100 rabbit anti CaV3.2 (Alomone Labs #ACC-025) or 1:100 rabbit anti CaV3.3 (Alomone Labs #ACC-009) for 24h, washed 3 times, incubated in secondary 1:500 anti-mouse Cy3 and 1:500 anti-rabbit AF647 antibody for another day, washed and imaged. T-type Ca2+ channel IHC was only successful when performed on fixed cells that had been induced to emigrate from an explant, without embedding or cutting; we did not observe any specific signal when native E11.5 guts were fixed, cryo-sectioned and labeled, most probably owing to degradation of the proteins when performing these steps.

2D migration assay

For the x60 experiments (Fig. S17), a E11.5 GCaMP midgut explant was cultured on a Petri dish with a culture surface-treated polymer coverslip bottom (Ibidi 81156), immobilized with a 500µL meniscus of 1% low-melting point agarose gel, and cultured one day in complemented medium with 10ng/mL GDNF. Numerous ENCCs migrated out on the coverslip and were imaged at x60 magnification at 1Hz, for 10 to 30min. Cell nuclei appeared as dark spots surrounded by brighter cytoplasm. They were tracked using the deep-learning Segment Anything Model 2 (SAM2) developed by Meta and implemented under QuPath70. Nucleus boundary and centroid tracking were very precise (Supplementary Movie 11). Nucleus centroid position was plotted together with the GCaMP signal obtained from ROI intensity measurements of the cytoplasm of the tracked cell. Kymographs were obtained with the ImageJ Reslice function performed along a rectangle encompassing the cell trajectory.

3D contractility assay

3D contractility experiments were performed after 1-2 day of culture in collagen gel seeded with beads (see above), at x20 magnification, acquiring z-stacks in the bulk of the gel in GFP (cells) and AF647 (beads) with a step of 2µm over a thickness of 30-50µm every minute for up to 120min. Drugs were added during the time-lapse and mixed very carefully to not perturb bead positions. The z & t stacks of the cells (GFP) and beads (AF647) were first zmax-projected to yield t-stacks (time-lapse). The bead t-stack was flat-field corrected (Biovoxxel toolbox under Fiji), smoothed (Gaussian blur radius 2), thresholded (Li Auto Threshold) and the initial bead positions were measured using the ImageJ Analyze Particles tool. These initial coordinated were fed into the Tracker plugin (developed by our colleague O. Cardoso) to yield the coordinates of each bead at each time point. Inconsistent tracking was filtered out by applying conditions on the maximum rate of displacement. The average displacement of beads from their initial position towards the ENCC migration front was computed.

Statistics and Reproducibility

All sample numbers indicated in this report correspond to different embryos (guts, biological replicates). Except for ls/ls experiments which were performed on 2 litters, data for all other experiments was collected from at least 3 different litters and technically replicated at least 3 times, i.e. they were performed on 3 different days with fresh samples following the same procedure each time. Experiments on ls/ls were performed blindly, i.e., the genotype was known after data collection and analysis. In all other experiments, blinding was not possible and not relevant to this study. The sample size range is n=5-23 and was determined according to the type and variability of experiment results. p-values reported correspond to statistical tests mentioned in the figure legends. All instances of the WMP test are two-tailed.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Essential data generated or analyzed during this study are included in the manuscript and supporting files. Source data are provided with this paper. Other non-essential data are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

Essential codes for calcium imaging analysis are provided as supporting files.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Chevalier et al. analyzed in mouse embryos how calcium activity, regulated by EDN3/EDNRB signaling, influences the migration of enteric neural crest cells migration of enteric neural crest cells (ENCCs), and what is its role in the pathogenesis of Hirschsprung disease (HD)? The authors applied multiple organ cultures and migration assays combined with calcium activity analysis, calcium imaging for enteric neural crest cells, and immunocytochemistry to characterize the developing ENS. Greater understanding of the ENS and emphasizing the complex molecular and mechanobiological pathways during ENCC differentiation is a great value to both the congenital neurointestinal diseases and the enteric neural crest biology field. The presented findings provide mechanistic insight into how calcium waves are required for the ENCC migration and differentiation in the developing hindgut.\n\nThe manuscript's topic is interesting and important in the context of hindgut ENS development. However, there are several major concerns in the present manuscript.\n\nInclude anti-Anti-CaV3.2 (CACNA1H) staining or in situ hybridization to demonstrate the expression of Ca2+ channels on E11.5 wavefront ENCCs.\n\nAccording to Hirst et al (2015) ENCCs do not require ion channel activity to migrate through the gut mesenchyme. Explain why your results is different.\n\nIf the 1mM ascorbic acid (specific CAV3.2 blocker) strongly reduced CA, how does the ascorbic acid affect ENCC migration? Why the effect of ascorbic acid on ENCC migration is missing from your results (see Fig. 3f).\n\nLine 23: This statement is not supported by your results: “CaV3.2 channel blockade results in an ENCC migration defect”. It is not proven that the HD-type aganglionosis was caused by the blockade of the CaV3.2 channel.\n\nDid you see CA in transmesenteric ENCCs?\n\nLine 58: typo “could at times”\n\nLine 62: migration front versus more proximal regions terminology should be changed according to Stavely et al., 2023; Development and Zhou B et al., 2024 to wavefront (these are the leading front of the ENCDCs) and trailing cells (these are the stationary ENCDCs) see also Druckenbrod and Epstein, 2007.\n\nLine 61-62: There is no apparent difference in the localization of CA and the immunoreactivity of Sox10 and Tuj1, likely due to technical difficulties with the Sox10 immunostaining. To better distinguish between Sox10 and Tuj1 expression, the authors should focus on the cecum, where the Sox10+/Tuj1- ENCDC wavefront is located at this developmental stage.\n\nFig1: What is the red color on image “a”? Tuj1 or Sox10? Add to the legend.\n\nFig S3: add proper labeling (midgut, hindgut, cecum) to mark the orientation of the embryonic gut samples\n\nFig S3: Are the different E11.5 samples in the same orientation?\n\nLine 82 and 85. Why was it important to change the concentration (10 fold) of EDN3 at stage E11.5 (1nM) to 10nM at E12.5?\n\nVideo 4: Can you mark the cells that retract their processes after BQ788 treatment?\n\nFig 2f: include high power images (inset) to show the morphology of the CTRL and BQ788-treated cells.\n\nLine 128: add the effect of ascorbic acid to Fig 3f.\n\nVideo S9: The time-lapse movie is too short to see the backflow of mesenchyme.\n\nLine 148: According to Barlow et al 2003 and Nagy and Goldstein 2006, EDN3 inhibits the migration of ENCCs in response to GDNF. Explain why your data using mouse ENCCs is different from the previously published results. What was the explant: midgut, ceca or hindgut?