RoboA reinforces planarian stem cell fate through FoxA and Anosmin1a

Wang, Kuang-Tse; Tsai, Fu-Yu; Chen, Yu-Chia; Judy, Catherine P.; Weathers, Indya E.; Özkan, Engin; Adler, Carolyn E.

doi:10.1038/s41467-026-68656-1

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Published: 23 January 2026

RoboA reinforces planarian stem cell fate through FoxA and Anosmin1a

Nature Communications volume 17, Article number: 1971 (2026) Cite this article

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Abstract

Whole-body regeneration requires adult stem cells with high plasticity to differentiate into missing cell types. Planarians possess a unique configuration of organs embedded in a vast pool of pluripotent stem cells. How stem cells integrate positional information with discrete fates remains unknown. Here, we use the planarian pharynx to define the cell fates that depend on the pioneer transcription factor FoxA. We find that Roundabout receptor RoboA suppresses aberrant pharynx cell fates by altering foxA expression, independent of the canonical ligand Slit. An RNAi screen for extracellular proteins identifies Anosmin1a as a potential partner of RoboA. Perturbing global patterning demonstrates that roboA/anosmin1a functions locally in the brain. By contrast, altering pharynx fate with foxA knockdown induces head-specific neurons in the pharynx, indicating a latent plasticity of stem cells. Our data links critical extracellular cues with cell fate decisions of highly plastic stem cells, ensuring the fidelity of organ regeneration.

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Introduction

Regeneration is defined by the ability of tissues to replenish specific cell types in discrete anatomical positions, ensuring both structural integrity and functional restoration. This process often relies on stem cells to sense surrounding tissues, which guides the subsequent replacement of necessary cell types. Because injuries are inherently unpredictable, stem cell plasticity, defined as the capability of adopting diverse fates in response to environmental stimuli, is thought to contribute to successful regeneration^1,2,3.

The planarian flatworm Schmidtea mediterranea is a classical model organism for studying whole-body regeneration⁴. This remarkable ability is driven by a population of pluripotent stem cells capable of giving rise to all cell types throughout the animal’s life^5,6. These stem cells are distributed across the body, while some organs are spatially restricted. Foundational studies in planarians have demonstrated that stem cells can give rise to either anterior or posterior tissues depending on their position relative to a wound^7,8,9,10,11. The context-dependent, flexible differentiation responses of stem cells suggest the existence of signaling pathways that mediate their differentiation.

Two systems that broadly influence stem cell fate and identity have been previously described. ‘Position control genes’ (PCGs), such as wnt and bmp, are regionally expressed by muscle cells¹². Their knockdown causes strong disruptions in axis patterning and duplications of organs^13,14,15, similar to homeotic transformations during development¹⁶. Although these findings highlight potential extracellular cues that define broad tissue boundaries, how stem cells receive these signals and adopt specific fates in the vicinity of organs is an open question. The ‘target zone’ model proposes that pre-existing organs can direct stem cell differentiation toward appropriate cell types^1,17,18, thus maintaining organ positioning and structure. While these systems work together to ensure regeneration fidelity, the specific extracellular signals that define the target zone and guide spatially precise stem cell differentiation remain unknown.

The pharynx is an organ with a unique anatomical position and known stem cell origin^19,20. It resides in a pouch in the middle of the animal body and consists of epithelium, muscles, and a radially-symmetric nerve net^19,20,21. Previous studies have demonstrated that the Forkhead transcription factor FoxA is essential for pharynx regeneration and the recovery of feeding behavior^22,23. Pharynx regeneration initiates from a subset of stem cells that express foxA transcripts, scattered around the pharynx²⁴. Together, these characteristics establish the pharynx as an excellent model for studying stem cell plasticity, where the stem cells differentiate into multiple cell types to restore a complex organ at a stereotypical position.

Prior work has identified genes responsible for inducing an abnormal pharynx either in the wrong position^13,15 or with disrupted anatomy²⁵. In particular, knockdown of the Roundabout receptor RoboA induces ectopic but nonfunctional pharynges²⁶. Although Robo receptors are best known for their roles in patterning the nervous system during development, they also regulate stem cell differentiation and tissue patterning in many organisms^{27,28,29,30,31}.

In this study, we capitalize on the roboA(RNAi) phenotype to explore the mechanisms that guide spatial restriction of stem cell fates to maintain organ identity. We characterize pharynx-specific lineages and find that roboA(RNAi) induces ectopic pharynx neurons (EPNs) and muscles in the brain of homeostatic animals, independently of slit and neural patterning. Further, we identify a homolog of the secreted protein Anosmin1a (Anos1a) as a potential signaling partner of RoboA. The pioneer factor FoxA is the primary determinant that toggles these fates within stem cells. This RoboA/Anos1a/FoxA triad acts downstream of global patterning. Taken together, our findings demonstrate that stem cells exhibit a latent plasticity that is suppressed by RoboA and Anos1a to reinforce organ identity mediated by the downstream transcription factor FoxA.

Results

roboA(RNAi) induces ectopic pharynx cells in the head

The single planarian pharynx is maintained by stem cells expressing the transcription factor foxA that are distributed in the surrounding parenchyma (Fig. 1a)³². How foxA⁺ stem cells and the pharynx are restricted to this region is unclear. Previous work showed that knockdown of roboA induces animals to regenerate supernumerary pharynges upon amputation²⁶. We hypothesized that this atypical anatomy might provide an inroad to elucidate the underlying mechanisms that normally confine pharynx fate and position. To understand which cell types in the pharynx arise abnormally in roboA(RNAi) animals, we analyzed the distribution of pharynx cells (see Supplementary Note and Supplementary Fig. 1) by in situ hybridization (ISH).

Fig. 1: roboA suppresses formation of EPNs and pharynx muscles in the brain. — **Fig. 1: *roboA* suppresses formation of EPNs and pharynx muscles in the brain.**

To determine whether pharynx number correlated with the severity of the injury, we varied the anterior-posterior (AP) amputation plane (Supplementary Fig. 2a). Using neuropeptide precursor-1 (npp-1) as a highly specific marker of pharynx neurons³³, we found that the number of ectopic pharynges increased when the amputation occurred closer to the pharynx (Supplementary Fig. 2b, c). However, we noticed that in all cases, aberrant npp-1 cells appeared in the brains of regenerating animals (Fig. 1b; Supplementary Fig. 2d). These npp-1-positive ‘ectopic pharynx neurons’ (EPNs) in head fragments were unexpected. The fragments that contained pre-existing brains (head fragments and AP1 body fragments) had more EPNs than fragments challenged to regenerate new brains (AP2 and AP3), raising the possibility that EPNs might have been present prior to amputation. Indeed, in homeostatic roboA(RNAi) animals, we detected EPNs in the head (Fig. 1c, d), indicating that EPNs arise independently of injury. To determine if roboA knockdown alters other pharynx cell types, we examined markers of pharynx muscle and epithelial cells in homeostatic animals (Supplementary Fig. 2e, f). We detected multiple pharynx neuron and muscle markers expressed with a similar brain-specific distribution (Fig. 1e). We only detected ectopic pharynx epithelial cells in ectopic pharynges (Supplementary Fig. 2f, g), but not in the brain (Fig. 1f). Furthermore, ectopic pharynges never expressed the pouch marker dd_1320, which may explain why ectopic pharynges are nonfunctional²⁶ (Fig. 1f; Supplementary Fig. 1f, g). In the brain, EPNs and pharynx muscle markers did not overlap (44 npp-1⁺ and 47 laminin⁺ cells in 6 animals) (Fig. 1g, h), suggesting that these are distinct cells, rather than cells with a mixed fate. Together, these results indicate that roboA restricts the emergence of multiple pharynx cell types to the proper anatomical region.

