Planarian regeneration and tissue turnover involve fate specification in pluripotent stem cells called neoblasts. Neoblasts select fates through the expression of fate-specific transcription factors, generating specialized neoblasts. Specialized neoblasts are spatially intermingled and can be dispersed broadly, frequently being present far from their target tissue. The post-mitotic progeny of neoblasts, serving as progenitors, can migrate and differentiate into mature cell types. Pattern formation is thus strongly influenced by the migratory assortment and differentiation of fate-specified progenitors in precise locations, which we refer to as progenitor targeting. This central step of pattern maintenance and formation, however, is poorly understood. Here, we describe a requirement for the conserved map3k1 gene in targeting, restricting post-mitotic progenitor differentiation to precise locations. RNAi of map3k1 causes ectopic differentiation of eye progenitors along their migratory path, resulting in dispersed, ectopic eye cells and eyes. Other neural tissues similarly display ectopic posterior differentiation, and ectopic pharynx cells emerge dispersed laterally and anteriorly in map3k1 RNAi animals. Ectopic differentiated cells are also found within the incorrect organs after map3k1 RNAi, and ultimately, teratomas form. These findings implicate map3k1 signaling in controlling the positional regulation of progenitor behavior – restricting progenitor differentiation to targeted locations in response to external cues in the local tissue environment.

Regeneration is the process by which organisms replace lost body parts. How cell-type production is tailored to match the identity of missing tissues is a central problem of regeneration. Here, we investigated the specificity of planarian stem-cell responses to the identity of missing tissues following injury. Proximal injury not affecting the mature tissue nonetheless drives increased cell incorporation into brain neurons, ventral nerve cords, pharynx muscle and neurons. Following direct injury, peripheral neurons show a spatially imprecise, generic amplification of incorporation relative to the injury; body-wall muscle incorporation was amplified, with decreased incorporation at wound-distal sites in favor of increased incorporation wound-proximally. By contrast, essentially no stem-cell division contributes to initial epidermal regeneration, instead post-mitotic progenitors supply the wound. Amplification of epidermal incorporation following injury does occur weeks after injury, including to uninjured regions. These results indicate that the identity of the missing mature tissue is not required in determining the stem-cell response to injury. We suggest that planarian regeneration specificity involves a combination of ongoing cell turnover, wound-associated amplification of stem-cells, and spatially broad neoblast specification zones.

Abstract Mechanistic accounts of brain function require a common coordinate system in which structural, molecular and functional data can be integrated and compared across individuals. The teleost genus Danionella is unique among vertebrates in retaining lifelong transparency, allowing non-invasive, cellular-resolution functional imaging across the entire adult brain. A reference atlas in this model would therefore provide a strong foundation for causal and comparative circuit studies. Here we present an integrated anatomical, molecular and functional reference brain for adult Danionella cerebrum as a standardised atlas resource. Using a transgenic nuclear fluorescence marker, whole-mount tissue clearing and high-resolution two-photon microscopy, we generated an average reference brain from 21 adult fish to create a common coordinate system. Whole-mount in situ hybridisation for 29 neuronal markers, complemented by tract annotation from structural imaging and tracer injections, enabled us to segment 203 neuroanatomical regions. We found pronounced sex differences in telencephalic, cerebellar and hindbrain nuclei, revealing sexually dimorphic organisation across multiple brain regions. All data and segmentations are made openly accessible, providing a community resource for studies of circuit function, molecular makeup and sexual dimorphism in an optically accessible adult vertebrate brain.

A complete cell-type transcriptome atlas of a vertebrate could promote understanding of animal cell-type composition, organization, and evolution. The miniaturized, transparent, and regenerative teleost Danionella cerebrum brings whole-vertebrate single-cell profiling experiments within reach. We performed region-stratified single-cell RNA sequencing across the adult Danionella body and mapped cell types and gene expression at single-cell resolution using spatial transcriptomics. We delineated spatially-distinct neural cell types based on their regional gene expression signatures. The body-wide atlas elucidated cell types harboring adult positional information, uncovered paedomorphic features, and revealed conserved body-region and appendage-specification programs in adult connective tissue. Comparative analyses revealed conserved neural cell types, and regeneration datasets uncovered expression dynamics during telencephalon regeneration. This whole-vertebrate transcriptome atlas yields a comprehensive resource for myriad questions in biology and neuroscience

The evolution and development of eyes are fundamental problems in biology, and numerous genetic and age-related degenerative eye diseases are still poorly understood. Planarians are flatworms that are able to fully regenerate functional eyes following injury, presenting a powerful model to study essential attributes of eye biology and regeneration. We performed single-cell eye transcriptomic analyses and large-scale RNA interference screening to define a hierarchical sequence of steps in eye regeneration and the genes that control each step in this process: from progenitor specification to differentiation into mature photoreceptors and melanin-pigmented optic cup cells, rhabdomere and dorsal projection formation in photoreceptors, eye morphogenesis (a self-organizing process where eyes trap progenitors and promote their differentiation), and interactions with the surrounding extracellular environment to produce a transparent region for light transmission. This hierarchical program defines roles for many conserved genes and establishes a framework for the regeneration of an entire organ.