\n\nLine 149 and Fig 3d. Include GDNF alone to prove the positive effect of 1nM EDN3+10ngGDNF on the ENCC migration distance.\n\nLines 169-170: The following sentence is not clear: “Several observations….”\n\nLine 171: It is just a speculation that the effect of the CA blockers indicates a loss of ENCC-ECM traction.\n\nLine 180-186: it is not related to the Results section.\n\nFig. 4. What are the green cells on a,b,c images? Are they isolated ENCCs or cells emigrated from the gut explants in response to GDNF?\n\nFig. 4: This figure is poorly presented in the Results section. a,b, c images are not introduced properly in the text.\n\nline 201: “ENCCS”\n\nTo increase the quantity of their work, the authors should include a more sensitive ENCC culture assay (e.g., culture of isolated Wnt1-GCaMP 11.5 or 12.5 dpc mouse intestine, either with FACS sorting or induce to emigrate from the gut explant in response to GDNF) to study with high-power microscopy and prove that modulation of Ca2+ activity affects the ENCC migration and cell contractility. You may include phalloidin staining to mark the cell skeleton of the cytoplasmic processes.\n\nFig.5. it is not clear why the schematic figure is important for the Discussion section.\n\nWhat is the relationship between β1-integrin signalling pathway, Ca2+-dependent cell adhesion molecules and Endothelin 3 activation or inhibition during ENCC migration?\n\nThe aganglionic segment of EDN3 mutant human and mouse is restricted to the distal colorectum. Compare Ca2+ frequency of the trailing and wavefront ENCCs in control and ls/ls E12.5 (dpc) mouse hindgut to demonstrate that CA is critical for hindgut ENCC migration and distal colorectum ENS formation.", "reviewer_2": "The manuscript by Chevalier et al is a well written and nicely illustrated description of a series of studies of murine embryonic gut explants which demonstrate a role for endothelin-3 (Edn3) / endothelin receptor B (Ednrb)-mediated calcium fluxes in the migratory activity of enteric neural crest cells (ENCCs). The study builds on previous data, appropriately cited in the manuscript, which indicate that calcium waves exist in ENCCs and that other members of the endothelin ligand / receptor family are known to influence cell contractility via calcium activation. The results provide novel insight into how Ednrb-signaling promotes colonization of the gut by ENCCs, discounts to some extent a prevailing counter hypothesis that the primary function of Endrb-signaling is to suppress differentiation and promote proliferation, and may lead to new approaches to Hirschsprung disease and other disorders of neural crest migration (e.g., piebaldism, melanoma, neuroblastoma).\n\nThe following concerns should be considered:\n\n1. Abstract, line 18 should read “… glia-derived neurotrophic factor (GDNF) / REarranged in Transfection receptor tyrosine kinase (RET) and the …”\n\n2. Abstract, 22: “trisphosphate-sensitive” (insert hyphen)\n\n3. Abstract, line 23: “EDNRB, Cl- channel, or CaV3.2-channel” (need to insert “channel” because EDNRB is not a channel)\n\n4. Main Text, line 39: “Vagal-derived” (insert hypen)\n\n5. Main Text, line 42: “1:5000 births” is missing the “s”. It is probably not correct to claim that HD is the most frequent neurocristopathy. Neurofibromatosis is 1:3000. Velocardiofacial syndrome is 1:3000. Consider stating “one of the most frequent neurocristopathies”.\n\n6. The Abstract and line 55 of the Main Text should indicate that this study was based on mice. For line 55, it would suffice to write “… ex-vivo embryonic mouse guts …”\n\n7. Main Text, line 60: Should read “7.5x10-4 +/- 2x10-4” (introduce missing X’s)\n\n8. Main Text, line 93: Technically, the ls allele is not a “knock-out” per se. The missense mutation predicts a protein that cannot be cleaved into mature Edn3. Functionally this is equivalent to complete loss of mature Edn3, but not abrogration of the unprocessed precursor protein which concievably might have other functions.\n\n9. Figure 1B and related data showing differences in Ca++ activity between E11.5 and E12.5 are fascinating and may warrant some contextual discussion in the manuscript. Specifically, the published work of Myun Shin and colleagues (Nature 1999;402:496; Nature Genet 2004;36:735), Coventry et al (Lab Invest 1994;71:82), and Drukenbrod Dev Biol 2005;287:125) collectively suggest that ENCC colonization of the cecum from the distal ileum may operate differently and possibly present a “developmental hurdle” which endothelin signaling helps negotiate. Whether endothelin signaling is operative after this hurdle is traversed is an open question, but the reduced Ca++ activity demonstrated between E11.5 and E12.5 lends some support to a model in which endothelin signals are most (solely?) important up until the ileocecum is traversed. Similarly, the seemingly dramatic change in response to exogenous Edn3 from “variable effects” at E11.5 to “an immediate increase of intracellular Ca++ across all ENCC’s” at E12.5 seems to correlate with some stage-specific change in the responsiveness of these cells, which correlates temporally with their crossing the cecum.\n\n10. Main Text, line 117 and Figure S9 and Figure S9 legend): It would be preferable to use the term “administration” or “introduction” in place of “injection”. The latter suggests something introduced into the cells or tissue directly.\n\n11. Main Text, line 118: “… in the presence of EDTA (Fig.3e, …” (EDTA is not shown in Figure 3e).\n\n12. Discussion, line 193: It seems inconsistent to regard ls/ls mice as edn3 “knock-outs” (see prior comment) and then state that they were first developed in the 1990s. As the authors recognize, the natural mutants of edn3 and ednrb were “developed” by mouse fanciers many decades earlier.\n\n13. Discussion, line 271: “Melanomas are” or “Melanoma is”.\n\n14. Discussion, last two sentences: The potential relevance of the current study to neural crest-related cancer is important. In addition, or perhaps in place of, melanoma, the authors might consider mentioning neuroblastoma, a pediatric malignancy arguably closer in cellular phenotype to ENCCs and with evidence that calcium signaling correlates with prognosis (see Lange I, Koster J, Koomoa DT. Calcium signaling regulates fundamental processes involved in Neuroblastoma progression. Cell Calcium. 2019 Sep;82:102052. doi: 10.1016/j.ceca.2019.06.006. Epub 2019 Jun 17. PMID: 31306997.)\n\n15. Figures S1 and S2: It would be helpful to modify the legend of Figure S1 to include the same terminology and abbreviations as used throughout the other figures. If I understand correctly, the minimum peak prominence was threshold for intensity and minimum peak-to-peak distance of 3 sec is for frequency threshold. Also, the last line of the legend should read “automated”, not “automatized.” IR is not defined in the legend for Figure S2.\n\n16. Figure S4 legend: Move space to read “Figure S4”. In the last line write “indicated (p values and red stars), unless …\"", "reviewer_3": "The authors present a technically rigorous study demonstrating that calcium activity in the embryonic mouse gut is partly driven by EDNRB signaling, with further mechanistic dissection implicating chloride channels and CaV3.2 (T-type) calcium channels. While the experimental detail and imaging approaches are impressive, the overall motivation of the study is insufficiently articulated.