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Boron nitride nanotubes (BNNTs) represent an extremely interesting class of nanomaterials, and recent findings have suggested a number of applications in the biomedical field. Anyhow, extensive biocompatibility investigations are mandatory before any further advancement toward preclinical testing. Here, we report on the effects of multiwalled BNNTs in freshwater planarians, one of the best-characterized in vivo models for developmental biology and regeneration research. Obtained results indicate that BNNTs are biocompatible in the investigated model, since they do not induce oxidative DNA damage and apoptosis, and do not show adverse effects on planarian stem cell biology and on de novo tissue regeneration. In summary, collected findings represent another important step toward BNNT realistic applications in nanomedicine.

The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea , using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms. An improved genome assembly for Schmidtea mediterranea shows that the genome is highly polymorphic and repetitive, and lacks multiple genes encoding core components of cell biological mechanisms. Genome of a regenerating worm The flatworm Schmidtea mediterranea is an important model for regeneration. Jochen Rink, Eugene Myers and colleagues report an improved genome assembly for the planarian S. mediterranea using long-read sequencing and a new genome assembler called MARVEL. They find that the S. mediterranea genome is highly polymorphic and repetitive, and includes a novel class of giant retroelements. This improved genome assembly provides a useful resource for studying regeneration and the evolution of cell plasticity.

Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. We used ionizing radiation and single-cell transplantation to identify neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known postmitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.

Longitudinal and circular muscle fibres have distinct regulatory roles during planarian regeneration. Fibres guide flatworm regeneration Correct positional information is critical to the successful regeneration of missing body parts, but how regeneration is accomplished is poorly understood. In planarians, it has been shown that inhibition of Wnt signalling leads to the regeneration of the wrong missing body parts and that other genes involved in correct body patterning during regeneration are expressed in muscle tissues. Peter Reddien and colleagues find that two different transcription factors are involved in the regeneration of longitudinal versus circular fibres, and that loss of circular fibres impairs the positioning of regenerating anterior parts. The ability to regenerate missing body parts exists throughout the animal kingdom. Positional information is crucial for regeneration, but how it is harboured and used by differentiated tissues is poorly understood. In planarians, positional information has been identified from study of phenotypes caused by RNA interference in which the wrong tissues are regenerated. For example, inhibition of the Wnt signalling pathway leads to regeneration of heads in place of tails 1 , 2 , 3 . Characterization of these phenotypes has led to the identification of position control genes (PCGs)—genes that are expressed in a constitutive and regional manner and are associated with patterning. Most PCGs are expressed within planarian muscle 4 ; however, how muscle is specified and how different muscle subsets affect regeneration is unknown. Here we show that different muscle fibres have distinct regulatory roles during regeneration in the planarian Schmidtea mediterranea . myoD is required for formation of a specific muscle cell subset: the longitudinal fibres, oriented along the anterior–posterior axis. Loss of longitudinal fibres led to complete regeneration failure because of defects in regeneration initiation. A different transcription factor-encoding gene, nkx1-1 , is required for the formation of circular fibres, oriented along the medial–lateral axis. Loss of circular fibres led to a bifurcated anterior–posterior axis with fused heads forming in single anterior blastemas. Whereas muscle is often viewed as a strictly contractile tissue, these findings reveal that different muscle types have distinct and specific regulatory roles in wound signalling and patterning to enable regeneration.

Issue Title: Development and Evolution of Stem Cells Planarians are members of the Platyhelminthes (flatworms). These animals have evolved a remarkable stem cell system. A single pluripotent adult stem cell type (\"neoblast\") gives rise to the entire range of cell types and organs in the planarian body plan, including a brain, digestive-, excretory-, sensory- and reproductive systems. Neoblasts are abundantly present throughout the mesenchyme and divide continuously. The resulting stream of progenitors and turnover of differentiated cells drive the rapid self-renewal of the entire animal within a matter of weeks. Planarians grow and literally de-grow (\"shrink\") by the food supply-dependent adjustment of organismal turnover rates, scaling body plan proportions over as much as a 50-fold size range. Their dynamic body architecture further allows astonishing regenerative abilities, including the regeneration of complete and perfectly proportioned animals even from tiny tissue remnants. Planarians as an experimental system, therefore, provide unique opportunities for addressing a spectrum of current problems in stem cell research, including the evolutionary conservation of pluripotency, the dynamic organization of differentiation lineages and the mechanisms underlying organismal stem cell homeostasis. The first part of this review focuses on the molecular biology of neoblasts as pluripotent stem cells. The second part examines the fascinating mechanistic and conceptual challenges posed by a stem cell system that epitomizes a universal design principle of biological systems: the dynamic steady state.[PUBLICATION ABSTRACT]