EPNs in the head arise from stem cells, not mispatterned neurons

Robo receptors are best known for their roles in nervous system patterning³⁴. Because regeneration of a new head requires significant neurogenesis and nervous system remodeling, one possibility is that the EPNs appearing in the brain arise downstream of abnormal patterning²⁶. To separate the appearance of EPNs from neural patterning defects, we examined the expression of the neural markers gluR1 and chat in homeostatic animals and did not detect major differences in brain morphology between roboA(RNAi) and control animals (Fig. 1i). We also immunostained the optic nerves with Arrestin to detect potential axon guidance defects (Fig. 1j). As expected, axons in the optic nerve in regenerating roboA(RNAi) animals were defasciculated and photoreceptor numbers were increased as compared to controls³⁵. Importantly, uninjured, homeostatic roboA(RNAi) animals had normally patterned optic nerves and brains, despite this treatment also resulting in EPN formation in the brain (Fig. 1j, k). To evaluate the impact of our knockdown, we performed quantitative RT-PCR and verified that knockdown animals had decreased roboA transcript levels (Supplementary Fig. 2h). Therefore, we conclude that roboA normally prevents the formation of EPNs independently of a mispatterned nervous system.

By in situ hybridization, roboA transcript appears enriched in the nervous system, but single-cell sequencing revealed a ubiquitous, low-level expression across all major cell types, including stem cells (Supplementary Fig. 3a, b). Between the brain lobes in wildtype animals, we identified piwi-1⁺ stem cells that co-expressed roboA, indicating that roboA is present in stem cells (Supplementary Fig. 3c). To determine whether the appearance of EPNs in roboA(RNAi) animals requires stem cells, we exposed animals to ionizing radiation, which eliminates all stem cells^36,37. Then we knocked down roboA and challenged head fragments to regenerate a new pharynx (Supplementary Fig. 3d). Three days later, we failed to detect EPNs in the heads of irradiated roboA(RNAi) animals (Supplementary Fig. 3e, f), while they did appear in unirradiated controls. Therefore, the formation of EPNs requires active stem cells.

foxA is required for pharynx neuron and epithelium but not muscle fates

The pioneer transcription factor foxA is required for pharynx regeneration through its expression in a subset of stem cells²². foxA⁺ stem cells are excluded from the brain and tail (Fig. 1a)³². Surprisingly, in the heads of roboA(RNAi) animals, we detected cells expressing both foxA and npp-1 among the EPNs (Fig. 2a). All npp-1-positive EPNs co-expressed foxA (59 cells from 3 animals), indicating that roboA normally suppresses foxA expression in the brain. To determine whether foxA is required for the appearance of EPNs, we knocked down roboA and foxA simultaneously. As compared to roboA knockdown alone, double knockdown animals had significantly reduced numbers of EPNs (Fig. 2b, c). By contrast, the number of laminin⁺ muscle cells significantly increased in double knockdown animals, indicating that ectopic pharynx muscle cells originate from stem cells independently of foxA. Together, these results suggest that roboA restricts stem cells from adopting a pharynx neuron fate in the brain by suppressing foxA expression.

Fig. 2: foxA is required for most pharynx fates. — **Fig. 2: *foxA* is required for most pharynx fates.**

The differential requirement of ectopic neurons and muscles in roboA(RNAi) animals prompted us to examine the necessity of foxA for pharynx cell types during normal pharynx regeneration. In single-cell RNAseq analysis of isolated pharynx libraries, foxA was primarily enriched in pharynx neurons and epithelium (Fig. 2d, e). To determine what cell fates require foxA during regeneration, we examined pharynx-specific markers in regenerating head fragments, which must form a new pharynx de novo. Seven days post-amputation, foxA(RNAi) animals completely lacked neuron (npp-1) and epithelial (dd_554 and dd_1320) markers but retained muscle cells (laminin) in the position where the pharynx should be forming (Fig. 2f). Together, we conclude that foxA is necessary for the production of pharynx neurons and epithelial cells, but not muscle cells (Fig. 2g). Moreover, our results suggest that pharynx neurons and muscles might arise from different stem cell subtypes, which are both suppressed by roboA in the head.

Anterior-posterior patterning occurs independently of roboA

Ectopic expression of foxA in the brain could arise from a global disruption in axial patterning. In planarians, axial patterning is controlled by position control genes (PCGs), which are expressed in defined anatomical positions across the animal¹². Certain PCGs (ndl-3, ptk-7, and wnt-p2) regulate body axis patterning and determine pharynx position. Their knockdown shifts the body axis posteriorly, expanding the domain of foxA expression and promoting ectopic pharynx growth posterior to the normal pharynx^13,15. To determine whether global patterning is altered by roboA knockdown, we examined the expression of sfrp-1, ndl-3, ptk-7, and wnt-p2 in homeostatic roboA(RNAi) animals. We detected no differences in their expression patterns (Supplementary Fig. 4). This indicates that the formation of EPNs in roboA(RNAi) animals are not due to defects in overall body patterning but instead arise from altered cell fates in the brain.

anos1a regulates pharynx neuron fates in concert with roboA

Our results suggest that RoboA, a transmembrane receptor, might act on stem cells to receive extracellular cues from the homeostatic brain. The canonical ligands of Robo are Slit proteins^34,38,39. To investigate whether RoboA functions via Slit, we verified the known knockdown phenotype of the sole slit gene in planarians, which disrupts midline patterning and causes animals to form abnormal eyes⁴⁰ (Supplementary Fig. 5a, b). No EPNs or ectopic pharynx muscles were detected in either homeostatic or regenerating slit(RNAi) animals, suggesting that roboA regulates stem cell differentiation in a slit-independent manner (Supplementary Fig. 5c).

RoboA is composed of five extracellular immunoglobulin domains, three fibronectin domains, and a transmembrane domain (Supplementary Fig. 5d). Robo receptors interact with the leucine-rich repeats of Slit proteins, mediating guidance and differentiation signals⁴¹. Thus, to identify alternative ligands for RoboA, we searched the S. mediterranea genome for proteins with a signal sequence or transmembrane domain along with immunoglobulin, fibronectin, or leucine-rich repeat domains, finding 190 gene candidates (Fig. 3c) (Supplementary Data). We screened 183 of these genes with RNAi, induced regeneration, and assessed animals 14 days later using ISH for EPNs. Knockdown of one gene, anosmin1a (anos1a), phenocopied roboA(RNAi) (Fig. 3b, c) by inducing supernumerary pharynges (Supplementary Fig. 6a, b) and EPNs (Supplementary Fig. 6c–e) in both regenerating and homeostatic animals. However, unlike roboA(RNAi) animals, anos1a(RNAi) animals did not cause the formation of ectopic pharynx muscle cells (Supplementary Fig. 6f). This suggests that anos1a specifically regulates neuronal differentiation in the brain.

Fig. 3: anos1a knockdown phenocopies roboA knockdown. — **Fig. 3: *anos1a* knockdown phenocopies *roboA* knockdown.**

Anosmin-1 proteins are secreted proteins that contain a whey acidic protein (WAP) domain and multiple fibronectin domains (Fig. 3d). They were first described in patients with X-linked Kallmann syndrome, where mutations in ANOS1 (also known as KAL-1) cause a disorder characterized by hypogonadotropic hypogonadism and anosmia^42,43. Our phylogenetic analysis identified anosmin-1 genes in the genomes of most model animals, as well as in the sea anemone Nematostella vectensis, but notably not in mouse or rat (Fig. 3e). S. mediterranea possess four anos1 homologs, which we rename here anosmin1a-d (Fig. 3d, e). A previous study identified anos1a expression in the anterior pole⁴⁴ and anos1b in the ventral epithelium (referred to as either anosmin-1 or kal-1)^26,45. anos1b, −1c, and −1d are expressed in the brain (Supplementary Fig. 6g), but their knockdown did not reproduce the roboA(RNAi) phenotype (data not shown).

