DSP1/CnB Regulates Planarian (Dugesia japonica) Cephalic Ganglia Regeneration

DSP1/CnB Regulates Planarian (Dugesia japonica) Cephalic Ganglia Regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article DSP1/CnB Regulates Planarian (Dugesia japonica) Cephalic Ganglia Regeneration Lili Gao, Mingyue Zheng, Ziyuzhu Kong, Yutong Li, Jiaxin Li, Fengtang Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9686603/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Regeneration and repair of the injured central nervous system is a major focus in biological research, yet many key questions remain unanswered, making it essential to explore the mechanisms underlying neural regeneration. Dorsal switch protein 1 (DSP1) plays critical roles in embryonic development and immune regulation in vertebrates, but its function in CNS regeneration has not been reported. In this study, we found that DSP1 was widely expressed in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. Knockdown of DSP1 promoted blastema growth at 1 dpa, without affecting the stem cell population. Silencing of DSP1 accelerated brain regeneration and significantly increased the number of daughter cells derived from stem cells, accompanied by downregulated expression of CnB , a Wnt signaling pathway gene. Notably, simultaneous knockdown of DSP1 and CnB promoted blastema regeneration. Double-knockdown DSP1 and CnB exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. These findings provide a theoretical basis for understanding the DSP1/CnB signaling axis in regulating neural regeneration in mammals, and have important implications for the development of drugs targeting neuronal regeneration. DSP1 Dugesia japonica cephalic ganglia regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Adult stem cells possess immeasurable medical value in the field of regenerative medicine, providing new strategies for the effective treatment of many major diseases. Neural stem cells serve as the cellular basis for neural self-repair and regeneration. Upon neural injury or neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease, the body can rely on endogenous stem cells to repair damaged tissues and generate new neurons and synapses (Heneka et al., 2015 ; Li and Clevers, 2010 ). Recent studies have shown that the mammalian central nervous system (CNS) can be repaired by endogenous neural stem cells (Naffaa, 2025 ; Yao X, 2025). However, due to constraints imposed by the intrinsic properties of stem cells and the internal microenvironment, their differentiation capacity is limited, resulting in extremely restricted regeneration in the adult mammalian central nervous system (Pilz et al., 2018 ; Taupin, 2011 ). Investigating the processes and molecular mechanisms by which adult stem cells participate in neural regeneration in vivo may represent an important direction for the future development of regenerative medicine. Therefore, understanding the mechanisms by which endogenous stem cells regulate neural regeneration is of great significance for neural regenerative medicine. Planarians, as typical representatives of flatworms, not only occupy an important position in animal evolution but also are well suited for investigating tissue regeneration and stem cell regulation owing to their extraordinary regenerative capacity. Upon amputation, each fragment can regenerate into a complete individual within approximately one week, and this robust regenerative ability is mainly attributed to the abundant population of stem cells (neoblasts), which account for about 25%–30% of the total cells (Zeng et al., 2018 ). Studies have demonstrated that neoblasts, as the only proliferative cell population in planarians, are classified into 12 subpopulations; among them, Nb2 represents a totipotent stem cell population, while Nb11 is a neural stem/progenitor cell population capable of proliferating and differentiating into the nervous system (Zeng et al., 2018 ). When planarians are injured, neoblasts are rapidly activated, enabling efficient regeneration of missing tissues and organs, including the CNS. The planarian CNS consists of a pair of inverted U‑shaped cephalic ganglia (brain), photoreceptors (eyes), and ventral nerve cords extending along the body length (Cebria, 2007 ). Research has indicated that the planarian brain comprises approximately 2,000–10,000 neurons, including classical neuronal types conserved in humans, such as dopaminergic, cholinergic, and GABAergic neurons, and these neuronal subtypes are precisely restored in terms of spatial location and number after each regeneration event (Nishimura et al., 2007a ; Nishimura et al., 2007b ; Nishimura et al., 2010 ; Nishimura et al., 2008 ). Therefore, planarians can serve as an excellent animal model for studying neural regeneration and stem cell biology. Studies have demonstrated that the proliferation, migration, and directed differentiation of stem cells during planarian neural regeneration are tightly regulated by a complex network of genes and signaling pathways. For instance, the Wnt signaling pathway mediates the establishment of the anterior-posterior axis in planarians, and downregulated expression of genes within this pathway can lead to posterior expansion of the brain (Kobayashi et al., 2007 ). The EGFR signaling pathway plays a crucial role in cell differentiation during planarian regeneration, with involvement in the early differentiation of brain neurons during head regeneration (Fraguas et al., 2014 ). Additionally, downregulation of Tec-1 expression results in an increased in several neuronal subtypes during both planarian regeneration and tissue homeostasis maintenance (Karge et al., 2020 ). As a key regulatory factor of neural precursor cells in planarians, Coe orchestrates the formation of dopaminergic, cholinergic, and GABAergic neurons during brain neural regeneration (Cowles et al., 2013 ; Cowles et al., 2014 ). Our previous studies have demonstrated that the Wnt/Ca²⁺ signaling pathway modulates the formation of GABAergic neurons during planarian brain regeneration; downregulation of genes associated with this pathway results in multiple regenerative abnormalities, such as hypoplastic regenerated brain nerves, failure of lateral nerve regeneration, and a decrease in GABAergic neurons—effects primarily mediated by the regulation of neural cell differentiation (Zhen et al., 2020 ). However, the molecular mechanisms underlying the directed differentiation of pluripotent stem cells into neural stem cells in planarians, as well as the processes by which neural stem cells further differentiate into neurons and glial cells, remain partly elucidated. DSP1 is a homolog of vertebrate HMGB1 (high mobility group protein 1), which is widely expressed in the nucleus and regulates transcription and chromatin reorganization via DNA binding (Bianchi et al., 2017 ). Studies have demonstrated that DSP1 can be passively released from dying cells or actively secreted by activated immune cells, intestinal cells, hepatocytes, and other cell populations (Tsung et al., 2007 ); as a damage-associated molecular pattern (DAMP) molecule, it initiates the innate immune response (Roh and Sohn, 2018 ). In Drosophila , nuclear DSP1 functions as a co-activator of the Dorsal protein to modulate Drosophila growth and development (Mahi Imam Mollah, 2025 ; Mosrin-Huaman et al., 1998 ). In Aedes aegypti , DSP1 facilitates chromatin remodeling of Toll-associated transcription factors, thereby enhancing their binding to promoters in response to immune challenges (de Mendonca Amarante et al., 2017 ). Upon bacterial attack in Spodoptera litura , DSP1 is released into the hemolymph and mediates various immune responses through PLA2 activation (Mollah et al., 2021 ). DSP1 is expressed throughout embryonic development: it is ubiquitously distributed in the first stage (cell blastoderm and germ band) and restricted to the central nervous system at the final stage. In adults, DSP1 protein is exclusively detected in the ovaries and brain (Mosrin-Huaman et al., 1998 ). Collectively, these findings indicate that DSP1 plays a critical role in embryonic development and immune regulation in vertebrates. However, whether DSP1 contributes to central nervous system regeneration remains unreported to date. In this study, we found that DSP1 was widely expressed in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. Knockdown of DSP1 promoted blastema growth at 1 day post regeneration, without affecting the stem cell population. Silencing of DSP1 accelerated brain regeneration and significantly increased the number of daughter cells derived from stem cell differentiation, accompanied by downregulated expression of CnB , a Wnt signaling pathway gene. Notably, simultaneous knockdown of DSP1 and CnB promoted blastema regeneration. These observations suggest that DSP1 / CnB regulates neural stem cells to sustain planarian cephalic ganglia regeneration. Materials and methods Planarians Dugesia japonica planarians were reared under controlled laboratory conditions: they were housed in spring water with a stable ambient temperature set at 20°C. To eliminate potential interference from recent feeding, all planarians were subjected to a mandatory fasting period of no less than 7 days before the start of any experimental procedures. For the regeneration experiments, individual planarians were transected to remove heads; the trunk fragments were then collected