\n\nIf the primary goal is to understand how calcium signaling is altered in the context of Hirschsprung disease (HSCR), additional data are needed from genetic models with known HSCR-associated mutations. The brief analysis in Edn3ls/ls mice is a good start, but all subsequent experiments are performed in wild-type mice using pharmacological blockers. This limits the study’s ability to mechanistically connect altered calcium dynamics to disease-relevant phenotypes.\n\nMore broadly, while the EDN3/EDNRB pathway’s role in ENS development is well-established, and the identification of CaV3.2’s involvement in ENCC bioelectric activity is novel, the functional link between CaV3.2 activity and HSCR pathogenesis remains speculative. The reliance on pharmacological inhibition without complementary genetic models (e.g., Cacna1h mutants, conditional knockouts) weakens causal interpretation. Likewise, the reported association of CACNA1H variants in HSCR is not supported by functional data or penetrance analyses.\n\nTo strengthen the manuscript and better position it within a disease framework, the authors should consider the following:\n\n1. Poorly Articulated Link to Hirschsprung Disease\n• The motivation for the study in the context of HSCR is weak and underdeveloped. While the authors state that EDN3/EDNRB and CACNA1H are involved in HSCR, they fail to:\n o Provide a clear hypothesis about how Ca²⁺ signaling defects cause aganglionosis.\n o Demonstrate in vivo relevance using proper genetic models with defined HSCR penetrance.\n\n2. Inadequate Validation of the CACNA1H-HSCR Link\n• The claim that CACNA1H SNPs contribute to HSCR is made based on one prior EWAS reference (Tang et al. 2017) without showing:\n o Whether this variant is functional (e.g., reduces CaV3.2 current or expression).\n o Whether CaV3.2 deficiency alone recapitulates aganglionosis (and they acknowledge the knockout mouse is viable).\n o If CACNA1H loss leads to reduced ENCC migration in vivo, not just in culture.\n\n3. Heavy Reliance on Pharmacological Inhibition Without Adequate Genetic Validation\n• Most of the mechanistic claims (e.g., CaV3.2 dependence) are based on pharmacology (Z944, ascorbic acid), which:\n o Are not entirely specific (Z944 also blocks CaV3.1).\n o May affect cell health (shown for some drugs).\n• No CaV3.2-specific knockdown/knockout experiments are provided to confirm specificity.\n• No rescue experiments (e.g., with CaV3.2 re-expression or EDNRB overexpression) are included to test causality.\n\n4. Migration Assays Lack Rigorous Quantification\n• The migration distances are modest and not accompanied by:\n o ENCC cell number quantification—is reduced migration due to fewer cells?\n o Proliferation or apoptosis assays to rule out cytotoxic effects of the drugs.\n o In vivo rescue or dose-response studies to support pharmacological conclusions.\n\n5. the rational for doses of END3 added is not well explained- eg it says “1 nM EDN3at E11.5 ……At stage E12.5, application of 10 nM EDN3” How were the doses calculated?\n\n6. some of the statements are incorrect- ‘EDN3 gene knockout model that develops Hirschsprung disease, the ls/ls mouse”. ls/ls is a Ednrb deficient mice not End3 knockout"}, "author_response": "We thank the referees for their constructive criticism and appreciation of our work. We\nhave now backed up our investigation with further experimental data and clarified points\nof concern, you will find point-by-point answers in green, with modification to the\nmanuscript in italic.\n\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Chevalier et al. analyzed in mouse embryos how calcium activity, regulated by\nEDN3/EDNRB signaling, influences the migration of enteric neural crest cells (ENCCs), and what\nis its role in the pathogenesis of Hirschsprung disease (HD)? The authors applied multiple organ\ncultures and migration assays combined with calcium activity analysis, calcium imaging for enteric neural crest cells,\nand immunocytochemistry to characterize the developing ENS. Greater\nunderstanding of the ENS and emphasizing the complex molecular and mechanobiological\npathways during ENCC differentiation is a great value to both the congenital neurointestinal\ndiseases and the enteric neural crest biology field. The presented findings provide mechanistic\ninsight into how calcium waves are required for the ENCC migration and differentiation in the\ndeveloping hindgut.\n\nThe manuscript's topic is interesting and important in the context of hindgut ENS development.\nHowever, there are several major concerns in the present manuscript.\n\nInclude anti-Anti-CaV3.2 (CACNA1H) staining or in situ hybridization to demonstrate the\nexpression of Ca2+ channels on E11.5 wavefront ENCCs.\n\nWe have performed additional experiments and now include IHCs for CaV3.1, CaV3.2 and\nCaV3.3 (new Fig.3). To our surprise ENCCs were positive for all three channel types, not just\nCaV3.2, as we had initially assumed based on the PCR data of Hirst et al. We therefore further\ntested whether SAK3, the only commercially available CaV3 agonist (it amplifies CaV3.1 and\nCaV3.3 currents), could enhance Ca2+ activity. We found that is does and added the results in\nFig.3b, Video S6 and Fig.S10e. We further found (new Fig.4) that SAK3 could potentiate the effect\nof GDNF in the collagen gel migration assay, much like EDN3 – a noteworthy result. We finally\ninvestigated whether SAK3 could “rescue” CA inhibition by BQ788: although it did increase CA, it\nremained at very low levels (~0.1 events/min/100 µm²) compared to native E11.5 guts\n(Fig.S14a,b). It also did not improve ENCC migration down the colon when applied together with\nBQ (new Fig.5).\n\nWe note that the CaV3 antibodies (Alomone Labs) worked well on ENCCs that had migrated from\nexplants, i.e., without embedding, freezing or cutting of the samples. On the contrary, we could\nnot detect specific signal from any of the 3 antibodies when the same IHC protocol was applied\nto frozen slices of native E11.5 gut. We describe this in the Materials & Methods.\n\nWe have summarized these findings in the revised version, indicating all changes to the initial\nversion of the manuscript in green. Thank you for suggesting this experiment, which rectifies our\nconclusions and puts us on the right track for future research.\n\nAccording to Hirst et al (2015) ENCCs do not require ion channel activity to migrate through the\ngut mesenchyme. Explain why your results is different.\n\nWe had addressed these differences previously but have now grouped them in a single paragraph\nof the discussion section to clarify:\n\n“T-type receptor blockade by mibefradil 1 µM was previously reported not to affect ENCC\nmigration28. It is probable that this concentration was insufficient to cause aganglionosis, because\nthe IC50 of mibefradil on isolated cells is 3 µM55; in our experience, effective, sustained blocking\nof channel activity in whole-gut cultures requires concentrations far above drug IC50 to yield a\nmigration phenotype. Hirst et al.28 also blocked Cl- channels with NPPB 100 µM from an only 100-\nfold stock solution in a non-specified solvent, and found no effect on migration or on ENCC\nsurvival. In our experiments, NPPB 100 µM disrupted CA and migration by systematically inducing\nENCC death (n=11/11, Fig.S15). The choice of NPPB solvent or inconsistencies in the actual\nconcentrations applied by Hirst et al. may explain the discrepancy. More generally, we were\nguided in our choices of pharmacological compounds and concentrations by the Ca2+ response,\nwhereas Hirst et al. applied these compounds “blindly” to wildtype guts.”