Wnt and TGFβ coordinate growth and patterning to regulate size-dependent behavior

Abstract

Differential coordination of growth and patterning across metazoans gives rise to a diversity of sizes and shapes at tissue, organ, and organismal levels. While tissue size and tissue function can be interdependent 1-5 , mechanisms coordinating size and function remain poorly understood. Planarians are regenerative flatworms that bi-directionally scale their adult body size 6,7 and asexually reproduce, via transverse fission, in a size-dependent manner 8-10 . This paradigm offers a robust context to address the gap in knowledge underlying the link between size and function. A novel planarian fission protocol revealed that progeny number and fission initiation frequency scale with parent size. Fission progeny size is fixed by previously unidentified mechanically vulnerable planes spaced at an absolute distance along the anterior-posterior (A/P) axis. An RNAi screen of A/P patterning genes uncovered TGFβ and Wnt signaling components as regulators of fission initiation frequency rather than fission plane position. Finally, inhibition of Wnt and TGFβ signaling during growth altered the patterning of mechanosensory neurons, a neural sub-population that is distributed in accordance with animal size and modulates fission behavior. Therefore, our study identifies a novel role for TGFβ and Wnt in regulating size-dependent behavior, uncovering an interdependence between patterning, growth, and neurological function. The infrequency of planarian fission behavior has largely precluded its mechanistic dissection. However, recently optimized animal husbandry techniques augmented fission activity 11-12 , permitting us to study the integration of animal size with fission behavior. Large planaria from recirculation culture exhibited robust and reproducible increases in fission activity when transitioned to static culture and starved (Fig. 1a, Video S1). Live imaging provided detailed characterization of the fission process. Planarians first elongate and adhere their posterior tissue to a substrate. Next, periodic body contractions concentrate body mass towards the head region while thinning out tissues immediately anterior to the adherent tail. After 20-40 minutes, progressive stretching ruptures connecting tissue with rapid recoil, separating anterior parent and posterior fission progeny (Video S1, Extended Data Fig. 1a). Observation of fission behavior in animals of increasing size showed that 1st posterior fission fragment length did not scale with parent length (Fig. 1b, d). Instead, larger animals produced additional progeny, each ~1mm in length, such that number of progeny after 2 weeks linearly correlated with parent size (Fig. 1c, e, Extended Data Fig 1. b-d). Thus, fission fragment size is fixed independent of A/P position or parent length. The frequency of fission fragment production – fission rate – did scale with animal length (Extended Data Fig. 1e, f) and both the time to 1st fission and time between sequential fission events was inversely related to parent size (Extended Data Fig. 1g-l). Automated webcam imaging of individual animals allowed us to generate timelines chronicling successful (upward displacement) and un-successful (downward displacement) fission attempts (Fig. 1f, Video S2). Fission attempts only occured in animals above 4-5mm in length, indicating a minimal size required for fission (Fig. 1g, h, Extended Data Fig. 2a, b). Furthermore, larger animals produced fission progeny more frequently due to more fission attempts (Fig. 1h, Extended Data Fig. 2 c, d), rather than higher rates of success (Fig. 1i). Altogether, these results confirm that planarian fission is a size-dependent behavior, with both progeny number and fission rate coupled to parent size. We tested the hypothesis that patterning cues are required to coordinate animal size and planarian fission. Genes from the Wnt 13-16 , TGFβ 17-19 , and Hh 20 signaling pathways that regulate A/P identity were screened using RNA-dependent genetic interference (RNAi) 21 (Fig. 2a, b, Extended Data Fig. 3a, b). Re-screening confirmed 6 presumptive activators of fission (ActR-1, smad2/3, β-catenin, Dsh-B, tsh, wnt11-6) and a presumptive inhibitor (APC) (Fig. 2c). Parent animals morphology was observed at days 0 and 14 of the fission assay and in regenerating tissue fragments. RNAi knockdown reproduced published A/P patterning defects in regenerating tissue fragments (Extended Data Fig. 4a), but few morphological defects were observed in parent animals (Fig. 2d). On day 0, β-catenin RNAi animals exhibited morphological abnormalities while other RNAi conditions were indistinguishable from controls. By day 14, RNAi of ActR-1 and smad2/3 elicited motility defects, but RNAi of Dsh-B, wnt11-6, tsh, and APC significantly altered fission rate without changes in morphology. In situ staining of the CNS, intestine, and muscle confirmed published A/P polarity regeneration phenotypes, but no gross morphological defects in parent RNAi animals (Extended Data Fig. 4b-d). Therefore, we conclude that Wnt and TGF-β signaling components modulate fission behavior independent of overt body plan re-polarization. Serendipitously, we discovered that compression of planaria reveals cryptic mechanically vulnerable planes that divide the animal at regularly spaced intervals along the A/P axis (Fig. 3a, b, Video S3. The number of these ‘compression planes’ scaled with animal size (Fig. 3b, c) and their position along the A/P axis overlapped with the position of fission planes (Fig. 3d). Furthermore, incomplete fission formed tears similar to those observed with compression (Extended Data Fig. 5a). Therefore, we conclude that compression planes are fission planes revealed by mechanical compression. Fission plane number and distribution correlated with animal length during tissue re-scaling and regeneration. Following starvation, animals reduced body length and lost fission planes to restore number and distribution (Extended Data Fig. 5b-d). To assay fission plane regeneration, we amputated animals around the pharynx such that 90% of fragments contained a single plane (Extended Data Fig.5e-g). One week after amputation, animals remodeled, doubled in length, and increased fission plane number (Extended Data Fig.5f-j). Subsequent feeding increased animal length and fission plane number (Extended Data Fig.5f-j). Absent feeding, animals exhibited little to no elongation or plane addition despite re-scaling and regenerating their other tissues (Extended Data Fig.5f-j). In summary, fission planes are pre-established in planarians and scale dynamically with animal size and form. Given the role of Wnt and TGFβ signaling in body patterning, we tested whether these genes regulate fission planes. RNAi-treated animals were mechanically compressed and the quantity and relative distribution of fission planes was measured (Fig. 3e-g). Surprisingly, while RNAi of ActR-1 and smad2/3 moderately reduced the number of fission planes, RNAi of Wnt signaling components had no effect on fission plane number or position (Fig. 3e, g, Extendended data Fig. 6a). Even knockdown of wnt11-6 through 3 rounds of amputation/regeneration failed to alter fission plane patterning (Figure 3f, g, Extendended data Fig. 6b). Hypomorphic RNAi knockdown of β-catenin, ActR-1, or smad2/3 revealed little or no effect on fission fragment size (Extended Data Fig. 6c-e), further supporting the conclusion that neither Wnt nor TGF-β signaling regulate fission behavior through the A/P patterning of fission planes. We tested whether Wnt and TGF-β signaling instread regulated the frequency of fission attempts. Using the automated webcam image capture system (Fig. 1f), we recorded fission behavior in RNAi-treated animals (Figure 4a). Remarkably, RNAi of β-catenin, ActR-1, smad2/3, and wnt11-6 reduced fission attempts, while RNAi of APC increased fission attempts (Fig. 4b-d, Extended Data Fig. 7a-l, Video S5, Video S6). RNAi of β-catenin and smad2/3, which resulted in observable morphological abnormalities, also significantly reduced fission success ratio (Fig. 2d, 4e, Extended Data Fig. 7k-n). Dsh-B RNAi reduced the fission success ratio without altering the number or frequency of fission attempts (Fig. 4d, e, Extended Data Fig. 7k-n). Finally, APC RNAi reduced the time between fission attempts by ~50%, and animals initiated fission attempts independent of remaining tissue, dramatically reducing their success ratio (Fig. 4e, Extended