and used for a series of analyses at predetermined time points (0, 1, 3, 5, 7, and 10 days post-amputation (dpa)). WISH (Whole mount in situ hybridization) WISH experiments were performed as previously described (Zhen et al., 2020 ). In brief, hybridization reactions were performed by incubating samples with the antisense RNA probe (1 ng/µL) at 56°C for 16 h. then, the samples were rinsed with maleic acid buffer, followed by a 2 h blocking step with 10% horse serum at room temperature. Next, antibodies diluted in Tween 20-supplemented maleic acid buffer (MABT) containing 10% horse serum were added for subsequent WISH (anti-DIG-AP, 1:2000, Roche). The samples were then observed under microscopy and recorded using CCD camera. RNA interference The dsRNAs corresponding to DSP1 and CnB were generated via the MEGAscript™ RNAi Kit purchased from Invitrogen (USA). Planarians assigned to the experimental group were immersed in a dsRNA solution at a concentration of 40 ng/µL for 6 h prior to their transfer into the designated culture medium. In parallel, planarians in the control group were treated with GFP-targeting dsRNA under the same conditions. Immunohistochemistry Immunostaining was conducted following our established protocol (Zhen et al., 2020 ). Briefly, planarians were euthanized in 2% HCl, fixed in paraformaldehyde at 37°C for 1 h, and dehydrated in anhydrous methanol. Samples were incubated with primary antibodies: Anti-SYNAPSIN (1:300, AB Company, USA) and Anti-phosphohistone-H3 (S10) (hereafter H3P) (1:200, Cell Signaling Technology, USA), followed by overnight incubation at 4°C with HRP-conjugated secondary antibodies (1:200, rabbit anti-mouse and goat anti-rabbit, EarthOx, USA). Immunofluorescent signals were visualized and captured using a laser scanning confocal microscope (Zeiss LSM900). Quantitative real-time PCR (qPCR) Briefly, total RNA was isolated using TRIzol reagent (Invitrogen, USA). qPCR amplifications were performed on a qTower 3G real-time PCR system (Analytikjena, Germany) with ChamQ Universal SYBR qPCR Master Mix (Q711–03, Vazyme, China) at an annealing temperature of 55°C. Relative expression levels, normalized to Gapdh , were calculated via the 2 ⁻ΔΔCt method. RNA-seq analysis Three days post the final dsRNA treatment; total RNA was isolated from three independent control and DSP1 RNAi groups using TRIzol reagent. RNA quality and concentration were assessed via an Agilent 2100 Bioanalyser and a NanoDrop ND-2000 spectrophotometer, respectively. Only high-quality RNA samples meeting the following criteria were used for library construction: OD₂₆₀/₂₈₀ ratio of 1.8–2.2, OD₂₆₀/₂₃₀ ratio ≥ 2.0, RNA integrity number (RIN) ≥ 6.5, 28S:18S ratio ≥ 1.0, and total yield > 1 µg. RNA sequencing data were analyzed on the Majorbio Cloud platform ( www.majorbio.com ). Heatmaps were generated using the R programming language combined with fastcluster software. For the target gene set, clustering was based on the log₂-transformed expression ratios between pairwise samples; inter-gene distances were computed via a corresponding algorithm and iteratively optimized to classify genes into distinct subclusters. Statistical analysis Statistical analyses were carried out using SPSS statistical software for Windows, version 16.0 (SPSS). Comparisons between groups were carried out using t-Tests. Data are presented as mean ± standard error of the mean (SEM). For all analyses, * p < 0.05 and ** p < 0.01 were considered statistically significant. Results Expression of DSP1 in the intact and regenerative planarians Using an optimized WISH protocol (see Materials and Methods), we detected robust DSP1 expression throughout the body in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia (arrows) (Fig. 1 A). To characterize its expression dynamics during regeneration, qPCR was performed at 1, 3, 5, 7, and 10 dpa on trunk and tail fragments. The results showed that the expression level of DSP1 exhibited dynamic changes during regeneration: it decreased at 1 and 3 dpa, increased at 5 dpa, and decreased again at 7 dpa. (*p < 0.05; **p < 0.01) (Fig. 1 B). WISH analysis further indicated that no positive signal was detected at the regeneration site at 1 dpa. Signals of DSP1 were predominantly restricted to the brain primordia of regenerating trunk and tail fragments at 3 dpa (arrows, Fig. 1 C) and, as regeneration proceeded, became increasingly concentrated in newly regenerated cephalic ganglia (Fig. 1 C). Together, these results suggest that DSP1 may play a critical role in the planarian cephalic ganglia regeneration. Knockdown of DSP1 causes accelerated planarian regeneration To clarify the physiological functions of DSP1 , we conducted RNAi using a soaking method that is widely used in planarian research [39]. As demonstrated in Fig. 2 A and B, three rounds of DSP1 RNAi treatment significantly reduced the expression of DSP1 in D. japonica . To examine the function of DSP1 in regeneration, planarians underwent head and tail segments cutting on the second day post the final RNAi treatment. The regenerated trunk fragments were examined at 1, 3, 5, 7 and 10 dpa. As shown in Fig. 2 C and D, compared with the control group, the blastema was significantly larger in DSP1 RNAi-treatment planarians at 1 dpa. At 3 dpa, the brain primordium in the DSP1 RNAi group was larger than that in the control group. Over time, regeneration in the RNAi group gradually became complete. At 10 dpa, head pigmentation had recovered to its original state, whereas the newly formed head tissue in the control group remained white. These results suggest that DSP1 may play an important role during planarian brain regeneration. To further investigate whether DSP1 promotes planarian regeneration, we examined the brain nerve regeneration using WISH ( Chat , TH ) and immunofluorescence (Anti-SYNAPSIN, the pan-neuronal marker anti-SYNORF1 (3C11) targeting synapsin was used to label the whole planarian CNS). As shown in Fig. S1 A, compared with the control group, brain nerve regeneration was more complete in the DSP1 RNAi group. Specifically, at 5 dpa, the regenerated brain nerves were larger and thicker in the DSP1 RNAi group. At 7dpa, the brain nerves were further enlarged, and lateral nerves emerged in the DSP1 RNAi group. At 10 dpa, although the brain nerves in the control group were also fully regenerated, those in the DSP1 RNAi group remained larger, with more distinct lateral nerves. WISH of Chat and TH showed that the regeneration of Chat + and TH + neurons in the DSP1 RNAi group was more complete (Fig. S1 B-E). These results further indicated that DSP1 could promote regeneration in planarians, particularly in cephalic ganglia regeneration. Knockdown of DSP1 not affect stem cells The abnormal phenotypes of regenerated cranial nerves in planarians may result from abnormalities in stem cells or neural stem cells. To explore DSP1 ’s role in planarian head regeneration, we assessed the expression of the neoblast marker DjPiwiA in regenerating trunk fragments via WISH and qPCR. As displayed in Fig. S2 A and B, knockdown of DSP1 had no significant effect on DjPiwiA expression at 1, 3, 5, 7, and 10 dpa compared with the control group. Subsequently, whether DSP1 was involved in the regulation of stem cell proliferation was further investigated by immunofluorescence detection of H3P + in regenerating planarians at 1, 2, 3 and 5 dpa after DSP1 RNAi treatment. As expected, knockdown of DSP1 did not alter cell proliferation levels in regenerating planarians (Fig. S2 C and 3D), indicating that silencing of DSP1 expression did not alters planarian stem cell counts, confirming that head regeneration anomalies arise independently of stem cell numbers. Downregulation of DSP1 led to a reduction in early progeny of neoblasts during planarian head regeneration To determine the role of DSP1 in differentiation in planarians, the expression of differentiation marker genes, including early progeny of neoblasts marker gene NB.21 , late progeny of neoblasts marker gene Agat-1 , neural stem cell ( Ston2 ), neurons ( Chat , GAD , TH , TPH and Neuropeptide F ), were chosen for analyses from the RNA-seq data in regenerated trunk fragments (1 dpa). The result showed that the expression of differentiation marker genes was upregulated following DSP1 RNAi (Fig. 3 E). Consistently, qPCR results confirmed that the expression patterns of multiple genes were consistent with those obtained from RNA-seq analysis (Fig. 3 D). As illustrated in Fig. 3 A and B, the expression of both the early progeny marker NB.21 and the late progeny marker Agat-1 was significantly elevated in DSP1 RNAi-treated planarians. To investigate the function of DSP1 in neuronal differentiation, we conducted WISH using the neural stem cell marker Ston2 . As shown in Fig. 3 C, the brain primordia in DSP1 RNAi-treated planarians exhibited a significant enlargement at 3 dpa compared with the control group, implying that neuronal differentiation was impaired in regenerating planarians after DSP1 RNAi treatment. RNA-seq analysis following knockdown of DSP1 To further elucidate how DSP1 modulates head regeneration in planarians, RNA-seq was conducted on planarians after DSP1 RNAi knockdown. A total of 915 differentially expressed genes (DEGs) were identified via RNA-seq profiling, with 696 transcripts upregulated and 219 downregulated (Fig. 4 A and B). KEGG enrichment analysis was subsequently performed to annotate signaling