To determine whether roboA and anos1a act in parallel or in the same pathway, we knocked both genes down simultaneously. We expected that if they act in parallel, we would detect increased numbers of EPNs as compared to single knockdowns (roboA;ctrl or anos1a;ctrl). Double knockdown of anos1a and roboA (anos1a;roboA) did not lead to a significant increase in the number of EPNs compared to the roboA;ctrl group, but is significantly increased when compared to the anos1a;ctrl group (Fig. 3f). We observed a similar trend where more laminin⁺ muscle cells in anos1a;roboA(RNAi) animals compared to roboA;ctrl, except that we detected no laminin⁺ cells in anos1a single knockdown animals as expected (Fig. 3f). Therefore, these results suggest that RoboA and Anos1a likely function within the same pathway, potentially as a ligand-receptor pair, although given the weak penetrance of the anos1a;ctrl knockdowns, there could be other possibilities. Because Anos1a does not appear to be required for inducing pharynx muscle cells, it may also act in a parallel pathway.

roboA-anos1a signaling acts locally in the head

To determine where anos1a might act in the animal, we examined anos1a transcript distribution. We found that it was expressed in the anterior pole, midline, pharynx, and parapharyngeal regions (Fig. 3g). Analysis of whole-animal single-cell RNA-sequencing data further showed that anos1a is expressed at very low levels, and enriched in muscle clusters (Supplementary Fig. 6h), particularly in pharynx muscle clusters (Supplementary Fig. 6i), but excluded from stem cells. Knockdown of roboA also induced ectopic anos1a⁺ cells in the heads of homeostatic animals, suggesting that these cells are likely to be ectopic pharynx muscle (Supplementary Fig. 6j). Our in situ hybridization shows that the parapharyngeal distribution of anos1a roughly mirrors foxA expression in this region, where foxA⁺ stem cells are located (Fig. 1a). To test whether anos1a is expressed in stem cells, we exposed animals to 60 Gy of ionizing radiation which depletes stem cells (Supplementary Fig. 6k). Following this treatment, anos1a expression was maintained, suggesting that anos1a is produced by cells other than stem cells.

Planarian stem cells have been shown to migrate over short and long distances^46,47,48. This observation raised the possibility that EPNs in roboA or anos1a knockdown animals might arise from foxA⁺ stem cells in the parapharyngeal region that migrate into the brain and differentiate abnormally, while parapharygeal anos1a suppresses the migration. To test this hypothesis, we surgically separated parapharyngeal tissues by head amputation and further prevented their regeneration through β-catenin knockdown (Fig. 4a). In this background, the entire animal is anteriorized by an overall reduction in Wnt signaling, resulting in a loss of parapharyngeal identity^7,14,49. We reasoned that if EPNs originate from migrating stem cells from the parapharyngeal region, they would not appear in β-catenin knockdown head fragments lacking parapharyngeal cells. Alternatively, if they arise from local stem cell differentiation in the head, EPNs would still appear.

**Fig. 4: RoboA and Anos1a act locally in the brain.**

We confirmed that this approach suppressed parapharyngeal identity, as foxA and anos1a expression were lost following β-catenin knockdown (Fig. 4b). In the double roboA;β-catenin knockdown context, the number of EPNs was significantly reduced in the anterior head (Fig. 4b, c), suggesting that either residual parapharyngeal stem cells contribute to the formation of EPNs, or that EPN formation requires a certain level of Wnt signaling. Interestingly, we still detected EPNs and foxA⁺ cells in posterior-facing brains, but significantly fewer than in the anterior, likely due to their smaller size. This trend was consistent in anos1a;β-catenin double knockdown animals (Fig. 4c). Together, these findings demonstrate that RoboA and Anos1a can act locally within the head to restrict stem cell differentiation, and EPNs arise wherever the brain is.

Depletion of the anterior pole is not sufficient to induce ectopic pharynx fates

Since roboA and anos1a suppress pharynx fates within the head, another potential source of Anos1a is the anterior pole, a group of muscle cells required for establishing anterior identity during regeneration^44,50. To test whether the anterior pole is required to suppress EPNs in the brain, we depleted it with a combination of surgical amputation and knockdowns of key anterior pole transcription factors, foxD, prep, or zicA^10,11,51,52. To ensure the removal of residual anterior pole signal, we surgically removed tissue anterior to the photoreceptors. If the anterior pole provides the inhibitory signal, we expected to observe the appearance of EPNs without any knockdown of roboA or anos1a. However, despite successful anterior pole depletion, as validated by lack of anos1a expression, these knockdown animals did not develop EPNs (Fig. 4d), indicating that Anos1a at the anterior pole is not the source of inhibition.

One possible explanation for the absence of EPNs following anterior pole depletion is that residual anos1a expression in the remaining brain tissue is sufficient to suppress EPN formation. To address this possibility, we forced regeneration of the entire brain by amputating as close to the pharynx as possible. Because knockdown of foxD, prep, or zicA results in generally smaller brain size, this may limit the potential induction and detection of EPNs. To overcome this limitation, we performed double knockdown experiments using nou-darake (ndk), a fibroblast growth factor receptor-like gene. ndk knockdown expands brain size⁵³ and can partially rescue anterior defects caused by prep knockdown⁵². In double knockdown animals (foxD;ndk, prep;ndk, or zicA;ndk), even though expression of the brain marker gluR1 was restored, we did not observe any EPNs (Fig. 4e). We conclude that the inhibitory signal that limits formation of EPNs does not originate in the anterior pole, but rather comes from elsewhere in the head.

foxA functions as a molecular switch that directs fate choice between pharynx and brain neurons

Our data reveals a latent fate choice of stem cells in the brain, which normally adopt brain-specific fates, to instead adopt pharynx-specific fates. When RoboA signaling is removed, stem cells reveal this potential by unleashing foxA expression. We hypothesized that stem cells in other parts of the body may also exhibit this potential. Because stem cells in the pharynx region normally induce pharynx-specific fates via FoxA (Fig. 5a), we tested whether inhibition of foxA could also reveal a latent potential of stem cells. To accomplish this, we compared the transcriptional profiles of brain and pharynx neurons, and further characterized how foxA knockdown could impact neuron fates.

**Fig. 5: Transcriptomic distinction of pharynx and head neurons.**

To identify unique molecular profiles for brain and pharynx neurons, we reanalyzed the published region-specific single-cell libraries⁵⁴, extracting neural-specific clusters (Fig. 5b). Pharynx neurons and brain neurons formed distinct clusters^54,55,56. Of 61 clusters, 17 are primarily composed of pharynx neurons, while 24 consist only of head neurons (Fig. 5c). As expected, pharynx neuron clusters had higher foxA expression (Fig. 5c, d). Differential gene expression analysis using the Wilcoxon rank sum test identified members of the polycystin (pkd) gene family as unique to either the head or pharynx (Fig. 5d). Consistent with a recent report and single-cell analysis, pkd2-2 and pkd2-3 show pharynx-specific expression, while other paralogues are predominantly expressed in auricles and brain regions (Fig. 5e)⁵⁶. These findings suggest that brain and pharynx neurons are molecularly distinct, with foxA playing a key role in defining these cell states.

With these markers in hand, we analyzed the impact of long-term foxA depletion on differentiation of specific neuron populations. In this context, foxA(RNAi) animals lose their pharynges and instead develop a dorsal outgrowth (Fig. 6a) enriched in neuronal markers such as pc2 and ndk²². We generated single-cell sequencing libraries from the parapharyngeal regions of foxA(RNAi) and control animals 21 days after RNAi administration (Fig. 6b). Both libraries were pooled, clustered and annotated (Supplementary Fig. 7a, b). The primary difference between control and foxA knockdown animals was the absence of pharynx epithelium and pouch cells (Supplementary Fig. 7a, b), consistent with the loss of epithelial marker staining in animals examined 7 days post-amputation (Fig. 2). Fractions of neurons in the control and foxA(RNAi) libraries were comparable, suggesting that foxA(RNAi) did not broadly impair neurogenesis (Supplementary Fig. 7b). Next, we examined neuron composition by calculating the fold change between the foxA(RNAi) cells relative to ctrl(RNAi) cells within each cluster (Supplementary Fig. 7c, left). We found that clusters enriched in foxA(RNAi) cells had higher expression of head-specific marker genes pkd2-1 and pkd2-4, while clusters enriched in controls expressed high foxA but lacked head-specific markers, as expected (Supplementary Fig. 7c, right). These findings suggest that foxA knockdown induces a broad transcriptional shift toward a brain-like profile, indicating that foxA functions as a central regulator of pharynx neuron differentiation, by integrating global inputs in stem cells.