Data Fig. 7i-n, Video S6). These findings demonstrate that Wnt and TGFβ signaling regulate the frequency of fission behavior. We hypothesized that Wnt and TGFβ signaling components might regulate fisison behavior through the planarian central nervous system (CNS). Double fluorescent in situ hybridization (FISH) with the CNS marker PC2 confirmed that Wnt and TGFβ fission regulators were detected in PC2+ cells in the anterior CNS (Extended Data Fig. 8a, b). Removal of anterior tissue containing the cephalic ganglia delayed the onset of fission behavior. (Extended Data Fig. 8c-f). Restoration of fission activity coincided with regeneration and re-establishment of anterior, PC2 co-localized, tsh expression (Extended Data Fig. 8g). Surprisingly, removal of anterior tissue containing just one cephalic ganglion did not alter total fissions produced (Extended Data Fig. 8c-f), indicating that half of the CNS is sufficient to initiate fission. Finally, RNAi against coe, a transcription factor essential for the patterning of the CNS 22,23 , dramatically reduced planarian fission (Extended Data Fig. 8h, i). Altogether, these data support a model in which an anterior CNS expressing Wnt and TGFβ signaling components regulates fission initiation. We hypothesized that polarity genes could modulate size-dependent behavior via size-dependent patterning of the CNS. To identify neuronal sub-populations regulating fission downstream of Wnt and TGFβ, we analyzed 17 neuronal markers 24-29 in small, medium, and large planaria and 10 markers in smad2/3 RNAi treated animals (Fig 4f, Extended Data Fig 9a, b). Patterning of pkd1L-2 +, gabrg3L-2 +, and sargasso-1 + mechanosensory neurons exhibited the clearest changes in both animals of increasing size and following smad2/3 RNAi treatment (Extended Data Fig 9a, b). In large animals, mechanosensory neurons are tightly restricted to the anterior and knockdown of either smad2/3 or wnt11-6 broadened their distribution akin to that of smaller animal (Fig 4g-l). RNAi against pkd1L-2 and gabrg3L-2 (homologous to cation and chloride channel genes, respectively) increased planarian fission activity (Fig 4m, n) and live imaging of gabrg3L-2 RNAi animals confirmed an increase in fission attempts without a reduction in fission success (Fig 4o-p, Extended Data Fig. 10, Video S7). These results indicate that mechanosensory neurons are differentially patterned during growth, inhibit fission behavior, and require Wnt and TGFβ for their appropriate patterning in accordance with animal size. Therefore, we conclude that Wnt and TGFβ signaling coordinates animal size and behavior via size-dependent patterning in the adult CNS. In conclusion, we used planaria as a model for the integration of size, patterning, and function and established fission as a robust, reproducible, and quantifiable size-dependent behavior (Fig. 1, Video S1). While previous studies have generated physical models for the process of transverse fission 9 , mechanisms coupling animal size and fission frequency have remained unknown. We discovered two independent mechanisms by which fission is coordinated with animal size in Schmidtea mediterranea. First, previously undescribed iterative structures patterned in accordance with A/P axis length couple animal size with the number of fission progeny produced (Fig. 3, Video S3). Second, the Wnt and TGFβ signaling pathways mediate size-dependent patterning of mechanosensory neurons, which regulate fission frequency (Fig. 4, Extended Data Fig. 9, 10). Thus, we demonstrate that differential patterning of key cell populations in accordance with tissue size provides a mechanistic link between animal growth and the acquisition or modulation of tissue function. Altogether, our results identify a novel role for Wnt and TGFβ patterning genes in regulating size-dependent behavior and show that developmental patterning cues coordinate tissue growth with size-dependent functions. Methods Animal Husbandry Clonal CIW4 Schmidtea mediterranea were maintained in 1X Montjuic salts as previously described. CIW4 animals were sourced from a large recirculation culture as previously reported 11 . In brief, animals are housed in three culture trays (96’ L × 24’ W × 12’ H) stacked vertically. Water is