pathways associated with downregulated DEGs, with the top 20 enriched pathways visualized in bubble plots (Fig. 4 C). The Wnt signaling pathway mediated the formation of the anteroposterior axis in planarians, and downregulation of genes in this pathway could lead to a complete cessation of neural regeneration (Bonar et al., 2022 ; Pascual-Carreras et al., 2023 ; Scimone et al., 2016 ). The Wnt signaling pathway (red box) has been given special attention (red box) (Fig. 4 C, Table S1 ). Among these transcriptional changes, calcineurin subunit B ( CnB ) was found to be downregulated, suggesting its potential requirement for planarian head regeneration. Co-knockdown of DSP1 and CnB led to a modest acceleration of planarian regeneration relative to single DSP1 knockdown. To investigate whether defects induced by DSP1 RNAi are associated with alterations in CnB signaling during planarian regeneration, we performed single-gene knockdown targeting either DSP1 or CnB , as well as co-knockdown of DSP1 and CnB via RNAi. Notably, CnB RNAi did not exert any significant effect on CNS regeneration in planarians (Fig. 5 and S3). As shown in Fig. 5 and S3, co-knockdown of DSP1 and CnB led to a modest acceleration of planarian regeneration relative to single DSP1 knockdown. Consistently, co-knockdown of DSP1 and CnB led to a larger brain nerve in planarians at 5, 7 and 10 dpa relative to single DSP1 knockdown (Fig. 6 A). And co-knockdown of DSP1 and CnB led to a larger brain primordium in planarians at 3 dpa relative to single DSP1 knockdown. (Fig. 6 B and C). Correspondingly, co-knockdown of DSP1 and CnB led to moderately more TH + , TPH + and GAD + neurons in regenerating planarians relative to single DSP1 knockdown (Fig. 7 ). Double-knockdown DSP1 and CnB exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. Taken together, our data indicate that DSP1 likely negative regulated neural stem cell differentiation through the positive regulation of CnB signaling pathways during planarian brain regeneration (Fig. 8 ). Discussions Planarians provide a unique experimental model for investigating stem cell and neural regeneration in vivo. In this study, we found that DSP1 was widely expressed in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. Knockdown of DSP1 promoted blastema growth at 1 dpa, with no obvious effect on the stem cell population. DSP1 silencing accelerated brain regeneration and significantly increased the number of daughter cells derived from stem cell differentiation, accompanied by downregulated expression of CnB , a Wnt signaling pathway gene. Notably, simultaneous knockdown of DSP1 and CnB resulted in a more pronounced phenotype that promotes blastema regeneration. Double-knockdown DSP1 and CnB exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. Our results indicate that DSP1 likely negative regulates neural stem cell differentiation through regulating CnB during planarian brain regeneration. DSP1, an insect ortholog of the vertebrate high mobility group box 1 (HMGB1) protein that was highly conserved across eukaryotic cells, was initially characterized in 1994 as a co-repressor of Dorsal in Drosophila melanogaster (Bianchi, 2009 ; Lehming et al., 1994 ). DSP1 is a protein encoded by the DSP1 gene, featuring a glutamine-rich N-terminal domain, an acidic C-terminal tail, and two HMG boxes (HMG box A and HMG box B) (Canaple et al., 1997 ). Studies have revealed that DSP1 was expressed in the ovaries and brain of adult Drosophila , underscoring its significance in embryonic development (Lehming et al., 1994 ). Consistent with previous studies, we found that DSP1 was expressed throughout the body in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. The results also showed that the expression level of DSP1 exhibited dynamic changes during planarian regeneration (Fig. 1 ). These results suggest that DSP1 may play a critical role in the planarian cephalic ganglia regeneration. DSP1 plays a critical role in DNA binding including transcription factors and chromatin remodeling complexes (Stros et al., 2007 ). Prior studies have shown that DSP1 interacts with Dorsal and modulates its transcriptional activity; Dorsal is a critical transcription factor that mediates dorso-ventral patterning during embryonic development in Drosophila (Brickman et al., 1999 ; Lehming et al., 1994 ). Additionally, HMGB1(the mammalian ortholog of DSP1) is linked to a range of neurodegenerative disorders, including Parkinson’s disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS) (Ikram et al., 2022 ). Overexpression of DSP1 in flies led to reduced climbing capacity and longevity, alongside NMJ abnormalities, smaller eyes, and decreased TH-positive neurons, demonstrating DSP1-induced neuronal toxicity (Baek et al., 2024 ). Distinct from previous findings, knockdown of DSP1 gave rise to abnormal regeneration including moderately larger blastema and brain primordia, multiple neurons ( TH , Chat , GAD and TPH -positive neurons) during regeneration compared with the control group at the same time points (Fig. 2 , 3 , 7 and S1). Downregulation of DSP1 did not affect changes in neoblasts, but led to abnormalities in progenitor cells of stem cells, such as an increase in neural stem cells (Fig. 3 and S1). These findings indicate that DSP1 likely modulates the normal regeneration of brain neural during planarian regeneration by regulating neural stem cells. In planarians, Wnt signal pathway regulated anteroposterior axis regeneration (β-catenin gradient), and non-canonical Wnt mediated mediolateral patterning and neural regeneration (Gurley et al., 2008 ; Petersen, 2023 ). Calcineurin (CaN) is a Ca²⁺/calmodulin-regulated eukaryotic serine/threonine phosphatase, forming a heterodimer with catalytic (A) and myristoylated (B) subunits (Rusnak and Mertz, 2000 ). CaN exhibits extensive subcellular localization within neurons (Jiang et al., 2012 ). CnB governs the transcription of neuronal migration-related genes via modulation of the NFAT signaling pathway, which is indispensable for proper cerebral cortex and cerebellum development (Kipanyula et al., 2016 ). Our results indicate a regulatory interaction between DSP1 and CnB , which is critical for enabling their essential functions in neural system physiology. Knockdown of DSP1 led to significant down-regulation of CnB expression in planarians (Fig. 3 E). Co-knockdown of DSP1 and CnB led to a modest acceleration of planarian regeneration relative to single DSP1 knockdown (Figs. 5 , 6 and 7 ). Double-knockdown DSP1 and CnB exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. In conclusion, our study identified planarian DSP1 as a conserved key regulator that modulates neural stem cell function via CnB to govern neuronal regeneration (Fig. 8 ). These findings a theoretical basis for understanding the DSP1/CnB signaling axis in regulating neural regeneration in mammals, and have important implications for the development of drugs targeting neuronal regeneration. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fundings This work was supported by the National Natural Science Foundation of China (32400391, 31701308), the Space Application System of China Manned Space Program (SCP03-01-05), the Natural Science Foundation of Shandong Province (ZR2024QC156, ZR2022QC026, ZR2025QC338). Author Contribution Study conception and design were carried out by Lili Gao and Mingyue Zheng, who also executed most of the experiments, data analysis, and manuscript co-preparation. Ziyuzhu Kong, Yutong Li and Jiaxin Li helped with analyzed data and data acquisition. Hui Zhen, Zhonghong Cao and Fengtang Yang oversaw the project, acquired funding, interpreted results, and revised the manuscript critically. All of the authors read, reviewed, and gave final approval of the manuscript. 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Front Biosci (Schol Ed) 3:178–190 Tsung A, Klune JR, Zhang X, Jeyabalan G, Cao Z, Peng X, Stolz DB, Geller DA, Rosengart MR, Billiar TR (2007) HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med 204:2913–2923 Yao X, Liu ZY, Jie Y, Wan J, Yang X P (2025) Engineered exosome-based treatment for peripheral nerve regeneration: a narrative review of clinical prospects. Adv Technol Neurosci 2, 135–143 Zeng A, Li H, Guo L, Gao X, McKinney S, Wang Y, Yu Z, Park J, Semerad C, Ross E et al (2018) Prospectively Isolated Tetraspanin(+) Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration. Cell 173 , 1593–1608 e1520 Zhen H, Deng H, Song Q, Zheng M, Yuan Z, Cao Z, Pang Q, Zhao B (2020) The Wnt/Ca(2+) signaling pathway is essential for the regeneration of GABAergic neurons in planarian Dugesia japonica. FASEB J 34:16567–16580 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx TableS1.csv Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9686603","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638716016,"identity":"4ee889ad-38cb-4e85-863a-7b2dc4a3250c","order_by":0,"name":"Lili Gao","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Gao","suffix":""},{"id":638716017,"identity":"2b0a4fed-3f0c-4f30-b238-027896b5079e","order_by":1,"name":"Mingyue Zheng","email":"","orcid":"","institution":"Qilu Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Zheng","suffix":""},{"id":638716018,"identity":"e41ec01d-2933-4b75-ad92-cc4dfb471286","order_by":2,"name":"Ziyuzhu Kong","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ziyuzhu","middleName":"","lastName":"Kong","suffix":""},{"id":638716019,"identity":"60c1a9fc-85a8-4467-857b-5f369dd77eb6","order_by":3,"name":"Yutong