Fig. 6: foxA dictates a fate switch between pharynx and brain neurons. — **Fig. 6: *foxA* dictates a fate switch between pharynx and brain neurons.**

To validate these findings, we examined the expression of several brain-specific markers in foxA(RNAi) animals with a dorsal outgrowth. Notably, these dorsal outgrowths had optic nerves and structured eye cups, indicating abnormal respecification to head-specific cell fates in the pharynx region (Fig. 6d). We also detected head-specific markers pkd2-1 and pkd2-4, along with brain-specific glutamic acid decarboxylase (gad) and gluR1 in the dorsal outgrowth, where they are otherwise never expressed (Supplementary Fig. 7c). These results suggest that foxA normally maintains pharynx fates and suppresses latent brain fates.

Collectively, our data suggests that FoxA is normally suppressed in the brain region by RoboA. Similar to the outgrowths observed in long-term foxA knockdown, roboA(RNAi) animals also form abnormal outgrowths, but only after decapitation, suggesting that massive tissue remodeling is necessary to drive uncontrolled growth (Supplementary Fig. 2b, c). Unlike the outgrowths in foxA(RNAi) animals, the outgrowths in roboA(RNAi) animals form in the neck region and exhibit hallmarks of both the brain and pharynx tissues²⁶. We observed both brain (opsin) and pharynx neurons (npp-1) in these outgrowths (Fig. 6e), suggesting that normal tissue identities have been mixed in roboA(RNAi) animals. This convergence supports the idea that stem cells retain a shared potential to adopt either brain or pharynx fates, with RoboA and FoxA acting as molecular switches that reinforce spatially appropriate fate decisions.

Discussion

Planarian stem cells can generate all the animal’s cell types⁶, suggesting that they exhibit extreme plasticity. This plasticity of adult stem cells has been lost in most animals, along with their ability to regenerate robustly. Thus, the mechanisms that enable stem cell plasticity, or how it might be regulated to coordinate faithful regeneration, remain unclear. While classical transplantation and irradiation studies established that planarian stem cells are functionally pluripotent, these approaches do not resolve the series of fate choices made by stem cells in different organ contexts. Our work demonstrates that stem cell differentiation is regulated by a new function for the Robo receptor that is independent of its role in axon guidance. Our study bridges the gap between the global signals known to broadly pattern the organism and downstream transcriptional responses driving individual cell fates that are influenced by their surroundings. This mechanism involves a previously unrecognized triad — Anos1a, RoboA, and FoxA — that refines stem cell fates to ensure that differentiation occurs in the appropriate anatomical position.

Local stem cell differentiation is controlled by position control genes (PCGs) and organ-maintenance signals (known as the target zone)¹. RoboA’s function aligns with the target zone concept by acting independently of PCGs to suppress pharyngeal fate in the brain, a downstream product of global axis signals. Because PCG expression and overall animal morphology remain intact in roboA(RNAi) animals, our findings refute the idea that roboA is a PCG as previously proposed¹. Instead, we define genes that suppress plasticity without broadly perturbing global patterning as ‘Fate-Reinforcing Genes’ (FRGs) (Fig. 7). In this framework, PCGs expressed in muscles define broad body regions. Downstream of PCGs, FRGs suppress inappropriate cell types, thereby maintaining proper organ positioning and maintenance. Interestingly, even in the absence of RoboA, anterior stem cells preferentially generate brain-specific neurons rather than pharynx neurons, further demonstrating that FRGs serve as modulators of cell identity rather than position determinants. This fine-tuning role highlights a previously underappreciated level of spatial control in stem cell fate decisions.

**Fig. 7: Model for stem cell plasticity modulated by RoboA and Anos1a in the brain.**

Recent studies have identified map3k1, a MAPK signaling component, as a regulator of differentiation in both brain and pharynx target zones^57,58. Knockdown of map3k1 leads to ectopic differentiation of brain and pharynx cells, producing outgrowths with mixed brain–pharynx features even under homeostasis, and without overt disruption of axis patterning. These knockdown phenotypes are similar to what we observe in roboA(RNAi) animals, suggesting that map3k1 may also function as an FRG. However, map3k1 restricts a broader range of fates as compared to roboA and anos1a. These differences suggest that extracellular signals act at distinct, possibly sequential, decision points to progressively constrain stem cell choices. The positions of these outgrowths also differ: map3k1 knockdown induces outgrowths in the brain region, but roboA and anos1a knockdown induces outgrowths in the neck region, and foxA knockdown induces outgrowths in the middle of the body, implying that different FRGs interact with the anterior–posterior axis in distinct ways. If additional FRGs that specify unique organs or body regions are identified, they will define a general framework that links extracellular regulators with downstream differentiation.

The mechanisms that determine when and how stem cell fates become committed remain poorly understood. In planarians, stem cells are heterogeneous, with subpopulations defined by fate-specific transcription factors (FSTFs)^23,59,60. One model proposes that these subpopulations arise in a cell cycle–dependent manner⁶¹: G1-phase stem cells are naïve and lack FSTFs, whereas G2-phase cells express FSTFs and give rise to lineage-committed progenitors. Because G2 cells are stochastically distributed across the body, this model implies that progenitors must migrate long distances to reach their final destinations⁴⁸. An alternative model posits that differentiation occurs in a hierarchical manner, with pluripotent stem cells gradually losing potential and sequentially acquiring FSTF expression^3,59,62. Regardless of whether differentiation occurs in a single step through cell-cycle transitions or through a series of fate decisions, our findings indicate that regional signals limit the available fate options. In the cell cycle–based model, instead of long-range migration, roboA/anos1a may suppress a transient foxA⁺ stem cell state specifically in the head. In the hierarchical model, roboA/anos1a may act at distinct decision points in progenitor lineages to restrict inappropriate fate acquisition.

Although Robo signaling is mediated through interaction with Slit ligands, our findings demonstrate that RoboA regulates stem cell differentiation in planarians independently of Slit, agreeing with the original finding of roboA and slit phenotypes^26,40. Molecular evidence from other systems has shown that Robo has a broader set of possible interactions beyond Slit. For example, Robo can interact with non-Slit proteins such as Netrin receptors and other Robo family members^29,63,64, homodimerize⁶⁵ or be cleaved to activate binding partners⁶⁶. Intriguingly, a recent interactome study suggests that the C. elegans Robo homolog Sax-3 can bind to multiple proteins in vitro, including Slit and Anos1⁶⁷. Our results contribute a previously unknown cellular context for Robo function, as well as a possible ligand for the Robo receptor.

ANOS1 mutations cause Kallmann syndrome, characterized by anosmia and hypogonadotropic hypogonadism, thought to result from impaired migration of olfactory and GnRH neurons^68,69,70. While the absence of Anos1 homologs in rat and mouse genomes complicates mechanistic studies, Anos1 modulates FGF signaling through interactions with FGF ligands and heparan sulfate, as observed in zebrafish⁷¹ and roundworms^72,73,74,75. A Robo mutation found in a Kallmann syndrome patient⁷⁶, combined with our findings identifying Anos1 as a potential RoboA ligand, suggests a previously unrecognized and conserved role for Robo-Anos1 interactions in neural differentiation, highlighting new molecular insights relevant to Kallmann syndrome.

Limitations of the study

Our model proposes that RoboA acts on stem cells, but we have only examined this with in situ hybridization, leaving the localization of RoboA and Anos1a elusive. Moreover, we cannot rule out whether de-differentiation or transdifferentiation accounts for the formation of EPNs, although there is no evidence for these types of fate transitions in planarians.

Methods

Animal husbandry

Schmidtea mediterranea asexual clonal line CIW4 was maintained in a recirculating water system supplemented with Montjuïc salts⁷⁷. Animals were fed beef liver and cleaned weekly. Before use, they were transferred to static culture supplemented with 50 µg/mL gentamicin, and starved for at least one week.

RNA interference

RNA interference was conducted by feeding animals with either bacterial-expressed or in vitro-synthesized double-stranded RNA (dsRNA). For bacterial RNAi, cDNA of the target genes was cloned into either the pJC53.2 or T4P vectors and transformed into HT115 competent cells⁷⁸. Cultures were grown in 2XYT broth at 37 °C with shaking at 200–250 rpm until reaching an OD600 of 0.6, followed by induction with 1 mM IPTG and additional shaking for 2 hours. Bacterial cultures were then pelleted, and every 50 mL culture pellet was resuspended in 125 µL of liver paste before being administered to the animals. For double RNAi, equal volumes of two bacterial cultures were mixed before pelleting. Bacterial RNAi was administered every other day for a total of four feedings for single gene knockdown and eight feeds for double gene knockdown unless otherwise indicated in the figure. All primer sequences are listed in Supplementary Data 1.