Differential coordination of growth and patterning across metazoans gives rise to a diversity of sizes and shapes at tissue, organ and organismal levels. Although tissue size and tissue function can be interdependent 1 – 5 , mechanisms that coordinate size and function remain poorly understood. Planarians are regenerative flatworms that bidirectionally scale their adult body size 6 , 7 and reproduce asexually, via transverse fission, in a size-dependent manner 8 – 10 . This model offers a robust context to address the gap in knowledge that underlies the link between size and function. Here, by generating an optimized planarian fission protocol in Schmidtea mediterranea , we show that progeny number and the frequency of fission initiation are correlated with parent size. Fission progeny size is fixed by previously unidentified mechanically vulnerable planes spaced at an absolute distance along the anterior–posterior axis. An RNA interference screen of genes for anterior–posterior patterning uncovered components of the TGFβ and Wnt signalling pathways as regulators of the frequency of fission initiation rather than the position of fission planes. Finally, inhibition of Wnt and TGFβ signalling during growth altered the patterning of mechanosensory neurons—a neural subpopulation that is distributed in accordance with worm size and modulates fission behaviour. Our study identifies a role for TGFβ and Wnt in regulating size-dependent behaviour, and uncovers an interdependence between patterning, growth and neurological function. A planarian fission protocol shows that the number of progeny and the frequency of fission initiation correlate with parent size, and TGFβ and Wnt signalling components are identified as regulators of fission behaviour.

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.

Many metazoans switch between asexual and sexual reproduction based on environmental changes, life cycle phases, or both. This reproductive strategy enables them to benefit from the features of both reproductive modes. In general, asexual reproduction is broadly divided into parthenogenesis and vegetative reproduction. As in parthenogenesis, individuals develop ovaries and lay eggs, the most significant event in switching from parthenogenesis to sexual reproduction is the production of testes. Meanwhile, in vegetative reproduction, individuals do not need germ cells themselves. Thus, they must post-embryonically develop and maintain germ cells derived from pluripotent cells as they switch from vegetative to sexual reproduction. The complicated mechanisms for controlling the postembryonic reproductive development remain unknown. The planarian Dugesia ryukyuensis can switch from vegetative to sexual reproduction by stimulating bioactive compounds called sex-inducing substances, which are widely conserved in Platyhelminthes, including parasitic flatworms. The two reproductive modes are facilitated by the presence of adult pluripotent stem cells, which generate any type of somatic tissue in the asexual state and produce and maintain hermaphroditic reproductive organs in the sexual state. In this study, using RNA sequencing analysis in experimental sexualization by sex-inducing substances, we identified four essential genes for sexualization. A common feature following the knockdown of the four essential genes was a blockage of testicular differentiation. One of the four essential genes was a gap junction gene, Dr-siri ( D ugesia r yukyuensis - s exual i nduction- r elated i nnexin). We suggest that the establishment of a testicular stem cell niche supported by Dr-siri protein is responsible for the breakthrough of dormancy in postembryonic reproductive development in planarian reproductive switching. Our findings suggest that the production of testes might be crucial for even switching from vegetative to sexual reproduction.

The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together, these data and analyses shed light on important physiological modifiers of morphological information in dictating species-specific shape, and reveal them to be a novel instructive input into head patterning in regenerating planaria.

Tissue regeneration is a complex process involving large changes in cell proliferation, fate determination, and differentiation. Mitochondrial dynamics and metabolism play a crucial role in development and wound repair, but their function in large-scale regeneration remains poorly understood. Planarians offer an excellent model to investigate this process due to their remarkable regenerative abilities. In this study, we examine mitochondrial dynamics during planarian regeneration. We find that knockdown of the mitochondrial fusion gene, opa1 , impairs both tissue regeneration and stem cell pluripotency. Interestingly, the regeneration defects caused by opa1 knockdown are rescued by simultaneous knockdown of the mitochondrial fission gene, drp1 , which partially restores mitochondrial dynamics. Furthermore, we discover that Mito low stem cells exhibit an enrichment of pluripotency due to their fate choices at earlier stages. Transcriptomic analysis reveals the delicate mitonuclear balance in metabolism and mitochondrial proteins in regeneration, controlled by mitochondrial dynamics. These findings highlight the importance of maintaining mitochondrial dynamics in large-scale tissue regeneration and suggest the potential for manipulating these dynamics to enhance stem cell functionality and regenerative processes. Mitochondrial dynamics in large-scale regeneration remain poorly understood. Here they show that the mitochondrial fusion-fission equilibrium can determine the pluripotency of planarian stem cells and that mitonuclear balance is critical for planarian regeneration.