\n\nIf the 1mM ascorbic acid (specific CAV3.2 blocker) strongly reduced CA, how does the ascorbic\nacid affect ENCC migration? Why the effect of ascorbic acid on ENCC migration is missing from\nyour results (see Fig. 3f).\n\nWe had just considered the immediate effect on Ca2+ activity of ascorbic acid. We have now\nadded n=7 samples with tracking of short- and long-term effects on Ca2+ activity (Fig.3b, Fig.S10d,\nFig.S12) and its effect on migration (Fig.5): we find that it significantly decreases the migration\ndistance covered by the ENCCs at J+1 compared to controls, although to a lesser degree than\nZ944, NFA or BQ.\n\nLine 23: This statement is not supported by your results: “CaV3.2 channel blockade results in an\nENCC migration defect”. It is not proven that the HD-type aganglionosis was caused by the\nblockade of the CaV3.2 channel.\n\nIndeed, we have changed the statement to “T-type channel blockade …” and wherever relevant\nin the manuscript.\n\nDid you see CA in transmesenteric ENCCs?\n\nYes we observe CA in transmesenteric ENCCs as well, we now point them out in Fig.S3 and in\nthe results section l.71 “CA was also present in transmesenteric ENCCs (Fig.S3).”\n\nLine 58: typo “could at times”\n\nWe modified : “Ca2+ transients could sometimes propagate …”\n\nLine 62: migration front versus more proximal regions terminology should be changed according\nto Stavely et al., 2023; Development and Zhou B et al., 2024 to wavefront (these are the leading\nfront of the ENCDCs) and trailing cells (these are the stationary ENCDCs) see also\nDruckenbrod and Epstein, 2007.\n\nWe modified this l.70 “it was significantly higher at the ENCC wavefront located at the ileo-cecal\njunction (ilcc) than in trailing ENCCs (Fig.1c), …”\n\nLine 61-62: There is no apparent difference in the localization of CA and the immunoreactivity of\nSox10 and Tuj1, likely due to technical difficulties with the Sox10 immunostaining. To better\ndistinguish between Sox10 and Tuj1 expression, the authors should focus on the cecum, where\nthe Sox10+/Tuj1- ENCDC wavefront is located at this developmental stage.\n\nWe had an issue with washing of the antibodies leading to these red aggregates. We repeated\nthis experiment, the new Video S2 shows clearly that CA correlates with Sox10, not with Tuj1.\n\nFig1: What is the red color on image “a”? Tuj1 or Sox10? Add to the legend.\n\nWe had specified in the legend : “Red: CA frequency heatmap shows concentration of activity at\nthe cecum and the ileum anti-mesenteric border (see also Fig.S3).” We used a finer spatial mesh\n(compared to the 250 µm² mesh used in the remainder of the report, see for example Fig.S1, S3)\nspecifically for this image, for presentation purposes.\n\nFig S3: add proper labeling (midgut, hindgut, cecum) to mark the orientation of the embryonic gut\nsamples\n\nFig S3: Are the different E11.5 samples in the same orientation?\n\nNo were are not all in the same orientation ; we corrected this for clarity and added labels\n\nLine 82 and 85. Why was it important to change the concentration (10 fold) of EDN3 at stage\nE11.5 (1nM) to 10nM at E12.5?\n\nWe tried both 1 and 10 nM at E12.5, but mentioned only the effect at 10 nM because it differed\nqualitatively from that observed at E11.5. We now include the data on 1 nM at E12.5 as well in\nFig.S5c and commented:\n\n“In stage E12.5 ileum, CA also increased threefold at 1 nM EDN3 (Fig.S5), and the application\nof 10 nM EDN3 induced an immediate increase of intracellular Ca2+ across all ENCCs (Fig.2b,\nVideo S3, Fig.S5).”\n\nVideo 4: Can you mark the cells that retract their processes after BQ788 treatment?\n\nFig 2f: include high power images (inset) to show the morphology of the CTRL and BQ788-\ntreated cells.\n\nWe added arrows to Video 4 showing the retracting cells, and zoomed-in images of cell\nmorphologies in Fig.2f.\n\nLine 128: add the effect of ascorbic acid to Fig 3f.\n\nWe added this as stated previously.\n\nVideo S9: The time-lapse movie is too short to see the backflow of mesenchyme.\n\nThe video needs to be loaded in ImageJ and the time-cursor tracked fast-forward& backward\nrepeatedly between frames 70 and 89, focusing on the areas indicated be the arrow; we clarified\nthis in the Supplementary Material description. You will see that the mesenchyme moves in the\nopposite direction to the ENCCs.\n\nLine 148: According to Barlow et al 2003 and Nagy and Goldstein 2006, EDN3 inhibits the\nmigration of ENCCs in response to GDNF. Explain why your data using mouse ENCCs is different\nfrom the previously published results. What was the explant: midgut, ceca or hindgut?\n\nWe have summarized our thoughts on this in this new paragraph of the discussion section:\n\n“We found that GDNF and EDN3 (or SAK3) have a synergistic action on ENCC migration from explants\n(Fig.3). Our results are in agreement with the findings of Bergeron et al.60. We did not observe that EDN3\nand GDNF were antagonistic, as has been reported in other studies with mouse25, rat61 and chicken\nembryos46. These studies were performed respectively at stages E10.5-E11, E13 and E8, at EDN3\nconcentrations of 100, 20 and 100 nM and after 16h, 2-3 days and 3 days of migration. It is difficult to\nfathom the reasons of this discrepancy but we stress that: 1°) migration after 16h25 is too scarce to be\nprecisely quantified, 2°) the EDN3 concentrations applied by other investigators are 1-2 orders of\nmagnitude higher than the maximum pro-CA and pro-migratory effect we report at 1 nM. 1 nM likely\nreflects the concentration encountered physiologically by ENCCs as they migrate down the gut\nmesenchyme. We found that CA was not significantly increased at 10 nM EDN3 compared to 1 nM (Fig.2a,\nFig.S5c), indicating a saturation effect. Administration of EDN3 10 nM at E12.5 gave rise to a single pan-\nENCC transient, which, although impressive, most likely never occurs physiologically.”\n\nLine 149 and Fig 3d. Include GDNF alone to prove the positive effect of 1nM EDN3+10ngGDNF\non the ENCC migration distance.\n\nYou surely meant edn3 alone (GDNF alone was already presented). We have performed new\nexperiments and added the result to the new Fig.4. Edn3 alone leads to a small, but significant\nincrease in migration, although not as marked as GDNF alone. This is consistent with the\nfindings of Bergeron et al.\n\nLines 169-170: The following sentence is not clear: “Several observations….”\n\nWe changed the punctuation and numbered the 3 observations to make it clearer.\n\n“Several observations indicated that Ca2+ oscillations could promote migration by enhancing cell\ncontractility, as in smooth muscle: 1°) …; 2°) … ; 3°) … “\n\nLine 171: It is just a speculation that the effect of the CA blockers indicates a loss of ENCC-\nECM traction.\n\nWe now include new traction force data (new Fig.6) showing that the T-type Ca2+ channel\nantagonist Z944 decreases the ENCC-ECM traction force similarly to BQ788.\n\nLine 180-186: it is not related to the Results section.\n\nWe have moved this to the Discussion section\n\nFig. 4. What are the green cells on a,b,c images? Are they isolated ENCCs or cells emigrated\nfrom the gut explants in response to GDNF?\n\nFig. 4: This figure is poorly presented in the Results section. a,b, c images are not introduced\nproperly in the text.