Screening of roboA ligands utilized in vitro-synthesized dsRNA. Most dsRNA was generated using T7-overhang primers (Supplementary Data 2) to amplify candidate genes from cDNA, and the PCR products were used as templates for in vitro transcription⁷⁹. The dsRNA was then mixed into a 4:1 liver-to-water paste, with 4 µg of dsRNA per 10 µL of liver. Exceptions to these in the screen were slit, roboA, roboB, roboC and roboD, which were cloned into pJC53.2 or T4P vectors and administered with bacterial feeding. Animals were fed every other day for a total of six feedings. Animals were either amputated 5 days after the final RNAi feeding and fixed 14 days later, or maintained without injury for 14 days, unless otherwise indicated in the figures. All experiments used the C. elegans gene unc-22 as a control.

For stem cell depletion, planarians were exposed to a dosage of 60 Gray radiation using a J.L. Shepherd & Associates Mark I-68 Irradiator. Following exposure, animals were rinsed immediately, transferred to fresh water, and fed RNAi food.

Genome-wide identification of IgSF, FN, and LRR domain containing proteins

Sequences derived from the dd-Smed-v6 transcriptome were utilized for gene annotation⁸⁰. Initially, transcripts including all isoforms, were translated into protein sequences and subsequently filtered to retain only those sequences with a minimum length of 100 amino acids. Conserved domain annotations were conducted using the SUPERFAMILY database through InterProScan (version 5.71)⁸¹. Proteins exhibiting immunoglobulin, fibronectin, or leucine-rich repeat domains underwent further analysis for the presence of signal peptides and transmembrane domains using DeepTMHMM (1.0.4)⁸². 196 proteins from 190 genes met these criteria, and the longest isoforms of each gene were further used for primer design. PCR failed for 15 of these genes.

qRT-PCR

For each biological replicate, 5 animals were homogenized in Trizol (Thermo Fisher 15596018) using Lysing Matrix D Tubes (MP Biomedicals 116913100) and a Bead Bug homogenizer (Benchmark). RNA was extracted according to the standard Trizol protocol. cDNA was synthesized using Superscript VILO (Life Technologies 11754250). PCR reactions were prepared with SYBR Green, and GAPDH was used as a control. Reactions were performed on an Applied Biosystems Viia7 Real-Time PCR System. Each biological replicate included three technical replicates, and Ct values were averaged. Data were analyzed using the Delta-Delta Ct method. All primers can be found in Supplementary Data 1.

In situ hybridization and immunostaining

In situ hybridizations were conducted^83,84 with some modifications. Fixed animals were rehydrated, bleached (5% formamide, 6% H₂O₂, 0.5% SDS in 0.5× SSC) for 1 hour, and treated with proteinase K [4 μg/ml in PBS with 0.3% Triton X-100 (PBSTx), Thermo Fisher 25530049] for 10 minutes, followed by a 10 min fixation in 4% formaldehyde. Probes were added and incubated at 56 °C for 16 hours following 2 h of pre-hybridization. Samples were washed twice in wash hybe (20 min each), followed by 1:1 wash hybe:2× SSC-0.1% Tween 20 (20 min), 2× SSC-0.1% Tween 20 (30 min), and 0.2× SSC-0.1% Tween 20 (30 minutes) at 56 °C, and then washed three times for 10 minutes each with MABT (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20, pH to 7.5 with NaOH) at room temperature. The animals were blocked in a solution of 0.5% Roche Western Blocking Reagent and 5% inactivated horse serum in MABT for 2 h at room temperature and incubated with antibody solution overnight at 4 °C. Antibody solution was made with either 1:3000 anti-DIG-AP (Roche 11093274910), 1:1000 anti-DIG-POD (Roche 11207733910), or 1:1000 anti-FITC-POD (Roche 11426346910) diluted in blocking solution. Tyramide conjugates were synthesized following the method outlined in ref. ⁸⁵. Tyramide signal amplification was achieved by incubating planarians for 5–10 min in a 1:1000 dilution of fluorophore-conjugated tyramide in 100 mM borate buffer (pH 8.5, 2 M NaCl, 0.003% H₂O₂, and 0.1 M Boric acid). For double FISH experiments, residual peroxidase activity was quenched by incubating the samples for at least 2 h in 1% sodium azide in PBSTx⁸⁴. After development, animals were soaked and mounted in ScaleA2 (4 M urea, 20% glycerol, 0.1% Triton X-100, 2.5% DABCO). Some hybridizations were performed using a CEM InSituPro Hybridization robot up to the development stage, using the same protocol.

In situ hybridization chain reaction (HCR) was performed following the manufacturer’s mouse embryo protocol (Molecular Instruments) with slight modifications⁸⁶. Animals were fixed, bleached, and treated with proteinase K as described above. Samples were pre-hybridized in HCR™ Probe Hybridization Buffer (v3.0) at 37 °C for 30 min, followed by overnight probe incubation at 37 °C with a 16 nM probe solution in HCR™ Probe Hybridization Buffer (v3.0). Probes were designed using insitu_probe_generator⁸⁷ and ordered from IDT (Supplementary Data 3). Samples were then washed four times at 37 °C for 15 min in HCR™ Probe Wash Buffer (v3.0), followed by two washes at room temperature for 5 min in 5× SSC with 0.1% Tween 20. Next, samples were incubated in HCR™ Amplifier Buffer (v3.0) at room temperature for 30 min. Fluorophore-conjugated hairpins (h1 and h2; 10 μL each of a 3 μM stock) were heated at 95 °C for 90 sec and then cooled to room temperature in a PCR machine. The cooled hairpins were added to 500 μL of HCR™ Amplifier Buffer (v3.0) to prepare the hairpin solution, and samples were incubated with this solution in the dark at room temperature overnight. The following day, samples were washed four times in 5× SSC with 0.1% Tween 20 and mounted in 80% glycerol.

For immunostaining, animals were fixed and bleached as for in situ hybridizations. 1% BSA was used for blocking and antibody solution. A mouse anti-Arrestin antibody was used at a 1:1000 dilution⁸⁸, and a goat-anti-mouse-Alexa Fluor 488 conjugated antibody (Thermo Fisher A11017) was used at a 1:2000 dilution.

Cell sorting and 10X single-cell library preparation

To generate single-cell libraries, 50 trunks from control(RNAi) or foxA(RNAi) animals were collected 21 days post-feeding. The tissues were dissociated into single-cell suspensions by mincing in CMFB buffer [calcium-magnesium-free solution with 1% BSA (400 mg/L NaH₂PO₄, 800 mg/L NaCl, 1200 mg/L KCl, 800 mg/L NaHCO₃, 240 mg/L glucose, 1% BSA, 15 mM HEPES, pH 7.3)] and gently rotating for 2 hours at room temperature. The cells were then centrifuged at 500 g for 5 minutes, resuspended, and passed through a 30 µm cell strainer (BD Biosciences, 340628) to remove clumps. The concentration of filtered cells was determined using a TC20 automated cell counter (Bio-Rad). After centrifugation, the cell concentration was adjusted to 100,000 cells/mL in a staining buffer [CMFB containing 0.4 µM Calcein-AM Thermofisher #C3100MP] and incubated with gentle rotation at room temperature for 20 minutes. Live cells were identified by Calcein-AM staining and sorted using a Sony MA900 Cell Sorter. A total of 100,000 cells were sorted and diluted to 1000 cells/µL for subsequent 10X library preparation. To aim for recovery of 10,000 cells after sequencing, 16,500 cells were loaded onto the 10X Genomics Chromium Controller for subsequent library preparation using Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1.

To deplete 16S sequence contamination from 10X libraries, Depletion of Abundant Sequences by Hybridization (DASH) method was used⁸⁹. Briefly, the cDNA library was treated with CRISPR/Cas9 targeting 16S at 37 °C overnight. Following CRISPR treatment, the cDNA was purified and eluted into 15 µL using AMPure beads (Beckman) according to the manufacturer’s protocol. The cDNA was then diluted to 30 µL and re-amplified with cDNA primers (R1 + TSO) for 10 cycles. After PCR amplification, the cDNA was processed according to the 10X Genomics protocol for enzymatic fragmentation and indexing. Libraries were pooled and sequenced on the Illumina NextSeq 2000 platform. To verify the removal of 16S cDNA, individual samples were analyzed using the Fragment Analyzer (Agilent).