recirculated through the system by a sump pump, which moves water through a chiller, a canister filter, a UV sterilizer, and the three housing trays. Water is then passed through two vertically stacked sieves and a set of filter/floss pads before being returned to the sump pump. Animals were pulled from this system and either placed directly into fission assays, starved for at least 7 days prior to tissue fixation for imaging, or transferred to a unidirectional flow system culture for controlled feeding or RNAi feeding experiments. Gene cloning and RNAi Feeding protocol Candidate genes analyzed in this study were cloned from a CIW4 cDNA library into a pPR-T4P vector as described (Supplemental Table 1) 20 . These served as template for in vitro dsRNA synthesis for RNAi feedings. Unc22 dsRNA was used for control RNA treatment. RNAi food was prepared by mixing 1 volume of dsRNA at 1600 ng/ml with 1.5 volumes of beef liver paste. For RNAi experiments targeting neuronal genes, 1 volume of dsRNA at 1400ng/ul was mized with 1 volume beef liver paste. The amount of food administered was 10ul of food per 1mm of worm length present in the worm flow container. Worms were allowed to feed for 6 to 10 hours with 2 rounds of light stimulation to facilitate additional consumption. Worms were fed every three days for a total of 3 RNAi feedings, unless otherwise specified. After RNAi feedings, worms were transferred to the relevant biological assay. Fission Assay A detailed protocol for fission induction has been made available through Protocol Exchange 10 . To induce fission, animals were removed from re-circulation culture or unidirectional flow system culture and washed 5-10 times with fresh 1X Montjuic salts. Individual animals were placed in 15cm tissue culture dishes with 50mL 1X Montjuic salts and their body length was measured. Representative images of d0 parents were captured using a Leica M205 microscope. Plates were stacked 6-12 dishes high and placed in a dark incubator at 20ºC. Daily, plates were removed from the incubator and fission fragments for each animal were counted and removed from the 15cm dish. For some experiments, images of fission fragments were taken on the day they were collected to allow for quantitation of fission fragment length. The 1X Montjuic salts in each individual dish was replaced weekly. For data analysis, the number of daily cumulative fissions were divided by initial body length and then normalized to the average of the control RNAi fissions. This normalized fission score for each day was converted to a heat color code. Daily scores for each individual worm were aligned in descending order along the y-axis and the average score of each column was calculated and used to sort individual worms in ascending order along the x-axis. The average fission score of each RNAi condition was then sorted in ascending order from left to right. This resulted in a heat map visualization ranking the effects of RNAi treatments on fission activity. Fission Plane Compression Assay Fission planes were revealed by compression between a plastic tissue culture dish and a glass coverslip (See Video S3). Animals were inverted with their ventral side up, compressed using four fingertips, then imaged. To ensure that all compression/fission planes were revealed for every animal, images were acquired sequentially using a Leica M205 microscope as each fission plane was revealed by mechanical compression. Position of fission planes and distance between fission planes was quantified using Fiji (https://fiji.sc/). Video depicting compression assay was captured with an iPhone 6 (Apple). Fluorescent whole mount in situ hybridization (FISH) For RNA expression analyses, fluorescent in situ hybridization was performed as previously described 30,31 . Antibodies were used in MABT containing 5% horse serum for FISH (Roche anti-DIG-POD 1:1000 and Roche anti-FLCN-POD 1:1000) or NBT/BCIP in situ hybridization (Roche anti-DIG-AP 1:1000). For double FISH, peroxidase activity was quenched between tyramide reactions using 100mM sodium azide for at least 1 hour at room temperature with agitation. Nuclear staining was performed using 1:1000 Hoescht 33342 (Invitrogen) in PBSTx (1X PBS with 0.5% Triton-X-100). Microscopy Images of