Eye loss occurs convergently in numerous animal phyla as an adaptation to dark environments. We investigate the cave planarian Girardia multidiverticulata (Gm), a representative species of the Spiralian clade, to study mechanisms of eye loss. We found that Gm, which was previously described as an eyeless species, retains rudimentary and functional eyes. Eyes are maintained in homeostasis and regenerated in adult planarians by stem cells, called neoblasts, through their fate specification to eye progenitors. The reduced number of eye cells in cave planarians is associated with a decreased rate of stem cell fate specification to eye progenitors during homeostasis and regeneration. Conversely, the homeostatic formation of new cells from stem cell-derived progenitors for other tissues, including for neurons, pharynx, and epidermis, is comparable between cave and surface species. These findings reveal a mode of evolutionary trait loss, with change in rate of fate specification in adult stem cells leading to tissue size reduction.

A central problem in regeneration is how the identity of new tissues is specified. A classic example is the head-versus-tail regeneration decision in planarians. notum is wound induced at anterior-facing planarian wounds, where it triggers head regeneration through inhibition of canonical Wnt signaling. This represents the earliest known asymmetric regeneration step between anterior-and posterior-facing wounds. Wound-induced notum is specific to longitudinal (anterior-posterior-axis oriented) muscle cells, suggesting these fibers might harbor polarity harnessed for the head-tail regeneration decision. The processes that occur within longitudinal muscle after injury for preferential notum activation at anterior-facing wounds are poorly understood. We utilized single-cell RNA sequencing to identify multiple wound-induced genes in longitudinal muscle cells and identified processes required for wound-induced notum asymmetry. Egalitarian-like-1 (Egal-1) is wound induced in longitudinal muscle and has some domain similarity with Drosophila Egalitarian, which facilitates asymmetric RNA localization. Both egal-1 RNAi animals and animals with destabilized microtubules (via colchicine or nocodazole treatment) show ectopic notum expression at posterior-facing wounds. We suggest that Egal-1 and microtubules are together required for longitudinal muscle fibers to promote planarian regeneration polarity.

Regeneration can involve the coordination of pattern formation in an outgrowth with the spatial pattern of pre-existing tissues, such as along body axes. Planarian adult axis patterning serves as a robust context for uncovering the mechanisms of such pattern integration. We investigated how the dorsal-ventral boundary (DVB), which surrounds the animal periphery at the dorsal-ventral (DV) median plane, regulates anterior-posterior (AP) axis growth and patterning. We define a spatial DVB gene expression atlas that includes genes encoding signaling, adhesion, and transcription factors. Wnt inhibition results in anterior positional information induction and ectopic head formation that is restricted to the DVB. DVB can be transplanted, and DVB identity can be experimentally induced at ectopic locations. Ectopic DVB is competent for anterior positional identity induction following Wnt inhibition, enabling the generation of animals with ectopic heads at experimentally dictated locations. DVB removal blocks the anteriorization that normally follows Wnt inhibition and prevents anterior positional information expression during head regeneration. Anterior positional information induction at the DVB after Wnt inhibition occurs independently from anterior pole formation, which promotes head patterning in regeneration. Our findings reveal a hierarchical model of pattern integration across body axes in which DV patterning is central by producing a DVB with competence to direct formation of large AP axis regions. This mechanism enables coordination of orthogonal positional information in the context of regeneration.

Some animals can regenerate large missing regions of their nervous system, requiring mechanisms to restore the pattern, numbers, and wiring of diverse neuron classes. Because injuries are unpredictable, regeneration must be accomplished from an unlimited number of starting points. Coordinated regeneration of neuron-glia architecture is thus a major challenge and remains poorly understood. In planarians, neurons and glia are regenerated from distinct progenitors. We found that planarians first regenerate neurons expressing a Delta-encoding gene, delta-2, at key positions in the central and peripheral nervous systems. Planarian glia are specified later from dispersed Notch-1-expressing mesoderm-like phagocytic progenitors. Inhibition of delta-2 or notch-1 severely reduced glia in planarians, but did not affect the specification of other phagocytic cell types. Loss of several delta-2-expressing neuron classes prevented differentiation of the glia associated with them, whereas transplantation of delta-2-expressing photoreceptor neurons was sufficient for glia formation at an ectopic location. Our results suggest a model in which patterned delta-2-expressing neurons instruct phagocytic progenitors to locally differentiate into glia, presenting a mechanism for coordinated regeneration of numbers and pattern of cell types.

