In vertebrates, the four transcription factors Sox2, c-Myc, Pou5f1 and Klf4 are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The in vitro forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts. Botryllus schlosseri is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified B. schlosseri orthologs of human Sox2 and c-Myc genes, as well as the closest homologs of the vertebrate-specific Pou5f1 gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of B. schlosseri using in situ hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover, Myc, SoxB1 and Pou3 were expressed in candidate stem cells residing in their niches, while Pou2 was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that Myc, SoxB1 and Pou3 may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.

The swimming larva represents the dispersal phase of ascidians, marine invertebrates belonging to tunicates. Due to its adhesive papillae, the larva searches the substrate, adheres to it, and undergoes metamorphosis, thereby becoming a sessile filter feeding animal. The larva anatomy has been described in detail in a few species, revealing a different degree of adult structure differentiation, called adultation. In the solitary ascidian Halocynthia roretzi, a species reared for commercial purposes, embryogenesis has been described in detail, but information on the larval anatomy is still lacking. Here, we describe it using a comparative approach, utilizing 3D reconstruction, as well as histological/TEM observations, with attention to its papillae. The larva is comparable to those of other solitary ascidians, such as Ciona intestinalis. However, it displays a higher level of adultation for the presence of the atrium, opened outside by means of the atrial siphon, and the peribranchial chambers. It does not reach the level of complexity of the larva of Botryllus schlosseri, a phylogenetically close colonial ascidian. Our study reveals that the papillae of H. roretzi, previously described as simple and conform, exhibit dynamic changes during settlement. This opens up new considerations on papillae morphology and evolution and deserves to be further investigated.

Allometry explores the relationship between an organism’s body size and its various components, offering insights into ecology, physiology, metabolism, and disease. The cell is the basic unit of biological systems, and yet the study of cell-type allometry remains relatively unexplored. Single-cell RNA sequencing (scRNA-seq) provides a promising tool for investigating cell-type allometry. Planarians, capable of growing and degrowing following allometric scaling rules, serve as an excellent model for these studies. We used scRNA-seq to examine cell-type allometry in asexual planarians of different sizes, revealing that they consist of the same basic cell types but in varying proportions. Notably, the gut basal cells are the most responsive to changes in size, suggesting a role in energy storage. We capture the regulated gene modules of distinct cell types in response to body size. This research sheds light on the molecular and cellular aspects of cell-type allometry in planarians and underscores the utility of scRNA-seq in these investigations.

Ascidians are sessile tunicates, that is, marine animals belonging to the phylum Chordata and considered the sister group of vertebrates. They are widespread in all the seas, constituting abundant communities in various ecosystems. Among chordates, only tunicates are able to reproduce asexually, forming colonies. The high regenerative potentialities enabling tunicates to regenerate damaged body parts, or the whole body, represent a peculiarity of this taxon. Here we review the methodological approaches used in more than a century of biological studies to induce regeneration in both solitary and colonial species. For solitary species, we refer to the regeneration of single organs or body parts (e.g., siphon, brain, gonad, tunic, viscera). For colonial species, we review a plethora of experiments regarding the surgical manipulation of colonies, the regeneration of isolated colonial entities, such as single buds in the tunic, or part of tunic and its circulatory system.

Cell type identity is controlled by gene regulatory networks (GRNs), where transcription factors (TFs) regulate target genes (TGs) via open chromatin regions (OCRs), often specific to one or multiple cell types. Classic GRN discovery using perturbations is laborious and not easily scalable across the tree of life. Single-cell transcriptomics enables cell type-resolved gene expression analysis, but integrating perturbation data remains difficult. Here, we investigate planarian stem cell differentiation by integrating single-cell transcriptomics and chromatin accessibility data. The integrated analysis identifies gene networks matching known TF interactions and highlights TFs that may drive differentiation across multiple cell types. Our data reveals at least two major cell type supergroups linked by their regulatory logic, including alx3-1 + cells, comprising muscle, neurons and secretory cells, and hnf4 + cells, comprising gut phagocytes, goblet cells and parenchymal cells. We validated our data demonstrating high overlap between predicted targets and experimentally validated differentially regulated genes. Overall, our study integrates TFs, TGs and OCRs to reveal the regulatory logic of planarian stem cell differentiation, showcasing a comprehensive catalogue of GRN computational inferences that will be key to study this process.

Stem cell niches are defined as the microenvironments where stem cells home, receiving stimuli defining their fate. In vertebrates, stem cell niches are stable and physically confined compartments. Botryllus schlosseri is an invertebrate colonial chordate where temporary stem cell niches have been identified in adult individuals that are cyclically resorbed and replaced by a new generation of clonal zooids. B. schlosseri also displays remarkable regenerative abilities, being capable of whole-body regeneration, but the cellular source of these processes is still unknown. Here we identified by means of a high-resolution morphological characterization a new putative stem cell niche in the ampullae of the circulatory system acting as a stem cell source during asexual reproduction. Stem cells of the ampullae travel via the circulatory system and contribute to the development of several organs and could explain where stem cells contributing to whole-body regeneration are stored. The ampullae niches are stable during the life cycle and regeneration of B. schlosseri , while additional niches of the zooid are dynamically established and colonised by circulating stem cells. Our results reveal an unprecedented dynamicity of stem cell niches in highly regenerative invertebrates.