Single-cell analysis

Sequencing reads of single-cell libraries were aligned to a customized genome file containing both the chromosomal-level genome (Smed_chr_ref_v1)⁹⁰ and 16S sequence (SMED30032887) by CellRanger (6.1.2)⁹¹. Data preprocessing, including normalization and variable feature identification, was conducted using the Seurat (5.0.0) package⁹² in R(4.1.1). Single-cell RNA-seq data matrices were read into Seurat objects using the Read10X function, and each dataset was normalized and processed to identify variable features.

The control(RNAi) and foxA(RNAi) datasets were combined into a single Seurat object and normalized using standard Seurat workflows. We used a previously published dataset⁵⁴ to assign cell type identities as a reference with Seurat’s FindTransferAnchors and TransferData functions. Before using it as a reference, the Fincher 2018 dataset’s gene annotations were converted from the dd-Smed-v4 transcriptome format to the SMESG gene model to match the datasets generated in this study⁸⁰. When multiple dd-Smed-v4 transcripts corresponded to the same SMESG gene, their read counts were aggregated. Cell type identities were predicted based on the “Major.cluster.description” annotations from the Fincher 2018 dataset, which define the main cell types. Cells predicted to be “Neural” were further extracted and subclustered by the first 20 Harmony coordinates used for clustering with the “FindClusters” function, set at a resolution of 2, employing the shared nearest neighbor (SNN) approach.

To assess the cell-type composition, cells were grouped based on their original sample type [control(RNAi) or foxA(RNAi)] and their predicted major cell type. Bar plots were then used to visualize and compare the relative abundance (log2 fold change) of each cell type between the control(RNAi) and foxA(RNAi) conditions, highlighting differences in cell-type fractions between the two experimental groups.

Image quantification

Imaging was done using a Leica M165F with a DFC7000T camera for live animals, a Keyence BZ-X800 microscope for colorimetric ISH. For fluorescent confocal images, full brains of animals were captured with a 40X water-immersion objective and magnification sufficient to capture the entire brain on a Zeiss 710 confocal microscope. Z-stacks represent the entire brain beginning at the first labelled cells. Cells were counted manually using ImageJ software (FIJI 2.14.0) and positive cells were determined by signal surrounding nuclei in a single z-slice.

Statistics and Reproducibility

PRISM-Graphpad (10.4.0) was used to perform one-way ANOVA with Tukey test or two-sided student’s t-tests as indicated in figure legends, except in cases where the variance was zero, making the Tukey test inapplicable. *p <0.05; **p <0.01; ***p <0.001; and ****p <0.0001. For all RNAi experiments, two independent biological replicates with at least 5 animals per condition were performed. Single-cell sequencing was performed once.

Phylogenetic analysis

Sequences for phylogenetic analysis were retrieved from the NCBI database, with accession numbers detailed in Supplementary Data 4. Planarian sequences were specifically obtained from the dd-Smed-v6 transcriptome⁸⁰. The retrieved sequences were translated into proteins and aligned using CLUSTALW with the SLOW/ACCURATE setting. Phylogenetic trees were inferred using the Maximum Likelihood method⁹³ with bootstrap support (n = 1000 replicates), implemented in IQ-TREE2 (version 2.2.6)⁹⁴. Final tree visualization and layout optimization were performed using FigTree (version 1.4.4).

Reporting summary

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

Data availability

All data are available upon request. The scRNA-seq data generated in this study have been deposited into the GEO database under Accession number GSE292456. Source data are provided with this paper.

Code availability

Codes have been deposited on Github (https://github.com/kw572/RoboA)⁹⁵.