Li","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yutong","middleName":"","lastName":"Li","suffix":""},{"id":638716020,"identity":"2a05a33d-a408-4c64-b2b7-08277d7faa2e","order_by":4,"name":"Jiaxin Li","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Li","suffix":""},{"id":638716021,"identity":"226b89ee-9ffd-49ad-b915-b06deb46f4ed","order_by":5,"name":"Fengtang Yang","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Fengtang","middleName":"","lastName":"Yang","suffix":""},{"id":638716022,"identity":"27a3df02-430a-419e-9c83-957a1b08a9c6","order_by":6,"name":"Zhonghong Cao","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhonghong","middleName":"","lastName":"Cao","suffix":""},{"id":638716023,"identity":"00719213-12d3-4cb6-ac7e-a0e279654051","order_by":7,"name":"Hui Zhen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDACCQYGZiDJw8bMfvBBQoUN8Vpk+Nl5kg0enEkjWguDjWQ/g5nkw7ZDhHXIz24+9riwzYLH4DBDWkUC2wEG/vbuBLxaGOccSzee2SYB1MJ47EYCzx0GiTNnN+DVwiyRYybNC9bCkHYjQeIZg4FELn4tbBL532BazAoSgCRBLTwSOWxgLZLNDGYMCQlEaJGQSDOT5jknwcPPzJMskXAgjYegX+RnJD+T5imrs2fjP37w489/NnL87b34tWC6lDTlo2AUjIJRMAqwAgCbPT56l2zMXwAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhen","suffix":""}],"badges":[],"createdAt":"2026-05-12 05:08:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9686603/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9686603/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109198717,"identity":"3d4a95f7-633d-48b1-b6d5-89b3d5d82761","added_by":"auto","created_at":"2026-05-13 13:29:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":741984,"visible":true,"origin":"","legend":"\u003cp\u003eSpatiotemporal expression of\u003cem\u003e DSP1\u003c/em\u003e in planarians\u003c/p\u003e\n\u003cp\u003e(A) Expression analysis of \u003cem\u003eDSP1\u003c/em\u003e in the intact planarians by WISH. Scale bars: 250 μm. (B) qPCR analysis of \u003cem\u003eDSP1\u003c/em\u003eexpression the regenerated planarians at 1, 3, 5, 7, 10 dpa. n = 5. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (C) Expression analysis of \u003cem\u003eDSP1\u003c/em\u003e at 1, 3, 5, 7 and 10 dpa. Scale bars: 250 μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/ad2f3af88ad8d2a19b63e50c.png"},{"id":109198750,"identity":"b3c420ea-d08d-41e7-935c-0d57a7ebafc2","added_by":"auto","created_at":"2026-05-13 13:30:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":367146,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of \u003cem\u003eDSP1\u003c/em\u003e significantly accelerates the regeneration process in planarians.\u003c/p\u003e\n\u003cp\u003e(A) Flowchart of RNAi and amputation schedules for this study. (B) qPCR analysis of RNAi efficiency after \u003cem\u003eDSP1\u003c/em\u003eRNAi. n = 5. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (C) Quantification of blastema regeneration area, n = 5. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ns, not significant. (D) Representative images of morphologic changes in the regenerated planarians at 1, 3, 5, 7 and 10 dpa after \u003cem\u003eDSP1\u003c/em\u003e RNAi. Scale bars: 250 μm. n = 20.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/2594552e19b776689227cfac.png"},{"id":109198730,"identity":"ec7cf8e5-0637-4c70-9f74-1c8f1801edb8","added_by":"auto","created_at":"2026-05-13 13:29:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":181106,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of \u003cem\u003eDSP1\u003c/em\u003e affects differentiation marker genes expression\u003c/p\u003e\n\u003cp\u003e(A) Expression of \u003cem\u003eNB.21 \u003c/em\u003eexamined by WISH in the regenerated planarians at 1 dpa and 3 dpa after \u003cem\u003eDSP1\u003c/em\u003e RNAi. Scale bar: 250 μm. (B) Expression of \u003cem\u003eAgat-1\u003c/em\u003eexamined by WISH in the regenerated planarians at 3 dpa and 5 dpa after \u003cem\u003eDSP1\u003c/em\u003eRNAi. Scale bar: 250 μm. (C) Expression of \u003cem\u003eSton2 \u003c/em\u003eexamined by WISH in the regenerated planarians at 3 dpa after \u003cem\u003eDSP1\u003c/em\u003e RNAi. Scale bar: 250 μm. (D) qPCR of the \u003cem\u003eNB.21\u003c/em\u003e, \u003cem\u003eAgat-1\u003c/em\u003e and \u003cem\u003eSton2 \u003c/em\u003ein the regenerated planarians at 3 dpa after \u003cem\u003eDSP1\u003c/em\u003e RNAi. n = 5. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (E) Heatmap of genes expression of \u003cem\u003eDSP1\u003c/em\u003e, \u003cem\u003eNB.21\u003c/em\u003e, \u003cem\u003eCnB\u003c/em\u003e, \u003cem\u003eGAD\u003c/em\u003e, \u003cem\u003eTH\u003c/em\u003e, \u003cem\u003eAgat-1\u003c/em\u003e, \u003cem\u003eTPH\u003c/em\u003e, \u003cem\u003eNeuropeptide F\u003c/em\u003e, \u003cem\u003eSton2\u003c/em\u003e, \u003cem\u003eChat\u003c/em\u003eand \u003cem\u003eOtxB\u003c/em\u003e in the regenerated planarians at 1 dpa after \u003cem\u003eDSP1\u003c/em\u003e RNAi, in comparison with that treated with control RNAi.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/99d88354d90ec7ffef686e7f.png"},{"id":109198751,"identity":"8289474d-9dc1-4432-8b93-6fb076f32ba4","added_by":"auto","created_at":"2026-05-13 13:30:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95288,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis after knockdown of \u003cem\u003eDSP1\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) The number of DEGs identified after \u003cem\u003eDSP1\u003c/em\u003e RNAi, with a threshold of the ratio change ≥2 and a q-value of \u0026lt;0.05. The red and blue columns represent up- and down-regulated genes in planarians after \u003cem\u003eDSP1\u003c/em\u003e RNAi. (C) KEGG enrichment analysis to identify signaling pathways involved in down-regulated DEGs after\u003cem\u003e DSP1\u003c/em\u003e RNAi.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/3c67ffe0d86ea2730e2c5021.png"},{"id":109198721,"identity":"50a205f1-e25f-42f6-8d45-d421182a97c4","added_by":"auto","created_at":"2026-05-13 13:29:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":725929,"visible":true,"origin":"","legend":"\u003cp\u003eCo-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e significantly accelerates the regeneration process in planarians.\u003c/p\u003e\n\u003cp\u003e(A) Representative images of morphologic changes in the regenerated planarians at 1, 3, and 5 dpa after Control RNAi, \u003cem\u003eDSP1\u003c/em\u003e RNAi, \u003cem\u003eCnB\u003c/em\u003e RNAi and \u003cem\u003eDSP1\u003c/em\u003e+\u003cem\u003eCnB \u003c/em\u003eRNAi. Scale bars: 250 μm. n = 20. (B) Quantification of blastema regeneration area, n = 5. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. ns, not significant.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/020cad7713b4eb0ed77c14b6.png"},{"id":109198719,"identity":"902e6c5e-0b23-4d4a-9811-449bc79f91ff","added_by":"auto","created_at":"2026-05-13 13:29:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":402701,"visible":true,"origin":"","legend":"\u003cp\u003eCo-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e significantly accelerates the brain nerve regeneration in planarians.\u003c/p\u003e\n\u003cp\u003e(A) Whole-mount immunohistochemistry with antibodies against SYNAPSIN in the regenerated planarians at 5, 7 and 10 dpa after either \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB \u003c/em\u003eRNAi alone or \u003cem\u003eDSP1\u003c/em\u003e+ \u003cem\u003eCnB \u003c/em\u003edouble RNAi. Scale bars: 100 μm. (B) FISH of \u003cem\u003eSton2\u003c/em\u003e in the regenerated planarians at 3 dpa after either \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB \u003c/em\u003eRNAi alone or \u003cem\u003eDSP1\u003c/em\u003e+ CnB double RNAi. Scale bar: 100 μm. (C) Quantification of blastema regeneration area of \u003cem\u003eSton2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells, n = 5. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/ffedd0da0249af1a5a5ebf80.png"},{"id":109198722,"identity":"ac9739ec-3655-4118-bb3f-e33d8cc244ab","added_by":"auto","created_at":"2026-05-13 13:29:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":537718,"visible":true,"origin":"","legend":"\u003cp\u003eWISH of different neurons\u003c/p\u003e\n\u003cp\u003e(A) WISH of \u003cem\u003eTH\u003c/em\u003e in the regenerated planarians at 7 dpa after either \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB \u003c/em\u003eRNAi alone or \u003cem\u003eDSP1\u003c/em\u003e+ CnB double RNAi. Scale bar: 250 μm. (B) Quantification of regeneration area of \u003cem\u003eTH\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells, n = 5. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (C) WISH of \u003cem\u003eTPH\u003c/em\u003e in the regenerated planarians at 7 dpa after either \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB \u003c/em\u003eRNAi alone or \u003cem\u003eDSP1\u003c/em\u003e+ CnB double RNAi. Scale bar: 250 μm. (D) Quantification of regeneration area of \u003cem\u003eTPH\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells, n = 5. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (E) WISH of \u003cem\u003eGAD\u003c/em\u003e in the regenerated planarians at 7 dpa after either \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB \u003c/em\u003eRNAi alone or \u003cem\u003eDSP1\u003c/em\u003e+ CnB double RNAi. Scale bar: 250 μm. (F) Quantification of regeneration area of \u003cem\u003eGAD\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e cells, n = 5. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/21875cf0ae8b94421f8171ea.png"},{"id":109198720,"identity":"6f06dfac-ea8c-4f1d-bc0c-4edcf7e8a353","added_by":"auto","created_at":"2026-05-13 13:29:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":238709,"visible":true,"origin":"","legend":"\u003cp\u003eProposed models illustrating the essential role of DSP1/CnB signaling in planarian cranial nerve regeneration\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDSP1\u003c/em\u003eas a conserved key regulator that modulates neural stem cell function via \u003cem\u003eCnB \u003c/em\u003eto govern neuronal regeneration\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/23e451780fb6a2ce34805b16.png"},{"id":109205539,"identity":"a83bf35d-1fa5-44a7-be41-25f14ed56387","added_by":"auto","created_at":"2026-05-13 15:05:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3299056,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/8bf5b47e-b6fa-4a1b-b205-7029f6a275fa.pdf"},{"id":109198749,"identity":"881e0dd3-fc43-477c-a072-af369e29eec1","added_by":"auto","created_at":"2026-05-13 13:30:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4324911,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/f382a3c2dc9d87b38b7b7c3f.docx"},{"id":109198731,"identity":"fdc089f0-925b-46f9-ad1c-a9d8761d3157","added_by":"auto","created_at":"2026-05-13 13:29:54","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":91978,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.csv","url":"https://assets-eu.researchsquare.com/files/rs-9686603/v1/c3db99684e1e86e091c72695.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"DSP1/CnB Regulates Planarian (Dugesia japonica) Cephalic Ganglia Regeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdult stem cells possess immeasurable medical value in the field of regenerative medicine, providing new strategies for the effective treatment of many major diseases. Neural stem cells serve as the cellular basis for neural self-repair and regeneration. Upon neural injury or neurodegenerative diseases such as Parkinson\u0026rsquo;s disease and Alzheimer\u0026rsquo;s disease, the body can rely on endogenous stem cells to repair damaged tissues and generate new neurons and synapses (Heneka et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Li and Clevers, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Recent studies have shown that the mammalian central nervous system (CNS) can be repaired by endogenous neural stem cells (Naffaa, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yao X, 2025). However, due to constraints imposed by the intrinsic properties of stem cells and the internal microenvironment, their differentiation capacity is limited, resulting in extremely restricted regeneration in the adult mammalian central nervous system (Pilz et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Taupin, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Investigating the processes and molecular mechanisms by which adult stem cells participate in neural regeneration in vivo may represent an important direction for the future development of regenerative medicine. Therefore, understanding the mechanisms by which endogenous stem cells regulate neural regeneration is of great significance for neural regenerative medicine.\u003c/p\u003e \u003cp\u003ePlanarians, as typical representatives of flatworms, not only occupy an important position in animal evolution but also are well suited for investigating tissue regeneration and stem cell regulation owing to their extraordinary regenerative capacity. Upon amputation, each fragment can regenerate into a complete individual within approximately one week, and this robust regenerative ability is mainly attributed to the abundant population of stem cells (neoblasts), which account for about 25%\u0026ndash;30% of the total cells (Zeng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Studies have demonstrated that neoblasts, as the only proliferative cell population in planarians, are classified into 12 subpopulations; among them, Nb2 represents a totipotent stem cell population, while Nb11 is a neural stem/progenitor cell population capable of proliferating and differentiating into the nervous system (Zeng et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). When planarians are injured, neoblasts are rapidly activated, enabling efficient regeneration of missing tissues and organs, including the CNS. The planarian CNS consists of a pair of inverted U‑shaped cephalic ganglia (brain), photoreceptors (eyes), and ventral nerve cords extending along the body length (Cebria, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Research has indicated that the planarian brain comprises approximately 2,000\u0026ndash;10,000 neurons, including classical neuronal types conserved in humans, such as dopaminergic, cholinergic, and GABAergic neurons, and these neuronal subtypes are precisely restored in terms of spatial location and number after each regeneration event (Nishimura et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007a\u003c/span\u003e; Nishimura et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007b\u003c/span\u003e; Nishimura et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nishimura et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, planarians can serve as an excellent animal model for studying neural regeneration and stem cell biology.\u003c/p\u003e \u003cp\u003eStudies have demonstrated that the proliferation, migration, and directed differentiation of stem cells during planarian neural regeneration are tightly regulated by a complex network of genes and signaling pathways. For instance, the Wnt signaling pathway mediates the establishment of the anterior-posterior axis in planarians, and downregulated expression of genes within this pathway can lead to posterior expansion of the brain (Kobayashi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The EGFR signaling pathway plays a crucial role in cell differentiation during planarian regeneration, with involvement in the early differentiation of brain neurons during head regeneration (Fraguas et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, downregulation of \u003cem\u003eTec-1\u003c/em\u003e expression results in an increased in several neuronal subtypes during both planarian regeneration and tissue homeostasis maintenance (Karge et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a key regulatory factor of neural precursor cells in planarians, \u003cem\u003eCoe\u003c/em\u003e orchestrates the formation of dopaminergic, cholinergic, and GABAergic neurons during brain neural regeneration (Cowles et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Cowles et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our previous studies have demonstrated that the Wnt/Ca\u0026sup2;⁺ signaling pathway modulates the formation of GABAergic neurons during planarian brain regeneration; downregulation of genes associated with this pathway results in multiple regenerative abnormalities, such as hypoplastic regenerated brain nerves, failure of lateral nerve regeneration, and a decrease in GABAergic neurons\u0026mdash;effects primarily mediated by the regulation of neural cell differentiation (Zhen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the molecular mechanisms underlying the directed differentiation of pluripotent stem cells into neural stem cells in planarians, as well as the processes by which neural stem cells further differentiate into neurons and glial cells, remain partly elucidated.\u003c/p\u003e \u003cp\u003eDSP1 is a homolog of vertebrate HMGB1 (high mobility group protein 1), which is widely expressed in the nucleus and regulates transcription and chromatin reorganization via DNA binding (Bianchi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Studies have demonstrated that DSP1 can be passively released from dying cells or actively secreted by activated immune cells, intestinal cells, hepatocytes, and other cell populations (Tsung et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2007\u003c/span\u003e); as a damage-associated molecular pattern (DAMP) molecule, it initiates the innate immune response (Roh and Sohn, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In \u003cem\u003eDrosophila\u003c/em\u003e, nuclear DSP1 functions as a co-activator of the Dorsal protein to modulate \u003cem\u003eDrosophila\u003c/em\u003e growth and development (Mahi Imam Mollah, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Mosrin-Huaman et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In \u003cem\u003eAedes aegypti\u003c/em\u003e, DSP1 facilitates chromatin remodeling of Toll-associated transcription factors, thereby enhancing their binding to promoters in response to immune challenges (de Mendonca Amarante et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Upon bacterial attack in \u003cem\u003eSpodoptera litura\u003c/em\u003e, DSP1 is released into the hemolymph and mediates various immune responses through PLA2 activation (Mollah et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). DSP1 is expressed throughout embryonic development: it is ubiquitously distributed in the first stage (cell blastoderm and germ band) and restricted to the central nervous system at the final stage. In adults, DSP1 protein is exclusively detected in the ovaries and brain (Mosrin-Huaman et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Collectively, these findings indicate that DSP1 plays a critical role in embryonic development and immune regulation in vertebrates. However, whether DSP1 contributes to central nervous system regeneration remains unreported to date.