live worms and regenerating fission fragments were acquired using a Leica M205 microscope. Confocal images were acquired on an LSM-700-Vis and stitching was performed in Fiji using built-in grid collection plugins. Live Imaging of Fission Behavior Videos of worms from two orthogonal views were acquired using 2 webcams (Logitech C910/920). Webcams were mounted using a variety of ring stands and test tube clamps. The imaging chamber was a clear plastic square lid obtained from a box of coverslips. Lighting of the chamber was achieved using a Volpi illuminator (NCL-150). Each camera was connected to its own computer running micro-manager (https://micro-manager.org/). The cameras were set-up in micro-manager using OpenCVgrabber to set the pixel density (1920×1080) and to acquire the images. The camera gain, exposure and all other settings were set using the Logitech Webcam Controller software (https://download01.logi.com/web/ftp/pub/video/lws/lws280.exe) Data was acquired using the Multi-Dimensional Acquisition mode of micro-manager. The two computers were synchronized for acquisition manually at the beginning of the experiment. For the high-throughput screening of fission behavior, worms were placed in 6 well dishes with cameras mounted above the plates using optics components (Thor Labs). Illumination was obtained using 4 LED ring lights (AmScope) mounted upside down and above the cameras to provide diffuse light. Image acquisition was performed using two different camera configurations: four cameras connected to one computer via a USB hub or one 4K camera connected to a USB port. In the four-camera configuration, images where captured sequentially from the cameras every 10 minutes. A script written in Python 3.6 (https://www.python.org/) was used as a wrapper for FFMPEG (https://www.ffmpeg.org/) to acquire images. The size of the images (1920×1080) and the pixel format (yuv420p) were set in the python script. The camera gain, exposure, and other settings where controlled with the Logitech Webcam Controller software (https://download01.logi.com/web/ftp/pub/video/lws/lws280.exe). The DirectShow framework was used to interface between the cameras and FFMPEG. In the single 4k camera setup, a 4096×2160 pixel image was captured every ten minutes from a Logitech BRIO webcam. The same python script was used as a wrapper for FFMPEG in this configuration. Quantification of Live Imaging Videos of individual animals were manually annotated. For each fission attempt the start time and completion time were recorded and the success or failure of the attempt was recorded. To depict fission behavior, a timeline was constructed and a numerical value was given to each frame of a video. A value of 0 was assigned to any frame in which no fission behavior was observed, a positive value was given to any frame during a successful fission attempt, and a negative value was given to any frame during a failed fission attempt (see Fig. 1f). A prolonged diagonal line in a timeline indicates a period where frames were not acquired due to failed communication between the image acquisition software and the webcam. Statistical Tests For all pair-wise comparisons, significance was tested using an un-paired Student t-test. GraphPad Prism was used to calculate Pearson correlation coefficients with a two-tailed 95% confidence interval and to perform linear regression analyses. Two-way ANOVA analysis was performed in Graphpad Prism to determine the significance or RNAi treatment over time. Data Availability Statement: Source data and construct sequences can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1356. All other data are available from the corresponding author upon reasonable request Code Availability Statement: Code for the Python 3.6 (https://www.python.org/) script used for a wrapper for FFMPEG (https://www.ffmpeg.org/) for the high-throughput recording of fission behavior is available at the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1356. Extended Data Extended Data Fig. 1: Characterization of planarian fission biology. a, Live imaging of large planarian worm during fission (representative of 12 experiments, see also Video 1). b, Imaging of single individual large planarian and regenerating progeny 0, 4, 8, and 