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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]

Planaria are a group of worms within the phylum Platyhelminthes (flatworms). Many species, including Schmidtea mediterranea , have the ability to regenerate their body from small pieces of tissue and are easy to keep in the laboratory, which makes them a prime model system for studying whole-body regeneration.

Metazoans function as individual organisms but also as “colonies” of cells whose single-celled ancestors lived and reproduced independently. Insights from evolutionary biology aboutmulticellular group formation help us understand the behavior of cells: why they cooperate, and why cooperation sometimes breaks down. Current explanations for multicellularity focus on two aspects of development which promote cooperation and limit conflict among cells: a single-cell bottleneck, which creates organisms composed of clones, and a separation of somatic and germ cell lineages, which reduces the selective advantage of cheating. However, many obligately multicellular organisms thrive with neither, creating the potential for withinorganism conflict. Here, we argue that the prevalence of such organisms throughout the Metazoa requires us to refine our preconceptions of conflict-free multicellularity. Evolutionary theory must incorporate developmental mechanisms across a broad range of organisms—such as unusual reproductive strategies, totipotency, and cell competition—while developmental biology must incorporate evolutionary principles. To facilitate this cross-disciplinary approach, we provide a conceptual overview from evolutionary biology for developmental biologists, using analogous examples in the well-studied social insects.

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

The remarkable regenerative abilities of flatworms are closely linked to neoblasts – adult pluripotent stem cells that are the only division-competent cell type outside of the reproductive system. Although the presence of neoblast-like cells and whole-body regeneration in other animals has led to the idea that these features may represent the ancestral metazoan state, the evolutionary origin of both remains unclear. Here we show that the catenulid Stenostomum brevipharyngium , a member of the earliest-branching flatworm lineage, lacks conventional neoblasts despite being capable of whole-body regeneration and asexual reproduction. Using a combination of single-nuclei transcriptomics, in situ gene expression analysis, and functional experiments, we find that cell divisions are not restricted to a single cell type and are associated with multiple fully differentiated somatic tissues. Furthermore, the cohort of germline multipotency genes, which are considered canonical neoblast markers, are not expressed in dividing cells, but in the germline instead, and we experimentally show that they are neither necessary for proliferation nor regeneration. Overall, our results challenge the notion that canonical neoblasts are necessary for flatworm regeneration and open up the possibility that neoblast-like cells may have evolved convergently in different animals, independent of their regenerative capacity. Adult pluripotent stem cells, called neoblasts, are considered a defining feature of all flatworms. This manuscript shows that canonical neoblasts are absent in an early branching flatworm lineage, raising important questions about the evolution of neoblasts.

Embryos in a particular phylum of the animal kingdom tend to most resemble one another at a stage in the middle of embryogenesis known as the phylotypic period; a transcriptional analysis of embryogenesis from single embryos of ten different phyla reveals that the transcripts expressed at the phylotypic stage (or mid-developmental transition) differ greatly between phyla, and a ‘phylum’ may be defined as a set of species sharing the same signals and transcription factor networks during the mid-developmental transition. Stage set for defining a phylum Embryos in a particular phylum tend to resemble one another closely at some point in the middle of embryogeny. This is known as the phylotypic stage, and it has been established that embryos at this stage tend to express a conserved set of genes that are evolutionarily older than the genes expressed before and after. This, however, only applies within a phylum, as Yanai and colleagues demonstrate in an analysis of transcriptomes from individual embryos of ten disparate phyla. Considered across the whole animal kingdom, the transcripts expressed at the phylotypic stage differ greatly between phyla and could be said to define the characters of a particular phylum. This work also provides an operational definition for a phylum as a set of species — with a common ancestor — that share the same molecular mechanisms at the phylotypic stage. Animals are grouped into ~35 ‘phyla’ based upon the notion of distinct body plans 1 , 2 , 3 , 4 . Morphological and molecular analyses have revealed that a stage in the middle of development—known as the phylotypic period—is conserved among species within some phyla 5 , 6 , 7 , 8 , 9 . Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals 10 . Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent ‘mid-developmental transition’ that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids. Light microscopy is a key tool in biomedical research. For perfect images, light needs to be able to pass through the sample, the material (or “mounting medium”) that holds the sample in place, and finally the image-detecting equipment in a straight line. However, in practice, light rays often deviate away from this line because they move at different speeds in different materials; how much the speed of light changes is related to a property called the refractive index of the material. This is exactly the effect that causes a stick stuck into water to look bent at the water’s surface. In light microscopy, mismatches in refractive index significantly reduce quality of the images that can be obtained. Live specimens are particularly challenging to image because different specimens have very different refractive indices compared to the mounting medium, which holds specimens in place but must also keep them alive. Although the addition of chemical compounds can theoretically match the refractive index of the mounting medium to that of the specimen, this approach has so far not been practical because such manipulations tend to kill the specimen. An important challenge has therefore been to identify a compound that can adjust, or “tune”, the refractive index of mounting media over a wide range, yet without harming the specimens. Now, Boothe et al. have identified a chemical called Iodixanol as an ideal and easy to use supplement for tuning the refractive index of water-based live imaging media. Adding Iodixanol to the mounting media did not appear to have any toxic effects on cell cultures, developing zebrafish embryos or regenerating planarian flatworms. Importantly, Boothe et al. found that Iodixanol significantly improved the quality of the images collected from all of these different specimens. It is important to stress that Iodixanol does not change the refractive index of the sample or cancel out refractive index differences within the sample – so it cannot render opaque specimens transparent. Nevertheless, Iodixanol supplementation is a simple and affordable technique to improve image quality in any live imaging application without having to resort to more expensive and highly specialized microscopes.