\n\nThe green cells (now in Fig.6a) are ENCCs emigrating from the explant (to the left, not seen in\nthe field-of-view). We have clarified this in the text\n\nl.220 “To quantify the force exerted by ENCCs on the ECM, we let ENCCs migrate from E11.5\nmidgut explants in a collagen gel seeded with fluorescent beads that served as fiducial markers\nof gel deformation (Fig.6a inset).”\n\nand in the legend of Fig.6:\n\n“ENCC 3D migration from a E11.5 midgut explant (to the left, not in the field of view)”\n\nand have added an inset scheme of the experiment in Fig.6a.\n\nline 201: “ENCCS”\n\nWe corrected this\n\nTo increase the quantity of their work, the authors should include a more sensitive ENCC\nculture assay (e.g., culture of isolated Wnt1-GCaMP 11.5 or 12.5 dpc mouse intestine, either\nwith FACS sorting or induce to emigrate from the gut explant in response to GDNF) to study\nwith high-power microscopy and prove that modulation of Ca2+ activity affects the ENCC\nmigration and cell contractility. You may include phalloidin staining to mark the cell skeleton of\nthe cytoplasmic processes.\n\nPerhaps it was not clear from the previous description of Fig.6, but that is precisely what we did\n(culture of isolated Wnt1-GCaMP 11.5 dpc mouse intestine induced to emigrate from the gut\nexplant in response to GDNF). The inclusion of beads in the gel and monitoring of their\ndisplacements to assess traction forces is novel in the enteric neural crest field.\n\nFig.5. it is not clear why the schematic figure is important for the Discussion section.\n\nThis figure (now Fig.7) is important because it summarizes the molecular route we have\nuncovered going from edn3 docking to EDNRB, to Ca2+ release and traction force generation\nnecessary for migration.\n\nWhat is the relationship between β1-integrin signalling pathway, Ca2+-dependent cell adhesion\nmolecules and Endothelin 3 activation or inhibition during ENCC migration?\n\nβ1-integrin link the cell cytoskeleton to the ECM, and thus allow the transmission of contractile\nforces generated by the ENCCs to the ECM. ENCC specific knock-out of β1-integrin induces a\nHirschsprung phenotype (Breau et al. 2006, Gazquez et al. 2016, Chevalier et al. 2021). Edn3\npromotes Ca2+ activity, which generates ENCC contractility (Fig.6). We do not discuss Ca2+-\ndependent cell adhesion molecules (cadherins) in the manuscript, beyond the observation that\nEDTA obviously perturbs them (Video S5).\n\nl.264: “ENCC contraction leads to an increased traction force to the extracellular-matrix, that is\nnecessary for their migration inside the gut mesenchyme. This force is the reason why\ninvestigators have found it indispensable to pin45 or embed the gut tract during ex-vivo migration\nassays46, as it otherwise leads to tissue shrinkage and improper migration. This force is\ntransmitted via β1-integrins, and ENCC-specific β1-integrin mutants have been shown to present\nwith a HD phenotype47–49.”\n\nThe aganglionic segment of EDN3 mutant human and mouse is restricted to the distal\ncolorectum. Compare Ca2+ frequency of the trailing and wavefront ENCCs in control and ls/ls\nE12.5 (dpc) mouse hindgut to demonstrate that CA is critical for hindgut ENCC migration and\ndistal colorectum ENS formation.\n\nWe compared the frequency between trailing and wavefront ENCCs in control samples (Fig.1c).\nWe cannot do the same at present for ls/ls, as we do not have a mouse line that presents both\nGCaMP and the ls/ls mutation.\n\nReviewer #2 (Remarks to the Author):\nThe manuscript by Chevalier et al is a well written and nicely illustrated description of a series of\nstudies of murine embryonic gut explants which demonstrate a role for endothelin-3 (Edn3) /\nendothelin receptor B (Ednrb)-mediated calcium fluxes in the migratory activity of enteric neural\ncrest cells (ENCCs). The study builds on previous data, appropriately cited in the manuscript,\nwhich indicate that calcium waves exist in ENCCs and that other members of the endothelin\nligand / receptor family are known to influence cell contractility via calcium activation. The\nresults provide novel insight into how Ednrb-signaling promotes colonization of the gut by\nENCCs, discounts to some extent a prevailing counter hypothesis that the primary function of\nEndrb-signaling is to suppress differentiation and promote proliferation, and may lead to new\napproaches to Hirschsprung disease and other disorders of neural crest migration (e.g.,\npiebaldism, melanoma, neuroblastoma).\n\nThe following concerns should be considered:\n\n1. Abstract, line 18 should read “… glia-derived neurotrophic factor (GDNF) / REarranged in\nTransfection receptor tyrosine kinase (RET) and the …”\n\n2. Abstract, 22: “trisphosphate-sensitive” (insert hyphen)\n\n3. Abstract, line 23: “EDNRB, Cl- channel, or CaV3.2-channel” (need to insert “channel”\nbecause EDNRB is not a channel)\n\n4. Main Text, line 39: “Vagal-derived” (insert hypen)\n\n5. Main Text, line 42: “1:5000 births” is missing the “s”. It is probably not correct to claim that HD\nis the most frequent neurocristopathy. Neurofibromatosis is 1:3000. Velocardiofacial syndrome\nis 1:3000. Consider stating “one of the most frequent neurocristopathies”.\n\n6. The Abstract and line 55 of the Main Text should indicate that this study was based on mice.\nFor line 55, it would suffice to write “… ex-vivo embryonic mouse guts …”\n\nThank you, we have corrected all these points, and also had to shorten the abstract to <150\nwords.\n\n7. Main Text, line 60: Should read “7.5x10-4 +/- 2x10-4” (introduce missing X’s)\n\n8. Main Text, line 93: Technically, the ls allele is not a “knock-out” per se. The missense\nmutation predicts a protein that cannot be cleaved into mature Edn3. Functionally this is\nequivalent to complete loss of mature Edn3, but not abrogration of the unprocessed precursor\nprotein which concievably might have other functions.\n\nWe corrected this:\n\n“We finally tested whether CA differed in an EDN3 missense mutation model that develops\nHirschsprung disease …”\n\n9. Figure 1B and related data showing differences in Ca++ activity between E11.5 and E12.5\nare fascinating and may warrant some contextual discussion in the manuscript. Specifically, the\npublished work of Myun Shin and colleagues (Nature 1999;402:496; Nature Genet\n2004;36:735), Coventry et al (Lab Invest 1994;71:82), and Drukenbrod Dev Biol 2005;287:125)\ncollectively suggest that ENCC colonization of the cecum from the distal ileum may operate\ndifferently and possibly present a “developmental hurdle” which endothelin signaling helps\nnegotiate. Whether endothelin signaling is operative after this hurdle is traversed is an open\nquestion, but the reduced Ca++ activity demonstrated between E11.5 and E12.5 lends some\nsupport to a model in which endothelin signals are most (solely?) important up until the\nileocecum is traversed. Similarly, the seemingly dramatic change in response to exogenous\nEdn3 from “variable effects” at E11.5 to “an immediate increase of intracellular Ca++ across all\nENCC’s” at E12.5 seems to correlate with some stage-specific change in the responsiveness of\nthese cells, which correlates temporally with their crossing the cecum.\n\nWe understand your thoughts, and point out the following:\n\n We find that CA inhibitors slow down migration between E11.5 and E11.5+1 (Fig.5).