Regeneration is widespread but sparsely distributed throughout the animal kingdom. Identifying factors that differentiate regenerative and non-regenerative organisms could enable approaches for improving regenerative outcomes in non-regenerative species. Constitutive adult positional information can be required for regeneration, but has been poorly characterized across animal species. Here, we generated positional gene expression atlases for the limbs of one regenerative (axolotl) and one non-regenerative (mouse) vertebrate. Regional gene expression signatures in both species are highly overlapping and mirror multiple developmental positional information patterns, particularly along the primary limb axis. These expression signatures are largely harbored in connective tissue, including diverse fibroblast types, in both organisms. We also identified species-specific regional expression patterns, including for Proxima , a novel gene encoding a secreted factor with strong positional expression in axolotl. Positional gene expression similar to developmental patterns also exists between fore and hindlimbs and along anterior-posterior and dorsal-ventral limb axes, but is notably stronger in axolotl than in mouse. Our results demonstrate that regenerative and non-regenerative vertebrate limbs share many, but not all, signatures of positional information. This work establishes regional atlases of adult vertebrate limbs and suggests that the connective tissue of regenerative and non-regenerative vertebrate limbs share a conserved signature of positional information that is stronger in regenerative species. ### Competing Interest Statement The authors have declared no competing interest. LEO Foundation, LF-OC-21-000784 Eleanor Schwartz Charitable Foundation Howard Hughes Medical Institute, https://ror.org/006w34k90, Investigator

In Caenorhabditis elegans, apoptosis is inhibited by the BCL-2 homolog CED-9. Although canonically anti-apoptotic, CED-9 has a poorly understood pro-apoptotic function. CED-9 is thought to inhibit apoptosis by binding to and inhibiting the pro-apoptotic C. elegans APAF-1 homolog CED-4. We show that CED-9 or CED-4 mutations located in their CED-9-CED-4 binding regions reduce apoptosis without affecting the CED-9 anti-apoptotic function. These mutant CED-9 and CED-4 proteins are defective in a CED-9-CED-4 interaction in vitro and in vivo, revealing that the known CED-9-CED-4 interaction is required for the pro-apoptotic but not for the anti-apoptotic function of CED-9. The pro-apoptotic CED-9-CED-4 interaction occurs at mitochondria. In mammals, BCL-2 family members can activate APAF-1 via cytochrome c release from mitochondria. The conserved role of mitochondria in CED-9/BCL-2-dependent CED-4/APAF-1 activation is notable and suggests that understanding how CED-9 promotes apoptosis in C. elegans could inform the understanding of mammalian apoptosis and how disruptions of apoptosis promote certain human disorders.

Turnover—constant component production and destruction—is ubiquitous in biology. Turnover occurs across organisms and scales, including for RNAs, proteins, membranes, macromolecular structures, organelles, cells, hair, feathers, nails, antlers, and teeth. For many systems, turnover might seem wasteful when degraded components are often fully functional. Some components turn over with shockingly high rates and others do not turn over at all, further making this process enigmatic. However, turnover can address fundamental problems by yielding powerful properties, including regeneration, rapid repair onset, clearance of unpredictable damage and errors, maintenance of low constitutive levels of disrepair, prevention of stable hazards, and transitions. I argue that trade-offs between turnover benefits and metabolic costs, combined with constraints on turnover, determine its presence and rates across distinct contexts. I suggest that the limits of turnover help explain aging and that turnover properties and the basis for its levels underlie this fundamental component of life.

Whole-body regeneration requires the ability to produce the full repertoire of adult cell types. The planarian Schmidtea mediterranea contains over 125 cell types, which can be regenerated from a stem cell population called neoblasts. Neoblast fate choice can be regulated by the expression of fate-specific transcription factors (FSTFs). How fate choices are made and distributed across neoblasts versus their post-mitotic progeny remains unclear. We used single-cell RNA sequencing to systematically map fate choices made in S/G2/M neoblasts and, separately, in their post-mitotic progeny that serve as progenitors for all adult cell types. We defined transcription factor expression signatures associated with all detected fates, identifying numerous new progenitor classes and FSTFs that regulate them. Our work generates an atlas of stem cell fates with associated transcription factor signatures for most cell types in a complete adult organism.