The planarian Schmidtea mediterranea is being studied as a model species for regeneration, but the assembly of planarian genomes remains challenging. Here, we report a high-quality haplotype-phased, chromosome-scale genome assembly of the sexual S2 strain of S. mediterranea and high-quality chromosome-scale assemblies of its three close relatives, S. polychroa , S. nova , and S. lugubris . Using hybrid gene annotations and optimized ATAC-seq and ChIP-seq protocols for regulatory element annotation, we provide valuable genome resources for the planarian research community and a first comparative perspective on planarian genome evolution. Our analyses reveal substantial divergence in protein-coding sequences and regulatory regions but considerable conservation within promoter and enhancer annotations. We also find frequent retrotransposon-associated chromosomal inversions and interchromosomal translocations within the genus Schmidtea and, remarkably, independent and nearly complete losses of ancestral metazoan synteny in Schmidtea and two other flatworm groups. Overall, our results suggest that platyhelminth genomes can evolve without syntenic constraints. Planarians are model systems for stem cells and regeneration. This study provides new genome assemblies of the model species S. mediterranea and 3 relatives and uses comparative genomics of planarians to reveal that synteny is not an evolutionary constraint in flatworm genome evolution.

We illustrate shape mode analysis as a simple, yet powerful technique to concisely describe complex biological shapes and their dynamics. We characterize undulatory bending waves of beating flagella and reconstruct a limit cycle of flagellar oscillations, paying particular attention to the periodicity of angular data. As a second example, we analyze non-convex boundary outlines of gliding flatworms, which allows us to expose stereotypic body postures that can be related to two different locomotion mechanisms. Further, shape mode analysis based on principal component analysis allows to discriminate different flatworm species, despite large motion-associated shape variability. Thus, complex shape dynamics is characterized by a small number of shape scores that change in time. We present this method using descriptive examples, explaining abstract mathematics in a graphic way.

Background Multiple RNA samples are frequently processed together and often mixed before multiplex sequencing in the same sequencing run. While different samples can be separated post sequencing using sample barcodes, the possibility of cross contamination between biological samples from different species that have been processed or sequenced in parallel has the potential to be extremely deleterious for downstream analyses. Results We present CroCo, a software package for identifying and removing such cross contaminants from assembled transcriptomes. Using multiple, recently published sequence datasets, we show that cross contamination is consistently present at varying levels in real data. Using real and simulated data, we demonstrate that CroCo detects contaminants efficiently and correctly. Using a real example from a molecular phylogenetic dataset, we show that contaminants, if not eliminated, can have a decisive, deleterious impact on downstream comparative analyses. Conclusions Cross contamination is pervasive in new and published datasets and, if undetected, can have serious deleterious effects on downstream analyses. CroCo is a database-independent, multi-platform tool, designed for ease of use, that efficiently and accurately detects and removes cross contamination in assembled transcriptomes to avoid these problems. We suggest that the use of CroCo should become a standard cleaning step when processing multiple samples for transcriptome sequencing.