\nENCCs at E11.5 are at the ileo-cecal junction and migrate through the cecum and then\nthe colon. It is possible that most of the slowing down linked to CA occurs in the cecum,\nand that for this reason the ENCC only barely reach the colon; is it equally possible that\nslowing down occurs both in the cecum and colon, we cannot tell from our data.\n\n Edn3 expression is highest in the cecum, and there is little expression in the hindgut at\nE12 (in-situ of Leibl et al., 1999), which indeed correlates with the lower CA we measure\nat the wavefront at E12.5 (compared to E11.5); this indeed supports a model where edn3\nis most necessary up until the cecum is traversed. Aganglionosis of the colon can result\nfrom slowing down of the migration front at any space or time-point, not necessarily from\nslower migration of the ENCCs specifically in the colon.\n\n Concerning the change “from “variable effects” at E11.5 to “an immediate increase of\nintracellular Ca++ across all ENCC’s” at E12.5”, we stress that the effect of edn3 1nM at\nE11.5 is almost systematic (10/12 samples) and quite dramatic (3-fold CA increase on\naverage). We do not know why edn3 10 nM induces a pan-ENCC Ca2+ wave at E12.5, but\nnot at E11.5. We doubt that it is related to ENCCs crossing the cecum, because these\npan-ENCC waves were found in regions proximal to the cecum (ileum but also jejunum).\nIt doesn’t seem to be related to the electric (gap-junctional) connectivity of the\nENCC network because pan-ENCC Ca2+ waves could be induced by ATPe at E11.5\n(Video S8). It doesn’t seem to be related to increased ion channel expression because\nspontaneous CA decreased with increasing age. EDNRB receptors at E12.5 may be less\nsaturated with endogenous edn3, and therefore react more sharply in response to\nexogenous edn3.\n\nWe have summarized some of these thoughts in the discussion:\n\n“2°) the EDN3 concentrations applied by other investigators are 1-2 orders of magnitude higher than the\nmaximum pro-CA and pro-migratory effect we report at 1 nM. 1 nM likely reflects the concentration\nencountered physiologically by ENCCs as they migrate down the gut mesenchyme. We found that CA was\nnot significantly increased at 10 nM EDN3 compared to 1 nM (Fig.2a, Fig.S5c), indicating a saturation\neffect. Administration of EDN3 10 nM at E12.5 gave rise to a single pan-ENCC transient, which, although\nimpressive, most likely never occurs physiologically. The pan-ENCC Ca2+ surge induced by exogenous EDN3\nmay occur only at E12.5 (and not at E11.5) because of a reduced saturation of EDNRB receptors by\nendogenous EDN3 at this stage, making it more sensitive to exogenous application.\n\nEdn3 expression is highest in the cecum, with little expression in the hindgut at E1226, which correlates\nwith reduced CA at the ENCC wavefront at E12.5 (Fig.1). These observations suggest that EDN3 may be\nmost necessary up until the cecum is traversed; slowing down of ENCCS by CA activity inhibitors in our ex-\nvivo assay (Fig.5) may have primarily affected migration through the cecum, resulting in a paucity of ENCCs\nin the colon. Colonic aganglionosis can result from slowed-down migration at any point of their journey\ndown the gut, not necessarily from slower migration in the colon. “\n\n10. Main Text, line 117 and Figure S9 and Figure S9 legend): It would be preferable to use the\nterm “administration” or “introduction” in place of “injection”. The latter suggests something\nintroduced into the cells or tissue directly.\n\nWe replaced the word “injection” by “administration” or “introduction” where relevant.\n\n11. Main Text, line 118: “… in the presence of EDTA (Fig.3e, …” (EDTA is not shown in Figure\n3e).\n\nWe removed the reference to Fig.3e.\n\n12. Discussion, line 193: It seems inconsistent to regard ls/ls mice as edn3 “knock-outs” (see\nprior comment) and then state that they were first developed in the 1990s. As the authors\nrecognize, the natural mutants of edn3 and ednrb were “developed” by mouse fanciers many\ndecades earlier.\n\nWe agree, we rephrased this as such:\n\n“As the roles of endothelins in blood pressure regulation became more prominent, mutant mice for\nEDN341,42 and EDNRB43 mouse were re-examined”\n\n13. Discussion, line 271: “Melanomas are” or “Melanoma is”.\n\nWe corrected this\n\n14. Discussion, last two sentences: The potential relevance of the current study to neural crest-\nrelated cancer is important. In addition, or perhaps in place of, melanoma, the authors might\nconsider mentioning neuroblastoma, a pediatric malignancy arguably closer in cellular\nphenotype to ENCCs and with evidence that calcium signaling correlates with prognosis (see\nLange I, Koster J, Koomoa DT. Calcium signaling regulates fundamental processes involved in\nNeuroblastoma progression. Cell Calcium. 2019 Sep;82:102052. doi:\n10.1016/j.ceca.2019.06.006. Epub 2019 Jun 17. PMID: 31306997.)\n\nThank you, we did not know about this literature, it is obviously relevant, we have added the\nfollowing references:\n\n“Melanoma7 and neuroblastoma59 are known to recapitulate many NCC traits. T-type Ca2+\nchannel upregulation is associated with melanoma aggressiveness60 while Ca2+ signaling is\naltered in neuroblastoma61”\n\n15. Figures S1 and S2: It would be helpful to modify the legend of Figure S1 to include the same\nterminology and abbreviations as used throughout the other figures. If I understand correctly,\nthe minimum peak prominence was threshold for intensity and minimum peak-to-peak distance\nof 3 sec is for frequency threshold. Also, the last line of the legend should read “automated”, not\n“automatized.” IR is not defined in the legend for Figure S2.\n\nWe have added the missing abbreviations in the legend of Fig.S1 and S2. Yes your\nunderstanding of the thresholds is correct.\n\n16. Figure S4 legend: Move space to read “Figure S4”. In the last line write “indicated (p values\nand red stars), unless …”\n\nWe have corrected this\n\nReviewer #2 (Remarks on code availability):\nCode was not part of this study.\n\nReviewer #3 (Remarks to the Author):\nThe authors present a technically rigorous study demonstrating that calcium activity in the\nembryonic mouse gut is partly driven by EDNRB signaling, with further mechanistic dissection\nimplicating chloride channels and CaV3.2 (T-type) calcium channels. While the experimental\ndetail and imaging approaches are impressive, the overall motivation of the study is\ninsufficiently articulated.\n\nIf the primary goal is to understand how calcium signaling is altered in the context of\nHirschsprung disease (HSCR), additional data are needed from genetic models with known\nHSCR-associated mutations. The brief analysis in Edn3ls/ls mice is a good start, but all\nsubsequent experiments are performed in wild-type mice using pharmacological blockers. This\nlimits the study’s ability to mechanistically connect altered calcium dynamics to disease-relevant\nphenotypes.\n\nMore broadly, while the EDN3/EDNRB pathway’s role in ENS development is well-established,\nand the identification of CaV3.2’s involvement in ENCC bioelectric activity is novel, the\nfunctional link between CaV3.2 activity and HSCR pathogenesis remains speculative. The\nreliance on pharmacological inhibition without complementary genetic models (e.g., Cacna1h\nmutants, conditional knockouts) weakens causal interpretation. Likewise, the reported\nassociation of CACNA1H variants in HSCR is not supported by functional data or penetrance\nanalyses.