12 days after fission induction (experiment repeated 50 times). c, d, A/P length of progeny and time to fission event since induction or previous fission (mean, SEM, n=50 animals). Fission fragments binned by position along the A/P axis (1st fission = most posterior). e, Schematic representation of fission induction and quantitative scoring system used to compare fission activity between different conditions. f, Cumulative fission fragments produced over 14 days by individual animals binned by parent size (n=10 per bin). g, h, Time to first fission event or time between sequential fission events for animals 6-8mm, 9-12mm, or 13-17mm in length. i, Raw parent length measurement of planarian individuals 6-8mm, 9-12mm, or 13-17mm in length. j, Time between 1st and 2nd fission events for animals 6-8mm, 9-12mm, or 13-17mm in length (mean, SEM, n= 139 independent measurements from 30 animals). k, l, Time between induction and first fission or between 1st and 2nd fission plotted relative to parent length, with Pearson correlation co-efficient, linear regression, and R2 value (n= 26 and 21 independent measurements from 30 animals). Statistics determined by two-sided t-test. Extended Data Fig. 2: Quantitation of fission behavior across a range of animal sizes. a, All individual timelines depicting fission activity over 9 days for animals ranging from 2mm (bottom) to 12mm (top) in length (n=39 animals). b, Number of successful fission attempts per animal relative to parent length. c, d, Number of fission attempts and time to 1st fission attempt for animals binned into small (2-5mm), medium (6-7mm), and large (8-12mm) groups (mean, SEM, n=16 small, 11 medium, 12 large animals). Extended Data Fig.3: Strategy for a targeted RNAi screen to identify regulators of fission. a, Detailed schematic of RNAi workflow. Animals are grown to an optimal size in the recirculation culture system and transferred to a flow system for RNAi feedings. After 3 RNAi feedings, worms were transferred to a 15cm dish and animal length recorded. The number of fissions were recorded daily for 14 days for each worm from each RNAi condition. For data analysis, the number of daily cumulative fissions were divided by initial body length and then normalized to the average of the control RNAi fissions. For data visualization, this normalized fission score for each day was converted to a heat color code. Daily scores for each individual worm were aligned in ascending order along the y-axis. The average score of each column is calculated and used to sort individual worms in ascending order along the x-axis. The average fission score of each RNAi condition was then sorted in ascending order from left to right. The result is a heat-map visualization ranking the effects of RNAi treatments on fission activity. b, Wnt, TGF-β, Hh signaling pathway diagrams focusing on components targeted for RNAi screen. Green arrows indicate positive interaction and red arrows indicate inhibitory interaction. Extended Data Fig. 4: Analysis of morphology and/or internal tissues in regenerating fragments and fissioning parents. a, Representative images of regenerating tissue fragments from different positions along the A/P axis at 15 days post amputation (dpa). Fraction of animals with pictured phenotype along with 1mm scale bar depicted below each image. b, c, In situ staining of CNS (PC2), intestine (porc) and muscle (t-mus) tissues at (b) day 15 of regeneration or (c) the fission assay. d, High resolution image of body wall musculature (t-mus) in control RNAi and smad2/3 or β-catenin RNAi treated animals. Representative images (n=7-13 animals). All images are oriented ventral side up with anterior on the left side. (Experiment performed a single time. Scale, 0.5mm) Extended Data Fig. 5: Effects of Growth, Starvation, and Regeneration on fission planes. a, Image of planaria after incomplete fission, revealing ventral tear identical to compression planes (observed greater than 5 independent times). b, Post-compression worms at 5dpf, 18dpf and 30dpf (5dpf image from same experiment as Fig. 3b; single experiment).c, d, Bidirectional plot (c) of compression planes versus animal length (n=25, mean, SD), and relative distribution (d) of planes at 5dpf, 18dpf, or 30dpf (n=28, 