\u003c/p\u003e \u003cp\u003eIn this study, we found that \u003cem\u003eDSP1\u003c/em\u003e was widely expressed in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. Knockdown of \u003cem\u003eDSP1\u003c/em\u003e promoted blastema growth at 1 day post regeneration, without affecting the stem cell population. Silencing of \u003cem\u003eDSP1\u003c/em\u003e accelerated brain regeneration and significantly increased the number of daughter cells derived from stem cell differentiation, accompanied by downregulated expression of \u003cem\u003eCnB\u003c/em\u003e, a Wnt signaling pathway gene. Notably, simultaneous knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e promoted blastema regeneration. These observations suggest that \u003cem\u003eDSP1\u003c/em\u003e/\u003cem\u003eCnB\u003c/em\u003e regulates neural stem cells to sustain planarian cephalic ganglia regeneration.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlanarians\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDugesia japonica\u003c/em\u003e planarians were reared under controlled laboratory conditions: they were housed in spring water with a stable ambient temperature set at 20\u0026deg;C. To eliminate potential interference from recent feeding, all planarians were subjected to a mandatory fasting period of no less than 7 days before the start of any experimental procedures. For the regeneration experiments, individual planarians were transected to remove heads; the trunk fragments were then collected and used for a series of analyses at predetermined time points (0, 1, 3, 5, 7, and 10 days post-amputation (dpa)).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWISH (Whole mount in situ hybridization)\u003c/h3\u003e\n\u003cp\u003eWISH experiments were performed as previously described (Zhen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In brief, hybridization reactions were performed by incubating samples with the antisense RNA probe (1 ng/\u0026micro;L) at 56\u0026deg;C for 16 h. then, the samples were rinsed with maleic acid buffer, followed by a 2 h blocking step with 10% horse serum at room temperature. Next, antibodies diluted in Tween 20-supplemented maleic acid buffer (MABT) containing 10% horse serum were added for subsequent WISH (anti-DIG-AP, 1:2000, Roche). The samples were then observed under microscopy and recorded using CCD camera.\u003c/p\u003e\n\u003ch3\u003eRNA interference\u003c/h3\u003e\n\u003cp\u003eThe dsRNAs corresponding to \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e were generated via the MEGAscript\u0026trade; RNAi Kit purchased from Invitrogen (USA). Planarians assigned to the experimental group were immersed in a dsRNA solution at a concentration of 40 ng/\u0026micro;L for 6 h prior to their transfer into the designated culture medium. In parallel, planarians in the control group were treated with GFP-targeting dsRNA under the same conditions.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunostaining was conducted following our established protocol (Zhen et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, planarians were euthanized in 2% HCl, fixed in paraformaldehyde at 37\u0026deg;C for 1 h, and dehydrated in anhydrous methanol. Samples were incubated with primary antibodies: Anti-SYNAPSIN (1:300, AB Company, USA) and Anti-phosphohistone-H3 (S10) (hereafter H3P) (1:200, Cell Signaling Technology, USA), followed by overnight incubation at 4\u0026deg;C with HRP-conjugated secondary antibodies (1:200, rabbit anti-mouse and goat anti-rabbit, EarthOx, USA). Immunofluorescent signals were visualized and captured using a laser scanning confocal microscope (Zeiss LSM900).\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eBriefly, total RNA was isolated using TRIzol reagent (Invitrogen, USA). qPCR amplifications were performed on a qTower 3G real-time PCR system (Analytikjena, Germany) with ChamQ Universal SYBR qPCR Master Mix (Q711\u0026ndash;03, Vazyme, China) at an annealing temperature of 55\u0026deg;C. Relative expression levels, normalized to \u003cem\u003eGapdh\u003c/em\u003e, were calculated via the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eThree days post the final dsRNA treatment; total RNA was isolated from three independent control and \u003cem\u003eDSP1\u003c/em\u003e RNAi groups using TRIzol reagent. RNA quality and concentration were assessed via an Agilent 2100 Bioanalyser and a NanoDrop ND-2000 spectrophotometer, respectively. Only high-quality RNA samples meeting the following criteria were used for library construction: OD₂₆₀/₂₈₀ ratio of 1.8\u0026ndash;2.2, OD₂₆₀/₂₃₀ ratio\u0026thinsp;\u0026ge;\u0026thinsp;2.0, RNA integrity number (RIN)\u0026thinsp;\u0026ge;\u0026thinsp;6.5, 28S:18S ratio\u0026thinsp;\u0026ge;\u0026thinsp;1.0, and total yield\u0026thinsp;\u0026gt;\u0026thinsp;1 \u0026micro;g. RNA sequencing data were analyzed on the Majorbio Cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.majorbio.com\" target=\"_blank\"\u003ewww.majorbio.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Heatmaps were generated using the R programming language combined with fastcluster software. For the target gene set, clustering was based on the log₂-transformed expression ratios between pairwise samples; inter-gene distances were computed via a corresponding algorithm and iteratively optimized to classify genes into distinct subclusters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were carried out using SPSS statistical software for Windows, version 16.0 (SPSS). Comparisons between groups were carried out using t-Tests. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). For all analyses, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e \u003cb\u003ein the intact and regenerative planarians\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing an optimized WISH protocol (see Materials and Methods), we detected robust \u003cem\u003eDSP1\u003c/em\u003e expression throughout the body in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia (arrows) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To characterize its expression dynamics during regeneration, qPCR was performed at 1, 3, 5, 7, and 10 dpa on trunk and tail fragments. The results showed that the expression level of \u003cem\u003eDSP1\u003c/em\u003e exhibited dynamic changes during regeneration: it decreased at 1 and 3 dpa, increased at 5 dpa, and decreased again at 7 dpa. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). WISH analysis further indicated that no positive signal was detected at the regeneration site at 1 dpa. Signals of \u003cem\u003eDSP1\u003c/em\u003e were predominantly restricted to the brain primordia of regenerating trunk and tail fragments at 3 dpa (arrows, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and, as regeneration proceeded, became increasingly concentrated in newly regenerated cephalic ganglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Together, these results suggest that \u003cem\u003eDSP1\u003c/em\u003e may play a critical role in the planarian cephalic ganglia regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e \u003cb\u003ecauses accelerated planarian regeneration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo clarify the physiological functions of \u003cem\u003eDSP1\u003c/em\u003e, we conducted RNAi using a soaking method that is widely used in planarian research [39]. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B, three rounds of \u003cem\u003eDSP1\u003c/em\u003e RNAi treatment significantly reduced the expression of \u003cem\u003eDSP1\u003c/em\u003e in \u003cem\u003eD. japonica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the function of \u003cem\u003eDSP1\u003c/em\u003e in regeneration, planarians underwent head and tail segments cutting on the second day post the final RNAi treatment. The regenerated trunk fragments were examined at 1, 3, 5, 7 and 10 dpa. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, compared with the control group, the blastema was significantly larger in \u003cem\u003eDSP1\u003c/em\u003e RNAi-treatment planarians at 1 dpa. At 3 dpa, the brain primordium in the \u003cem\u003eDSP1\u003c/em\u003e RNAi group was larger than that in the control group. Over time, regeneration in the RNAi group gradually became complete. At 10 dpa, head pigmentation had recovered to its original state, whereas the newly formed head tissue in the control group remained white. These results suggest that \u003cem\u003eDSP1\u003c/em\u003e may play an important role during planarian brain regeneration.