References

Reddien, P. W. The Cellular and Molecular Basis for Planarian Regeneration. Cell 175, 327–345 (2018).
Article CAS PubMed PubMed Central Google Scholar
Tanaka, E. M. & Reddien, P. W. The cellular basis for animal regeneration. Dev. Cell 21, 172–185 (2011).
Article CAS PubMed PubMed Central Google Scholar
Adler, C. E. & Sánchez Alvarado, A. Types or States? Cellular Dynamics and Regenerative Potential. Trends Cell Biol. 25, 687–696 (2015).
Article CAS PubMed PubMed Central Google Scholar
Ivankovic, M. et al. Model systems for regeneration: planarians. Development 146, dev167684. (2019).
Pearson, B. J. Finding the potency in planarians. Commun. Biol. 5, 970 (2022).
Article PubMed PubMed Central Google Scholar
Wagner, D. E., Wang, I. E. & Reddien, P. W. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration. Science 332, 811–816 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Gurley, K. A., Rink, J. C. & Sánchez Alvarado, A. Beta-catenin defines head versus tail identity during planarian regeneration and homeostasis. Science 319, 323–327 (2008).
Article ADS CAS PubMed Google Scholar
Petersen, C. P. & Reddien, P. W. Smed-betacatenin-1 is required for anteroposterior blastema polarity in planarian regeneration. Science 319, 327–330 (2008).
Article ADS CAS PubMed Google Scholar
Molina, M. D. & Cebrià, F. Decoding stem cells: An overview on planarian stem cell heterogeneity and lineage progression. Biomolecules 11, 1532 (2021).
Article CAS PubMed PubMed Central Google Scholar
Vogg, M. C. et al. Stem cell-dependent formation of a functional anterior regeneration pole in planarians requires Zic and Forkhead transcription factors. Dev. Biol. 390, 136–148 (2014).
Article CAS PubMed Google Scholar
Vásquez-Doorman, C. & Petersen, C. P. zic-1 Expression in Planarian neoblasts after injury controls anterior pole regeneration. PLoS Genet 10, e1004452 (2014).
Article PubMed PubMed Central Google Scholar
Witchley, J. N., Mayer, M., Wagner, D. E., Owen, J. H. & Reddien, P. W. Muscle cells provide instructions for planarian regeneration. Cell Rep. 4, 633–641 (2013).
Article CAS PubMed PubMed Central Google Scholar
Scimone, M. L., Cote, L. E., Rogers, T. & Reddien, P. W. Two FGFRL-Wnt circuits organize the planarian anteroposterior axis. Elife 5, e12845 (2016).
Gurley, K. A. et al. Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response. Dev. Biol. 347, 24–39 (2010).
Article CAS PubMed PubMed Central Google Scholar
Lander, R. & Petersen, C. P. Wnt, Ptk7, and FGFRL expression gradients control trunk positional identity in planarian regeneration. Elife 5, e12850 (2016).
Lewis, E. B. A gene complex controlling segmentation in Drosophila. Nature 276, 565–570 (1978).
Article ADS CAS PubMed Google Scholar
Atabay, K. D., LoCascio, S. A., de Hoog, T. & Reddien, P. W. Self-organization and progenitor targeting generate stable patterns in planarian regeneration. Science 360, 404–409 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Hill, E. M. & Petersen, C. P. Positional information specifies the site of organ regeneration and not tissue maintenance in planarians. Elife 7, e33680 (2018).
Cebrià, F., Bueno, D., Reigada, S. & Romero, R. Intercalary muscle cell renewal in planarian pharynx. Dev. Genes Evol. 209, 249–253 (1999).
Article PubMed Google Scholar
Bueno, D., Espinosa, L., Baguñà, J. & Romero, R. Planarian pharynx regeneration in regenerating tail fragments monitored with cell-specific monoclonal antibodies. Dev. Genes Evol. 206, 425–434 (1997).
Article CAS PubMed Google Scholar
Baguñà, J. & Ballester, R. The nervous system in planarians: Peripheral and gastrodermal plexuses, pharynx innervation, and the relationship between central nervous system structure and the acoelomate organization: NERVOUS SYSTEM IN PLANARIANS. J. Morphol. 155, 237–252 (1978).
Article PubMed Google Scholar
Adler, C. E., Seidel, C. W., McKinney, S. A. & Sánchez Alvarado, A. Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. Elife 3, e02238 (2014).
Article PubMed PubMed Central Google Scholar
Scimone, M. L., Kravarik, K. M., Lapan, S. W. & Reddien, P. W. Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Rep. 3, 339–352 (2014).
Article CAS Google Scholar
Bohr, T. E., Shiroor, D. A. & Adler, C. E. Planarian stem cells sense the identity of the missing pharynx to launch its targeted regeneration. Elife 10, e68830 (2021).
Article CAS PubMed PubMed Central Google Scholar
Fraguas, S., Barberán, S. & Cebrià, F. EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis. Dev. Biol. 354, 87–101 (2011).
Article CAS PubMed Google Scholar
Cebrià, F. & Newmark, P. A. Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians. Development 134, 833–837 (2007).
Article PubMed Google Scholar
Grieshammer, U. et al. SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev. Cell 6, 709–717 (2004).
Article CAS PubMed Google Scholar
Harburg, G. et al. SLIT/ROBO2 signaling promotes mammary stem cell senescence by inhibiting Wnt signaling. Stem Cell Rep. 3, 385–393 (2014).
Article CAS Google Scholar
Koch, A. W. et al. Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5. B. Dev. Cell 20, 33–46 (2011).
Article CAS Google Scholar
Biteau, B. & Jasper, H. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila. Cell Rep. 7, 1867–1875 (2014).
Article CAS PubMed PubMed Central Google Scholar
Borrell, V. et al. Slit/Robo signaling modulates the proliferation of central nervous system progenitors. Neuron 76, 338–352 (2012).
Article CAS PubMed PubMed Central Google Scholar
Koinuma, S., Umesono, Y., Watanabe, K. & Agata, K. Planaria FoxA (HNF3) homologue is specifically expressed in the pharynx-forming cells. Gene 259, 171–176 (2000).
Article CAS PubMed Google Scholar
Collins, J. J. et al. Genome-wide analyses reveal a role for peptide hormones in planarian germline development. PLoS Biol. 8, e1000509 (2010).
Article PubMed PubMed Central Google Scholar
Blockus, H. & Chédotal, A. Slit-Robo signaling. Development 143, 3037–3044 (2016).
Article CAS PubMed Google Scholar
Scimone, M. L. et al. Muscle and neuronal guidepost-like cells facilitate planarian visual system regeneration. Science 368, eaba3203 (2020).
Article CAS PubMed PubMed Central Google Scholar
Wagner, D. E., Ho, J. J. & Reddien, P. W. Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis. Cell Stem Cell 10, 299–311 (2012).
Article CAS PubMed PubMed Central Google Scholar
Eisenhoffer, G. T., Kang, H. & Sánchez Alvarado, A. Molecular analysis of stem cells and their descendants during cell turnover and regeneration in the planarian Schmidtea mediterranea. Cell Stem Cell 3, 327–339 (2008).
Article CAS PubMed PubMed Central Google Scholar
Tong, M., Jun, T., Nie, Y., Hao, J. & Fan, D. The Role of the Slit/Robo Signaling Pathway. J. Cancer 10, 2694–2705 (2019).
Article CAS PubMed PubMed Central Google Scholar
Kidd, T., Bland, K. S. & Goodman, C. S. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785–794 (1999).
Article CAS PubMed Google Scholar
Cebrià, F., Guo, T., Jopek, J. & Newmark, P. A. Regeneration and maintenance of the planarian midline is regulated by a slit orthologue. Dev. Biol. 307, 394–406 (2007).
Article PubMed PubMed Central Google Scholar
Aleksandrova, N. et al. Robo1 Forms a Compact Dimer-of-Dimers Assembly. Structure 26, 320–328.e4 (2018).
Article CAS PubMed PubMed Central Google Scholar
Franco, B. et al. A gene deleted in Kallmann’s syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 353, 529–536 (1991).
Article ADS CAS PubMed Google Scholar
Legouis, R. et al. The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell 67, 423–435 (1991).
Article CAS PubMed Google Scholar
Li, D. J., McMann, C. L. & Reddien, P. W. Nuclear receptor NR4A is required for patterning at the ends of the planarian anterior-posterior axis. Elife 8, e42015 (2019).
Wurtzel, O., Oderberg, I. M. & Reddien, P. W. Planarian Epidermal Stem Cells Respond to Positional Cues to Promote Cell-Type Diversity. Dev. Cell 40, 491–504.e5 (2017).
Article CAS PubMed PubMed Central Google Scholar
Sahu, S. et al. Ongoing repair of migration-coupled DNA damage allows planarian adult stem cells to reach wound sites. Elife 10, e63779 (2021).
Guedelhoefer, O. C. 4th & Sánchez Alvarado, A. Amputation induces stem cell mobilization to sites of injury during planarian regeneration. Development 139, 3510–3520 (2012).
Article CAS PubMed PubMed Central Google Scholar
Park, C., Owusu-Boaitey, K. E., Valdes, G. M. & Reddien, P. W. Fate specification is spatially intermingled across planarian stem cells. Nat. Commun. 14, 7422 (2023).
Article ADS PubMed PubMed Central Google Scholar
Petersen, C. P. & Reddien, P. W. A wound-induced Wnt expression program controls planarian regeneration polarity. Proc. Natl. Acad. Sci. Usa. 106, 17061–17066 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Petersen, C. P. & Reddien, P. W. Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration. Science 332, 852–855 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Scimone, M. L., Lapan, S. W. & Reddien, P. W. A forkhead transcription factor is wound-induced at the planarian midline and required for anterior pole regeneration. PLoS Genet 10, e1003999 (2014).
Article PubMed PubMed Central Google Scholar
Felix, D. A. & Aboobaker, A. A. The TALE class homeobox gene Smed-prep defines the anterior compartment for head regeneration. PLoS Genet 6, e1000915 (2010).
Article PubMed PubMed Central Google Scholar
Cebrià, F. et al. FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. Nature 419, 620–624 (2002).
Article ADS PubMed Google Scholar
Fincher, C. T., Wurtzel, O., de Hoog, T., Kravarik, K. M. & Reddien, P. W. Cell type transcriptome atlas for the planarian Schmidtea mediterranea. Science 360, eaaq1736 (2018).
King, H. O., Owusu-Boaitey, K. E., Fincher, C. T. & Reddien, P. W. A transcription factor atlas of stem cell fate in planarians. Cell Rep. 43, 113843 (2024).
Article CAS PubMed PubMed Central Google Scholar
Ross, K. G. et al. The role of polycystic kidney disease-like homologs in planarian nervous system regeneration and function. Integr. Org. Biol. 6, obae035 (2024).
Article CAS PubMed PubMed Central Google Scholar
Lo, K. C. & Petersen, C. P. Map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration. PLoS Genet 21, e1011457 (2025).
Article CAS PubMed PubMed Central Google Scholar
Canales, B. I.-I., King, H. O. & Reddien, P. W. Map3k1 is required for spatial restriction of progenitor differentiation in planarians. eLife https://doi.org/10.7554/elife.106439.1.(2025)
Roberts-Galbraith, R. H. & Newmark, P. A. On the organ trail: insights into organ regeneration in the planarian. Curr. Opin. Genet. Dev. 32, 37–46 (2015).
Article CAS PubMed Google Scholar
Zhu, S. J. & Pearson, B. J. Neo)blast from the past: new insights into planarian stem cell lineages. Curr. Opin. Genet. Dev. 40, 74–80 (2016).
Article CAS PubMed Google Scholar
Raz, A. A., Wurtzel, O. & Reddien, P. W. Planarian stem cells specify fate yet retain potency during the cell cycle. Cell Stem Cell 28, 1307–1322.e5 (2021).
Article CAS PubMed PubMed Central Google Scholar
Zeng, A. et al. Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration. Cell 173, 1593–1608.e20 (2018).
Article CAS PubMed PubMed Central Google Scholar
Yu, T. W., Hao, J. C., Lim, W., Tessier-Lavigne, M. & Bargmann, C. I. Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a Netrin-independent UNC-40/DCC function. Nat. Neurosci. 5, 1147–1154 (2002).
Article CAS PubMed Google Scholar
Jaworski, A. et al. Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2. Science 350, 961–965 (2015).
Article CAS PubMed Google Scholar
Barak, R. et al. Structural Principles in Robo Activation and Auto-inhibition. Cell 177, 272–285.e16 (2019).
Article CAS PubMed Google Scholar
Qu, Z., Zhang, A. & Yan, D. Robo functions as an attractive cue for glial migration through SYG-1/Neph. Elife 9, e57921 (2020).
Nawrocka, W. I. et al. Nematode extracellular protein interactome expands connections between signaling pathways. bioRxiv 2024 07, 602367 (2024).
Google Scholar
García-González, D. et al. Anosmin-1 over-expression increases adult neurogenesis in the subventricular zone and neuroblast migration to the olfactory bulb. Brain Struct. Funct. 221, 239–260 (2016).
Article PubMed Google Scholar
Kallmann, F., Schoenfeld, W. A. & Barrera, S. E. The genetic aspects of primary eunuchoidism. Am. J. Ment. Defic. 203, 236 (1944).
Google Scholar
Schwanzel-Fukuda, M., Bick, D. & Pfaff, D. W. Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Brain Res. Mol. Brain Res. 6, 311–326 (1989).
Article CAS PubMed Google Scholar
Wang, J. et al. Anosmin1 shuttles fgf to facilitate its diffusion, increase its local concentration, and induce sensory organs. Dev. Cell 46, 751–766.e12 (2018).
Article CAS PubMed PubMed Central Google Scholar
Hudson, M. L., Kinnunen, T., Cinar, H. N. & Chisholm, A. D. C. elegans Kallmann syndrome protein KAL-1 interacts with syndecan and glypican to regulate neuronal cell migrations. Dev. Biol. 294, 352–365 (2006).
Article CAS PubMed Google Scholar
Hu, Y. et al. Novel mechanisms of fibroblast growth factor receptor 1 regulation by extracellular matrix protein anosmin-1. J. Biol. Chem. 284, 29905–29920 (2009).
Article CAS PubMed PubMed Central Google Scholar
Bülow, H. E., Berry, K. L., Topper, L. H., Peles, E. & Hobert, O. Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1. Proc. Natl. Acad. Sci. Usa. 99, 6346–6351 (2002).
Article ADS PubMed PubMed Central Google Scholar
Tecle, E., Diaz-Balzac, C. A. & Bülow, H. E. Distinct 3-O-sulfated heparan sulfate modification patterns are required for kal-1-dependent neurite branching in a context-dependent manner in Caenorhabditis elegans. G3 (Bethesda) 3, 541–552 (2013).
Article CAS PubMed PubMed Central Google Scholar
Zhu, Z. et al. Identification of ROBO1/2 and SCEL as candidate genes in Kallmann syndrome with emerging bioinformatic analysis. Endocrine 67, 224–232 (2020).
Article CAS PubMed Google Scholar
Merryman, M. S., Sánchez Alvarado, A. & Jenkin, J. C. Culturing Planarians in the Laboratory. Methods Mol. Biol. 1774, 241–258 (2018).
Article CAS PubMed Google Scholar
Adler, C. E. & Sánchez Alvarado, A. Systemic RNA interference in planarians by feeding of dsRNA containing bacteria. Methods Mol. Biol. 1774, 445–454 (2018).
Article CAS PubMed Google Scholar
Rouhana, L. et al. RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics. Dev. Dyn. 242, 718–730 (2013).
Article CAS PubMed PubMed Central Google Scholar
Rozanski, A., Moon, H. K. & Brandl, H. PlanMine 3.0—improvements to a mineable resource of flatworm biology and biodiversity. Nucleic acids 47, D812–D820 (2019).
Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240 (2014).
Article CAS PubMed PubMed Central Google Scholar
Hallgren, J. et al. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. bioRxiv 2022 04, 487609 (2022).
Google Scholar
Pearson, B. J. et al. Formaldehyde-based whole-mount in situ hybridization method for planarians. Dev. Dyn. 238, 443–450 (2009).
Article PubMed PubMed Central Google Scholar
King, R. S. & Newmark, P. A. In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev. Biol. 13, 8 (2013).
Article CAS PubMed PubMed Central Google Scholar
Hopman, A. H., Ramaekers, F. C. & Speel, E. J. Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification. J. Histochem. Cytochem. 46, 771–777 (1998).
Article CAS PubMed Google Scholar
Choi, H. M. T. et al. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, dev165753 (2018).
Article PubMed PubMed Central Google Scholar
Kuehn, E. et al. Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii. J. Exp. Zool. B Mol. Dev. Evol. 338, 225–240 (2022).
Article PubMed Google Scholar
Akiyama, Y., Agata, K. & Inoue, T. Coordination between binocular field and spontaneous self-motion specifies the efficiency of planarians’ photo-response orientation behavior. Commun. Biol. 1, 148 (2018).
Article PubMed PubMed Central Google Scholar
Wang, K.-T. & Adler, C. E. CRISPR/Cas9-based depletion of 16S ribosomal RNA improves library complexity of single-cell RNA-sequencing in planarians. BMC Genomics 24, 625 (2023).
Article CAS PubMed PubMed Central Google Scholar
Guo, L. et al. Island-specific evolution of a sex-primed autosome in a sexual planarian. Nature 606, 329–334 (2022).
Article ADS CAS PubMed PubMed Central Google Scholar
Zheng, G. X. Y. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015).
Article CAS PubMed PubMed Central Google Scholar
Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Article CAS PubMed Google Scholar
Minh, B. Q. et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020).
Article CAS PubMed PubMed Central Google Scholar
Wang, K.-T. et al RoboA reinforces planarian stem cell fate through FoxA and Anosmin1a. RoboA. https://doi.org/10.5281/zenodo.18143260 (2026).