\n\nWe thank the reviewer for the overall impression and comments. We have reformulated the\nmanuscript to make our motivation clearer: our primary aim was not to understand how calcium\nsignaling is altered in Hirschsprung disease, but to understand the role and molecular\nmechanisms of calcium signaling during physiological migration. This required the establishment\nof\nestablishment\nnew methodological tools (Ca2+ imaging and analysis protocol, 3D traction force assay). Our\nconclusions pertaining to Hirschsprung disease are perspectives resulting from this work, which\nmotivate further investigations with dedicated mouse models as you point out, for future research\nprojects. The mostly-pharmacological investigation we present is a necessary first step before\nmoving on to the more financially and time-demanding developments of conditional knockouts.\nThis is only the second study to address Ca2+ signaling in enteric neural crest cells, after the\nseminal first observation of these transients by Hao et al. in 2017. We have added a wealth of\ninformation:\n\n Most of this activity occurs as single-cell events, and not as multicellular waves. The focus\non multicellular waves in Hao et al.’s work did not allow them to grasp the fundamental\nmechanisms of ENCC electrical activity, nor its precise spatial and temporal dependence\n(Fig.1).\n\n We revealed that edn3/EDNRB (Fig.2), T-type Ca2+ channels and Cl- channels (Fig.3) are\nthe main drivers of Ca2+ activity; that purinergic signaling, which was presented as the\nmain source of Ca2+ waves in Hao et al.’s work, is merely a modulator. We also now point\nout more clearly in the discussion why the pharmacological investigation of Hirst et al.\n(2015) on the role of ion channels in ENCC migration overlooked the major role played by\nT-type Ca2+ channels and Cl- channels.\n\n We now understand that this Ca2+ activity translates to increased cell contractility (Fig.6),\nwhich is necessary for their migration down the gut mesenchyme (Fig.4&5). Previous\ninvestigations had failed to establish a clear link between Ca2+ activity and migration. The\n3D traction force assay we present in Fig.6 is the first to be applied in the enteric neural\ncrest field and the results are, in our opinion, fascinating, because they explain the\nfundamental biophysical mechanism by which edn3/EDNRB enhances ENCC migration.\n\nWe reflected these considerations in the manuscript in the abstract (mildering our claim regarding\nHD and CACNA1H, and also shortening it to get below the 150 word limit), introduction (l.52-60),\ndiscussion (l.291-306), outlining all changes in green.\n\nTo strengthen the manuscript and better position it within a disease framework, the authors should\nconsider the following:\n\n1. Poorly Articulated Link to Hirschsprung Disease\n• The motivation for the study in the context of HSCR is weak and underdeveloped. While the\nauthors state that EDN3/EDNRB and CACNA1H are involved in HSCR, they fail to:\n o Provide a clear hypothesis about how Ca²⁺ signaling defects cause aganglionosis.\n\nWe clarified this in the discussion section, and our argument if further bolstered by new\nmeasurements showing that Z944 decreases the traction force (new Fig.6c,e):\n\nl.260: “We reveal here that the immediate action of EDN3 on ENCCs is in fact nearly identical to its effect\non smooth muscle tone: it triggers Ca2+ activity (Fig.1,2), via a very similar molecular route (Fig.3,7) to\nvSMC, and the increased cytosolic Ca2+ oscillations enhance migration (Fig.4,5) by inducing cell contractility\n(Fig.6). vSMC contraction leads to vessel constriction; ENCC contraction leads to an increased traction force\nto the extracellular-matrix, that is necessary for their migration inside the gut mesenchyme. This force is\nthe reason why investigators have found it indispensable to pin45 or embed the gut tract during ex-vivo\nmigration assays46, as it otherwise leads to tissue shrinkage and improper migration. The traction force is\ntransmitted via β1-integrins, and ENCC-specific β1-integrin mutants have been shown to present with a\nHD phenotype47–49. Ca2+ signaling defects reduce the traction force of the ENCCs to the extracellular matrix,\nlowering their migration speed, resulting in colonic aganglionosis. Our investigation shows that ENCCs are\nakin to miniature muscles that contract and crawl in response to a constrictor peptide, endothelin 3.”\n\n o Demonstrate in vivo relevance using proper genetic models with defined HSCR penetrance.\n\nThis will be investigated in a future project.\n\n2. Inadequate Validation of the CACNA1H-HSCR Link\n• The claim that CACNA1H SNPs contribute to HSCR is made based on one prior EWAS\nreference (Tang et al. 2017) without showing:\n o Whether this variant is functional (e.g., reduces CaV3.2 current or expression).\n o Whether CaV3.2 deficiency alone recapitulates aganglionosis (and they acknowledge the\nknockout mouse is viable).\n\nWe have now added n=7 samples cultured with the specific CaV3.2 blocker ascorbic acid, with\ntracking of short- and long-term effects on Ca2+ activity (Fig.3b, Fig.S10d, Fig.S12) and migration\n(Fig.5): we found that ascorbic acid significantly decreases the migration distance covered by\nENCCs at J+1 compared to controls, although to a lesser degree than Z944, NFA or BQ.\n\nFollowing comments by another reviewer, we have added IHCs for CaV3.1, Cav3.2 and CaV3.3\nchannels, which show (new Fig.3) that all three channel types are expressed by ENCCs, not just\nCaV3.2 as we had initially thought based on the PCR data of Hirst et al. We therefore further\ntested whether SAK3, the only commercially available CaV3 agonist (it amplifies CaV3.1 and\nCaV3.3 currents), could enhance Ca2+ activity. We found that is does, and added the results in\nFig.3b and Fig.S10e. We further found (new Fig.4) that SAK3 could potentiate the effect of GDNF\nin the collagen gel migration assay, much like EDN3. We finally investigated whether SAK3 could\nrescue CA inhibition by BQ788 : although it did increase CA, it remained at very low levels (~0.1\nevents/min/100 µm²) compared to native E11.5 guts. It also did not improve ENCC migration\ndown the colon when applied together with BQ (new Fig.5).\n\nIn light of these new results, we have shifted the focus from CaV3.2 to T-type channels in general.\nEWAS studies however only point to a link with CaV3.2. Tang et al. do not specify whether the\nvariant affected channel current or expression. We reflect this in the abstract and discussion\nsection, and, because we indeed do not have ENCC-specific KO models at this stage, nuanced\nour claim regarding CACNA1H and HD:\n\nl.307: “CaV3.1 and CaV3.2 expression in E11.5 ENCCs has been previously measured by PCR28;\nCaV3.2 was found to be ~640 times more expressed than CaV3.1. Our IHC, CA and migration\nassays indicate that all three CaV3 channel types are present and functional in ENCCs. The\nCaV3.1 & CaV3.3 agonist SAK3 promoted CA (Fig.3) and ENCC migration (Fig.4); the CaV3.2\nantagonist ascorbic acid reduced CA (Fig.3) and ENCC migration (Fig.5). Very interestingly, a\nsingle-nucleotide polymorphism (SNP) of the CACNA1H gene encoding CaV3.2 has been\nuncovered in a recent HD exome-wide association study15. Although it is not known whether this\npoint mutation altered CaV3.2 expression or function, our findings suggest it alters Ca2+ signaling.