\u003c/p\u003e \u003cp\u003eTo further investigate whether \u003cem\u003eDSP1\u003c/em\u003e promotes planarian regeneration, we examined the brain nerve regeneration using WISH (\u003cem\u003eChat\u003c/em\u003e, \u003cem\u003eTH\u003c/em\u003e) and immunofluorescence (Anti-SYNAPSIN, the pan-neuronal marker anti-SYNORF1 (3C11) targeting synapsin was used to label the whole planarian CNS). As shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, compared with the control group, brain nerve regeneration was more complete in the \u003cem\u003eDSP1\u003c/em\u003e RNAi group. Specifically, at 5 dpa, the regenerated brain nerves were larger and thicker in the \u003cem\u003eDSP1\u003c/em\u003e RNAi group. At 7dpa, the brain nerves were further enlarged, and lateral nerves emerged in the \u003cem\u003eDSP1\u003c/em\u003e RNAi group. At 10 dpa, although the brain nerves in the control group were also fully regenerated, those in the \u003cem\u003eDSP1\u003c/em\u003e RNAi group remained larger, with more distinct lateral nerves. WISH of \u003cem\u003eChat\u003c/em\u003e and \u003cem\u003eTH\u003c/em\u003e showed that the regeneration of \u003cem\u003eChat\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTH\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e neurons in the \u003cem\u003eDSP1\u003c/em\u003e RNAi group was more complete (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-E). These results further indicated that \u003cem\u003eDSP1\u003c/em\u003e could promote regeneration in planarians, particularly in cephalic ganglia regeneration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e \u003cb\u003enot affect stem cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe abnormal phenotypes of regenerated cranial nerves in planarians may result from abnormalities in stem cells or neural stem cells. To explore \u003cem\u003eDSP1\u003c/em\u003e\u0026rsquo;s role in planarian head regeneration, we assessed the expression of the neoblast marker \u003cem\u003eDjPiwiA\u003c/em\u003e in regenerating trunk fragments via WISH and qPCR. As displayed in Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and B, knockdown of \u003cem\u003eDSP1\u003c/em\u003e had no significant effect on \u003cem\u003eDjPiwiA\u003c/em\u003e expression at 1, 3, 5, 7, and 10 dpa compared with the control group. Subsequently, whether \u003cem\u003eDSP1\u003c/em\u003e was involved in the regulation of stem cell proliferation was further investigated by immunofluorescence detection of H3P\u003csup\u003e+\u003c/sup\u003e in regenerating planarians at 1, 2, 3 and 5 dpa after \u003cem\u003eDSP1\u003c/em\u003e RNAi treatment. As expected, knockdown of \u003cem\u003eDSP1\u003c/em\u003e did not alter cell proliferation levels in regenerating planarians (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC and 3D), indicating that silencing of \u003cem\u003eDSP1\u003c/em\u003e expression did not alters planarian stem cell counts, confirming that head regeneration anomalies arise independently of stem cell numbers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDownregulation of\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e \u003cb\u003eled to a reduction in early progeny of neoblasts during planarian head regeneration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the role of \u003cem\u003eDSP1\u003c/em\u003e in differentiation in planarians, the expression of differentiation marker genes, including early progeny of neoblasts marker gene \u003cem\u003eNB.21\u003c/em\u003e, late progeny of neoblasts marker gene \u003cem\u003eAgat-1\u003c/em\u003e, neural stem cell (\u003cem\u003eSton2\u003c/em\u003e), neurons (\u003cem\u003eChat\u003c/em\u003e, \u003cem\u003eGAD\u003c/em\u003e, \u003cem\u003eTH\u003c/em\u003e, \u003cem\u003eTPH\u003c/em\u003e and \u003cem\u003eNeuropeptide F\u003c/em\u003e), were chosen for analyses from the RNA-seq data in regenerated trunk fragments (1 dpa). The result showed that the expression of differentiation marker genes was upregulated following \u003cem\u003eDSP1\u003c/em\u003e RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Consistently, qPCR results confirmed that the expression patterns of multiple genes were consistent with those obtained from RNA-seq analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B, the expression of both the early progeny marker \u003cem\u003eNB.21\u003c/em\u003e and the late progeny marker \u003cem\u003eAgat-1\u003c/em\u003e was significantly elevated in \u003cem\u003eDSP1\u003c/em\u003e RNAi-treated planarians. To investigate the function of \u003cem\u003eDSP1\u003c/em\u003e in neuronal differentiation, we conducted WISH using the neural stem cell marker \u003cem\u003eSton2\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the brain primordia in \u003cem\u003eDSP1\u003c/em\u003e RNAi-treated planarians exhibited a significant enlargement at 3 dpa compared with the control group, implying that neuronal differentiation was impaired in regenerating planarians after \u003cem\u003eDSP1\u003c/em\u003e RNAi treatment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq analysis following knockdown of\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further elucidate how \u003cem\u003eDSP1\u003c/em\u003e modulates head regeneration in planarians, RNA-seq was conducted on planarians after \u003cem\u003eDSP1\u003c/em\u003e RNAi knockdown. A total of 915 differentially expressed genes (DEGs) were identified via RNA-seq profiling, with 696 transcripts upregulated and 219 downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). KEGG enrichment analysis was subsequently performed to annotate signaling pathways associated with downregulated DEGs, with the top 20 enriched pathways visualized in bubble plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The Wnt signaling pathway mediated the formation of the anteroposterior axis in planarians, and downregulation of genes in this pathway could lead to a complete cessation of neural regeneration (Bonar et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pascual-Carreras et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scimone et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The Wnt signaling pathway (red box) has been given special attention (red box) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among these transcriptional changes, calcineurin subunit B (\u003cem\u003eCnB\u003c/em\u003e) was found to be downregulated, suggesting its potential requirement for planarian head regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-knockdown of\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCnB\u003c/b\u003e \u003cb\u003eled to a modest acceleration of planarian regeneration relative to single\u003c/b\u003e \u003cb\u003eDSP1\u003c/b\u003e \u003cb\u003eknockdown.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether defects induced by \u003cem\u003eDSP1\u003c/em\u003e RNAi are associated with alterations in \u003cem\u003eCnB\u003c/em\u003e signaling during planarian regeneration, we performed single-gene knockdown targeting either \u003cem\u003eDSP1\u003c/em\u003e or \u003cem\u003eCnB\u003c/em\u003e, as well as co-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e via RNAi. Notably, \u003cem\u003eCnB\u003c/em\u003e RNAi did not exert any significant effect on CNS regeneration in planarians (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and S3). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and S3, co-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e led to a modest acceleration of planarian regeneration relative to single \u003cem\u003eDSP1\u003c/em\u003e knockdown. Consistently, co-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e led to a larger brain nerve in planarians at 5, 7 and 10 dpa relative to single \u003cem\u003eDSP1\u003c/em\u003e knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). And co-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e led to a larger brain primordium in planarians at 3 dpa relative to single \u003cem\u003eDSP1\u003c/em\u003e knockdown. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C). Correspondingly, co-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e led to moderately more \u003cem\u003eTH\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eTPH\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGAD\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e neurons in regenerating planarians relative to single \u003cem\u003eDSP1\u003c/em\u003e knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Double-knockdown \u003cem\u003eDSP1\u003c/em\u003eand \u003cem\u003eCnB\u003c/em\u003e exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. Taken together, our data indicate that \u003cem\u003eDSP1\u003c/em\u003e likely negative regulated neural stem cell differentiation through the positive regulation of \u003cem\u003eCnB\u003c/em\u003e signaling pathways during planarian brain regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e "},{"header":"Discussions","content":"\u003cp\u003ePlanarians provide a unique experimental model for investigating stem cell and neural regeneration in vivo. In this study, we found that \u003cem\u003eDSP1\u003c/em\u003e was widely expressed in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. Knockdown of \u003cem\u003eDSP1\u003c/em\u003e promoted blastema growth at 1 dpa, with no obvious effect on the stem cell population. \u003cem\u003eDSP1\u003c/em\u003e silencing accelerated brain regeneration and significantly increased the number of daughter cells derived from stem cell differentiation, accompanied by downregulated expression of \u003cem\u003eCnB\u003c/em\u003e, a Wnt signaling pathway gene. Notably, simultaneous knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e resulted in a more pronounced phenotype that promotes blastema regeneration. Double-knockdown \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. Our results indicate that \u003cem\u003eDSP1\u003c/em\u003e likely negative regulates neural stem cell differentiation through regulating \u003cem\u003eCnB\u003c/em\u003e during planarian brain regeneration.