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Acknowledgements

We would like to thank members of the Adler laboratory for insight on this project; T. Inoue for kindly sharing the Arrestin antibody; and T. Tumbar for critical reading of the manuscript. We thank the Cornell University Biotechnology Resource Center’s Flow Cytometry (RRID:SCR_021740), Imaging (RRID:SCR_021741), and Genomics (RRID:SCR_021727) cores for equipment and resources used in this project. This work was funded by a National Institutes of Health grant R01GM139933 to C.E.A., a Cornell University Stem Cell Program fellowship (to K-T.W), Cornell University Center for Vertebrate Genomics Scholarship (to K-T.W.) and a Taiwan Ministry of Education scholarship (to K-T.W.), and a T32 GM144292 (to I.E.W.).

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Authors and Affiliations

Department of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, NY, USA
Kuang-Tse Wang, Fu-Yu Tsai, Catherine P. Judy & Carolyn E. Adler
Department of Genetics, Harvard Medical School, Boston, MA, USA
Yu-Chia Chen
Department of Orthopedic Research, Boston Children’s Hospital, Boston, MA, USA
Yu-Chia Chen
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, USA
Indya E. Weathers & Engin Özkan
Institute for Neuroscience, The University of Chicago, Chicago, IL, USA
Indya E. Weathers & Engin Özkan
Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA
Indya E. Weathers & Engin Özkan

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Kuang-Tse Wang
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Fu-Yu Tsai
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Yu-Chia Chen
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Catherine P. Judy
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Indya E. Weathers
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Engin Özkan
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Carolyn E. Adler
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Contributions

K-T.W., Y-C.C. and C.E.A. conceived the study. C.E.A. supervised the research and acquired funding. K-T.W., Y-C.C., F-Y.T. and C.P.J. performed the ISH, RNAi, qRT-PCR and imaging experiments. K-T.W. generated the sequencing data and performed all statistical analyses. I.E.W. and E.O. performed biochemical assays. K-T.W. and C.E.A. wrote the original draft. All authors edited, read, and approved the manuscript.

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Correspondence to Carolyn E. Adler.

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Wang, KT., Tsai, FY., Chen, YC. et al. RoboA reinforces planarian stem cell fate through FoxA and Anosmin1a. Nat Commun 17, 1971 (2026). https://doi.org/10.1038/s41467-026-68656-1

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Received: 17 April 2025
Accepted: 13 January 2026
Published: 23 January 2026
Version of record: 24 February 2026
DOI: https://doi.org/10.1038/s41467-026-68656-1