\nThis motivates further research using dedicated mouse models to better understand the causes\nof neurocristopathies. We note that the CaV3.2 -/- mouse is viable56,57, although the mutation\ninduces pre-natal lethality58. It is possible that the surviving CaV3.2 -/- embryos develop\ncompensatory Ca2+ influx mechanisms, a common behavior when only one of several protein\nisoforms is knocked-out59.”\n\n o If CACNA1H loss leads to reduced ENCC migration in vivo, not just in culture.\n\nThis will be investigated in a future project with dedicated ENCC-specific knock outs of the\ndifferent (or multiple) CaV3 channels.\n\n3. Heavy Reliance on Pharmacological Inhibition Without Adequate Genetic Validation\n• Most of the mechanistic claims (e.g., CaV3.2 dependence) are based on pharmacology (Z944,\nascorbic acid), which:\n o Are not entirely specific (Z944 also blocks CaV3.1).\n\nAs explained above, we have, in the light of our new IHC and pharmacological results, modified\nour claim from the sole involvement of CaV3.2 to that of all three CAV3 channels.\n\n o May affect cell health (shown for some drugs).\n• No CaV3.2-specific knockdown/knockout experiments are provided to confirm specificity.\n• No rescue experiments (e.g., with CaV3.2 re-expression or EDNRB overexpression) are\nincluded to test causality.\n\nThis involves genetic means which we currently do not have at our disposal.\n\n4. Migration Assays Lack Rigorous Quantification\n• The migration distances are modest and not accompanied by:\n\nThe migration distances in the cecum & colon in control conditions, 0.7-1.2 mm are in the same\nrange as reported for 1-day culture of E11.5 guts (Hao et al. 2017), and the migration distances\nafter 3 days in collagen gel are in the same range (0.1-0.7 mm) as reported by Bergeron et al.\n2015 for identical culture conditions.\n\n o ENCC cell number quantification—is reduced migration due to fewer cells?\n o Proliferation or apoptosis assays to rule out cytotoxic effects of the drugs.\n\nENCC proliferation and migration are undoubtedly linked – they cannot invade the colon or the\ncollagen gel if they do not also proliferate (“Cell proliferation drives neural crest cell invasion of\nthe intestine”, Simpson et al., Dev. Biol., 2007). As an example, in the gel migration assay, the\nspherical halo measures up to 600 µm radius, whereas the initial explant typically measures only\n100-200 um. A x3-6 increase in radius means a x9-36 time increase in volume occupied by\nENCCs, which can only be accounted for by proliferation. We found it more meaningful to present\nmacroscopic migration distances (linear in the colon, spherical in the gel) rather than perform\nproliferation IHCs which would be more variable (because it only samples a small region of each\ngut) and dependent on where the section is performed. We reflected these considerations in the\ndiscussion section:\n\nl.273: “It is likely that cytosolic Ca2+ oscillations are also involved in the long-term effects of\nEDN3/EDNRB on ENCC proliferation. ENCC proliferation and migration are inexorably linked –\nENCC cannot invade the colon or a collagen gel if they do not also proliferate54. Here, we reported\nmacroscopic migration distances (linear in the colon, spherical in the gel) rather than local\nproliferation rates.”\n\nGCaMP directly allows to assess cytotoxicity because dead cells appear bright (they become\npermeable to Ca2+) and rounded. We now quantify in Fig.S16 the percentage of dead ENCCs in\nall chemical conditions investigated in Fig.5. The fraction of dead cells was <7% in 49/54 samples,\nand none of the fractions were significantly different (Mann-Whitney test and Student t-test) from\nthe control group, although BQ788 tended to induce higher average cell death.\n\n o In vivo rescue or dose-response studies to support pharmacological conclusions.\n\nZ944 could be injected to pregnant mice at ENCC migration stages, however it would be difficult\nto rule out side-effects of this treatment, as T-type Ca2+ channels are expressed in many other\ncell types, in particular in the brain.\n\nConsidering rescue studies, we have reported in the manuscript three approaches: BQ +SAK3,\nBQ + 4-AP and BQ + ARL and/or ATPe. None of these approaches allowed to recover Ca2+\nactivity or migration post EDNRB-blockade by BQ788. SAK3 was however able to mimic the pro-\nmigratory effect of EDN3 in the gel migration assay, a very interesting result (new Fig.4).\n\n5. the rational for doses of END3 added is not well explained- eg it says “1 nM EDN3at E11.5\n……At stage E12.5, application of 10 nM EDN3” How were the doses calculated?\n\nWe tried both 1 and 10 nM at E11.5 and at E12.5, but mentioned only the effect at 10 nM for\nE12.5 because it differed qualitatively from that observed at E11.5. We now include the data on\n1 nM at E12.5 as well in Fig.S5c and commented in the result section:\n\n“In stage E12.5 ileum, CA also increased threefold at 1 nM EDN3 (Fig.S5), and the application\nof 10 nM EDN3 induced an immediate increase of intracellular Ca2+ across all ENCCs (Fig.2b,\nVideo S3, Fig.S5).”\n\n6. some of the statements are incorrect- ‘EDN3 gene knockout model that develops\nHirschsprung disease, the ls/ls mouse”. ls/ls is a Ednrb deficient mice not End3 knockout\n\nls/ls mice present a missense mutation in the EDN3 gene. We agree they are not knock-outs\nper-se, and have reformulated:\n\nl.103: “We finally tested whether CA differed in an EDN3 missense mutation model that\ndevelops Hirschsprung disease …”\n\nl.254: “As the roles of endothelins in blood pressure regulation became more prominent, mutant\nmice for EDN341,42 and EDNRB43 mouse were re-examined”\n\nReviewer #3 (Remarks on code availability):\nThere is no code involved"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for clearly addressing each of the comments. I have no further questions or remarks.", "reviewer_2": "The revised version of this manuscript adequately addresses all of the concerns raised in my original review. I remain impressed with the findings which collectively support the hypothesis that asynchronous Ca+-flux mediated contractions of enteric neural crest cells are influenced by EDN3/ENDRB signaling and mediated by CaV3 and Cl- channels. I believe this is significant because, as the authors point out, it unifies the functions of ENDRB signaling in ENCCs with a more established role in mature smooth muscle contractile regulation. I have no strong reservations but offer the following comments:\n\n1. The immunohistochemistry results shown in Figure 3 seem to show similar diffuse labeling of all the cells. While this may reflect specific labeling with each of the antibodies used, it would be helpful to show that some cell type known not to express the CaV protein of interest is immunonegative.\n\n2. I have trouble following the logic of this sentence (line 308): \"We note that the CaV2 -/- mouse is viable, although the mutation induces prenatal lethality.\" Is the intended meaning fetal versus post-natal viability. Please clarify.\n\nMinor typo: line 72: ENCC-specific (add hyphen)", "reviewer_3": "All my concerns have been addressed. I don't have any additional comments"}, "author_response": "We thank the referees for their constructive criticism and appreciation of our work. We\nhave now backed up our investigation with further experimental data and clarified points\nof concern, you will find point-by-point answers in green, with modification to the\nmanuscript in italic."}]

Decision letter