\u003c/p\u003e \u003cp\u003eDSP1, an insect ortholog of the vertebrate high mobility group box 1 (HMGB1) protein that was highly conserved across eukaryotic cells, was initially characterized in 1994 as a co-repressor of Dorsal in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (Bianchi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lehming et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). DSP1 is a protein encoded by the \u003cem\u003eDSP1\u003c/em\u003e gene, featuring a glutamine-rich N-terminal domain, an acidic C-terminal tail, and two HMG boxes (HMG box A and HMG box B) (Canaple et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Studies have revealed that \u003cem\u003eDSP1\u003c/em\u003e was expressed in the ovaries and brain of adult \u003cem\u003eDrosophila\u003c/em\u003e, underscoring its significance in embryonic development (Lehming et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Consistent with previous studies, we found that \u003cem\u003eDSP1\u003c/em\u003e was expressed throughout the body in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. The results also showed that the expression level of \u003cem\u003eDSP1\u003c/em\u003e exhibited dynamic changes during planarian regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggest that \u003cem\u003eDSP1\u003c/em\u003e may play a critical role in the planarian cephalic ganglia regeneration.\u003c/p\u003e \u003cp\u003eDSP1 plays a critical role in DNA binding including transcription factors and chromatin remodeling complexes (Stros et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Prior studies have shown that DSP1 interacts with Dorsal and modulates its transcriptional activity; Dorsal is a critical transcription factor that mediates dorso-ventral patterning during embryonic development in Drosophila (Brickman et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Lehming et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Additionally, HMGB1(the mammalian ortholog of DSP1) is linked to a range of neurodegenerative disorders, including Parkinson\u0026rsquo;s disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS) (Ikram et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overexpression of DSP1 in flies led to reduced climbing capacity and longevity, alongside NMJ abnormalities, smaller eyes, and decreased TH-positive neurons, demonstrating DSP1-induced neuronal toxicity (Baek et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Distinct from previous findings, knockdown of \u003cem\u003eDSP1\u003c/em\u003e gave rise to abnormal regeneration including moderately larger blastema and brain primordia, multiple neurons (\u003cem\u003eTH\u003c/em\u003e, \u003cem\u003eChat\u003c/em\u003e, \u003cem\u003eGAD\u003c/em\u003e and \u003cem\u003eTPH\u003c/em\u003e-positive neurons) during regeneration compared with the control group at the same time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and S1). Downregulation of \u003cem\u003eDSP1\u003c/em\u003e did not affect changes in neoblasts, but led to abnormalities in progenitor cells of stem cells, such as an increase in neural stem cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S1). These findings indicate that \u003cem\u003eDSP1\u003c/em\u003e likely modulates the normal regeneration of brain neural during planarian regeneration by regulating neural stem cells.\u003c/p\u003e \u003cp\u003eIn planarians, Wnt signal pathway regulated anteroposterior axis regeneration (β-catenin gradient), and non-canonical Wnt mediated mediolateral patterning and neural regeneration (Gurley et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Petersen, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Calcineurin (CaN) is a Ca\u0026sup2;⁺/calmodulin-regulated eukaryotic serine/threonine phosphatase, forming a heterodimer with catalytic (A) and myristoylated (B) subunits (Rusnak and Mertz, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). CaN exhibits extensive subcellular localization within neurons (Jiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). CnB governs the transcription of neuronal migration-related genes via modulation of the NFAT signaling pathway, which is indispensable for proper cerebral cortex and cerebellum development (Kipanyula et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our results indicate a regulatory interaction between \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e, which is critical for enabling their essential functions in neural system physiology. Knockdown of \u003cem\u003eDSP1\u003c/em\u003e led to significant down-regulation of \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eCnB\u003c/span\u003e expression in planarians (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Co-knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e led to a modest acceleration of planarian regeneration relative to single \u003cem\u003eDSP1\u003c/em\u003e knockdown (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Double-knockdown \u003cem\u003eDSP1\u003c/em\u003eand \u003cem\u003eCnB\u003c/em\u003e exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration.\u003c/p\u003e \u003cp\u003eIn conclusion, our study identified planarian \u003cem\u003eDSP1\u003c/em\u003e as a conserved key regulator that modulates neural stem cell function via \u003cem\u003eCnB\u003c/em\u003e to govern neuronal regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings a theoretical basis for understanding the DSP1/CnB signaling axis in regulating neural regeneration in mammals, and have important implications for the development of drugs targeting neuronal regeneration.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003ch2\u003eFundings\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32400391, 31701308), the Space Application System of China Manned Space Program (SCP03-01-05), the Natural Science Foundation of Shandong Province (ZR2024QC156, ZR2022QC026, ZR2025QC338).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eStudy conception and design were carried out by Lili Gao and Mingyue Zheng, who also executed most of the experiments, data analysis, and manuscript co-preparation. Ziyuzhu Kong, Yutong Li and Jiaxin Li helped with analyzed data and data acquisition. Hui Zhen, Zhonghong Cao and Fengtang Yang oversaw the project, acquired funding, interpreted results, and revised the manuscript critically. All of the authors read, reviewed, and gave final approval of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaek SE, Kwon Y, Yoon JW, Kim HS, Yang JY, Lee DS, Yeom E (2024) The overexpression of DSP1 in neurons induces neuronal dysfunction and neurodegeneration phenotypes in Drosophila. Mol Brain 17:43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBianchi ME (2009) HMGB1 loves company. 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FASEB J 34:16567\u0026ndash;16580\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DSP1, Dugesia japonica, cephalic ganglia, regeneration","lastPublishedDoi":"10.21203/rs.3.rs-9686603/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9686603/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegeneration and repair of the injured central nervous system is a major focus in biological research, yet many key questions remain unanswered, making it essential to explore the mechanisms underlying neural regeneration. Dorsal switch protein 1 (DSP1) plays critical roles in embryonic development and immune regulation in vertebrates, but its function in CNS regeneration has not been reported. In this study, we found that \u003cem\u003eDSP1\u003c/em\u003e was widely expressed in the intact planarian, predominantly in parenchyma, retropharyngeal and cephalic ganglia. Knockdown of \u003cem\u003eDSP1\u003c/em\u003e promoted blastema growth at 1 dpa, without affecting the stem cell population. Silencing of \u003cem\u003eDSP1\u003c/em\u003e accelerated brain regeneration and significantly increased the number of daughter cells derived from stem cells, accompanied by downregulated expression of \u003cem\u003eCnB\u003c/em\u003e, a Wnt signaling pathway gene. Notably, simultaneous knockdown of \u003cem\u003eDSP1\u003c/em\u003e and \u003cem\u003eCnB\u003c/em\u003e promoted blastema regeneration. Double-knockdown \u003cem\u003eDSP1\u003c/em\u003eand \u003cem\u003eCnB\u003c/em\u003e exacerbate the phenotypes, suggesting that the two signal systems function maybe dependently in the process of brain regeneration. These findings provide a theoretical basis for understanding the DSP1/CnB signaling axis in regulating neural regeneration in mammals, and have important implications for the development of drugs targeting neuronal regeneration.\u003c/p\u003e","manuscriptTitle":"DSP1/CnB Regulates Planarian (Dugesia japonica) Cephalic Ganglia Regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 13:29:40","doi":"10.21203/rs.3.rs-9686603/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ea65b66-8d4d-4466-a540-946f04a28e72","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"submitted","content":"Molecular Neurobiology","date":"2026-05-12T04:52:06+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T13:29:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 13:29:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9686603","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9686603","identity":"rs-9686603","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}