Although demographic studies have failed to find evidence of aging in certain animal species, classic evolutionary theories of aging struggle to explain how evolution could favor agelessness in such cases. Here, we develop mathematical models of the disposable soma theory to identify conditions in which agelessness would be evolutionarily favored. For any given type of damage that could accumulate and cause age-accelerating mortality risk, we find that evolution could select for its complete removal if the mortality risk it poses is severe enough and its repair does not pose too large of a penalty to reproduction. Environmental factors such as extrinsic mortality and the form of population density-dependent regulation also play a large role in determining the optimal rate of aging and whether agelessness should be evolutionarily favored. However, in a system with multiple sources of damage and multiple independent repair processes, avoiding aging is rarely evolutionarily favorable. Pleiotropic repair processes, such as those that could be present in asexual fissioning organisms, make agelessness more likely but do not guarantee it. Our results indicate that agelessness could be favored by evolution in narrow contexts but that multiple types of damage and repair make agelessness unlikely to arise in sufficiently complex organisms.

Regeneration requires mechanisms for producing a wide array of cell types. Neoblasts are stem cells in the planarian Schmidtea mediterranea that undergo fate specification to produce over 125 adult cell types. Fate specification in neoblasts can be regulated through expression of fate-specific transcription factors. We utilize multiplexed error-robust fluorescence in situ hybridization (MERFISH) and whole-mount FISH to characterize fate choice distribution of stem cells within planarians. Fate choices are often made distant from target tissues and in a highly intermingled manner, with neighboring neoblasts frequently making divergent fate choices for tissues of different location and function. We propose that pattern formation is driven primarily by the migratory assortment of progenitors from mixed and spatially distributed fate-specified stem cells and that fate choice involves stem-cell intrinsic processes.

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell-generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell-like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.

Regeneration and tissue homeostasis require accurate production of missing cell lineages. Cell production is driven by changes to gene expression, which is shaped by multiple layers of regulation. Here, we find that the ubiquitous mRNA base-modification, m6A, is required for proper cell fate choice and cellular maturation in planarian stem cells (neoblasts). We mapped m6A-enriched regions in 7,600 planarian genes and found that perturbation of the m6A pathway resulted in progressive deterioration of tissues and death. Using single-cell RNA sequencing of >20,000 cells following perturbation of the m6A pathway, we identified an increase in expression of noncanonical histone variants, and that inhibition of the pathway resulted in accumulation of undifferentiated cells throughout the animal in an abnormal transcriptional state. Analysis of >1,000 planarian gene expression datasets revealed that the inhibition of the chromatin modifying complex NuRD had almost indistinguishable consequences, unraveling an unappreciated link between m6A and chromatin modifications. Our findings reveal that m6A is critical for planarian stem cell homeostasis and gene regulation in tissue maintenance and regeneration.

Regeneration often involves the formation of a blastema, an outgrowth or regenerative bud formed at the plane of injury where missing tissues are produced. The mechanisms that trigger blastema formation are therefore fundamental for regeneration. Here, we identify a gene, which we named equinox, that is expressed within hours of injury in the planarian wound epidermis. equinox encodes a predicted secreted protein that is conserved in many animal phyla. Following equinox inhibition, amputated planarians fail to maintain wound-induced gene expression and to subsequently undergo blastema outgrowth. Associated with these defects is an inability to reestablish lost positional information needed for missing tissue specification. Our findings link the planarian wound epidermis, through equinox, to regeneration of positional information and blastema formation, indicating a broad regulatory role of the wound epidermis in diverse regenerative contexts.

The capacity for regeneration is broad in the animal kingdom. Planarians are flatworms that can regenerate any missing body part and their regenerative powers have combined with ease of experimentation to make them a classic regeneration model for more than a century. Pluripotent stem cells called neoblasts generate missing planarian tissues. Fate specification happens in the neoblasts, and this can occur in response to regeneration instructions in the form of positional information. Fate specification can lead to differentiating cells in single steps rather than requiring a long lineage hierarchy. Planarians display constitutive expression of positional information from muscle cells, which is required for patterned maintenance of tissues in tissue turnover. Amputation leads to the rapid resetting of positional information in a process triggered by wound signaling and the resetting of positional information is required for regeneration. These findings suggest a model for planarian regeneration in which adult positional information resets after injury to regulate stem cells to bring about the replacement of missing parts.