18, 31, 15, and 19 animals). e, Schematic of experiment tracking establishment of fission planes during tissue regeneration. f, g, Representative images and bidirectional plot of compression planes versus animal length (mean, SD) following amputation (1dpa, n=15 animals), regeneration (8dpa, n=19 animals), and growth (Fed 14dpa and 25dpa, n= 12 and 32 animals) or de-growth (Starved 25dpa, n=15 animals) (single experiment). h-j, Graph of (h) animal length, (i) # of compression planes (mean, SEM), and (j) relative distribution of planes following amputation (1dpa, n=15 animals), regeneration (8dpa, n=19 animals), and growth (Fed 14dpa and 25dpa, n= 12 and 32 animals) or de-growth (Starved 25dpa, n=15 animals). Extended Data Fig. 6: Effects of Wnt/TGF-β signaling component RNAi on fission planes. a, b, Relative plane distribution following RNAi treatment (n=20 and 10 animals). c, representative images of progeny within 24 hours of fission and of remaining parent tissue at day 28 after fission induction for animals treated with control, β-catenin, ActR-1, or smad2/3 RNAi. (experimentally independently performed twice. Scale, 1mm). d, e, Length of the first fission progeny or all subsequent progeny in animals treated with control, β-catenin, ActR-1, or smad2/3 RNAi (mean, SEM, n= 85 fission fragments from 36 animals). p-value calculated with 2-way ANOVA Interaction Factor (a, b) or two-sided t-test (d, e). Extended Data Fig. 7: Wnt and TGFβ signaling components regulate the frequency of fission initiation.1 a-h, All individual timelines depicting fission activity over 9-10 days for animals treated with (a,g) Control, (b) ActR-1, (c) smad2/3, (d) β-catenin, (e) DshB, (f) APC, or (h) wnt11-6 RNAi. i-n, Graphs depicting (i, j) the time between sequential fission attempt, (k, l) the number of successful fission attempts, (m,n) the number of unsuccessful fission attempts in animals fed dsRNA targeting fission regulators (mean, SEM, n= 421 fission events from 116 animals). Animals were given either 3 (a-f, i, k, m) or 18 (g,h,j,l,n) dsRNA feedings. Batched experiments are plotted separately. p-value calculated with two-sided t-test. Extended Data Fig. 8: The planarian anterior CNS regulates fission. a, Whole brain imaging of PC2 and fission regulator gene expression detected by double fluorescent in situ hybridization (n=2-4 animals; experiment independently repeated; Scale, 100μm). b, Single cell co-expression of PC2 and fission regulators in the posterior branches of the anterior CNS. (n= 3-5 animals; Scale bar, 50μm) (n=3-5). c, Fission induction in intact, 100% head amputated, or 50% head amputated animals over a 9-day observation period (n=12 animals). d-f, Plot of (d) total number of fission progeny over 9 days, (e) time between fission induction and 1st fission, (f) and time between 1st and 2nd fission for intact, 100% head amputated, or 50% head amputated animals (mean, SEM, n= 94 fission events from 36 animals). g, Regeneration time course in 100% head amputated animals showing recovery of anterior gene expression of PC-2 co-localized with teashirt. (n=4-5 animals; experiment performed a single time; Scale, 500μm). h, Heatmaps depicting fission activity following coe RNAi treatment. Normalized cumulative fissions over time are displayed for individual worms from each RNAi condition (n=12 animals). i, Representative parent images on days 0 and 14 of the fission assay (n=12, experiment independently performed twice; Scale, 1mm). p-value calculated with two-sided t-test (d-f) or 2-WAY ANOVA (h). Extended Data Fig. 9: Comparison of neuronal subpopulations in animals of increasing size and following smad2/3 RNAi treatment a, Representative images of neuronal marker staining in small, medium and large animals (n=3-5 animals; 1 experiment). b, Representative images of a subset of neuronal markers analyzed in smad2/3 RNAi-treated animals (n=3-5 animals; 1 experiment). Scale, 0.5mm. Extended Data Fig. 10: Gabrg3L-2 negatively regulates the frequency of fission initiation. a-b, All individual timelines depicting fission activity over 9 days for animals treated with (a) Control or (b) gabrg3L-2 RNAi (n=recordings of 10 animals combined from two independent experiments). Supplementary Material 1 2 3 4 5 6 7 8 9