Extracellular vesicles promote proliferation in an animal model of regeneration.

Extracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarian Schmidtea mediterranea had similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up more quickly by S/G2 neoblasts than G1 neoblasts/early progeny and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced upregulation of neoblast-associated transcripts. In addition, EV injection increased the number of F-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenable in vivo model for the study of EV function during regeneration.

Intestine-enriched apolipoprotein b orthologs are required for stem cell progeny differentiation and regeneration in planarians.

Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem cell-driven regeneration are unclear. Planarian flatworms readily restore missing tissue due to injury-induced activation of pluripotent somatic stem cells called neoblasts. Here, we identify two intestine-enriched orthologs of apolipoprotein b, apob-1 and apob-2, which mediate transport of neutral lipid stores from the intestine to target tissues including neoblasts, and are required for tissue homeostasis and regeneration. Inhibition of apob function by RNAi causes head regression and lysis in uninjured animals, and delays body axis re-establishment and regeneration of multiple organs in amputated fragments. Furthermore, apob RNAi causes expansion of the population of differentiating neoblast progeny and dysregulates expression of genes enriched in differentiating and mature cells in eight major cell type lineages. We conclude that intestine-derived lipids serve as a source of metabolites required for neoblast progeny differentiation.

An Emerging Frontier in Intercellular Communication: Extracellular Vesicles in Regeneration.

Regeneration requires cellular proliferation, differentiation, and other processes that are regulated by secreted cues originating from cells in the local environment. Recent studies suggest that signaling by extracellular vesicles (EVs), another mode of paracrine communication, may also play a significant role in coordinating cellular behaviors during regeneration. EVs are nanoparticles composed of a lipid bilayer enclosing proteins, nucleic acids, lipids, and other metabolites, and are secreted by most cell types. Upon EV uptake by target cells, EV cargo can influence diverse cellular behaviors during regeneration, including cell survival, immune responses, extracellular matrix remodeling, proliferation, migration, and differentiation. In this review, we briefly introduce the history of EV research and EV biogenesis. Then, we review current understanding of how EVs regulate cellular behaviors during regeneration derived from numerous studies of stem cell-derived EVs in mammalian injury models. Finally, we discuss the potential of other established and emerging research organisms to expand our mechanistic knowledge of basic EV biology, how injury modulates EV biogenesis, cellular sources of EVs in vivo, and the roles of EVs in organisms with greater regenerative capacity.

Cell-type diversity and regionalized gene expression in the planarian intestine.

Proper function and repair of the digestive system are vital to most animals. Deciphering the mechanisms involved in these processes requires an atlas of gene expression and cell types. Here, we applied laser-capture microdissection (LCM) and RNA-seq to characterize the intestinal transcriptome of Schmidtea mediterranea, a planarian flatworm that can regenerate all organs, including the gut. We identified hundreds of genes with intestinal expression undetected by previous approaches. Systematic analyses revealed extensive conservation of digestive physiology and cell types with other animals, including humans. Furthermore, spatial LCM enabled us to uncover previously unappreciated regionalization of gene expression in the planarian intestine along the medio-lateral axis, especially among intestinal goblet cells. Finally, we identified two intestine-enriched transcription factors that specifically regulate regeneration (hedgehog signaling effector gli-1) or maintenance (RREB2) of goblet cells. Altogether, this work provides resources for further investigation of mechanisms involved in gastrointestinal function, repair and regeneration.

The human body has a limited ability to regenerate and repair itself after major injuries. By contrast, flatworms ­ most notably planarians such as Schmidtea mediterranea ­ have exceptional regenerative abilities and can regrow large parts of their bodies. Regrowing body parts is a complex process involving the coordinated creation of many different types of cells, and thus an important first step in understanding tissue regeneration is to develop a detailed catalog of cell types in that tissue. Laser capture microdissection, or LCM for short, is a technology used to isolate and study subregions or even individual cells from within a tissue. This approach can help to identify different cell types and to examine what makes them unique. LCM can be used to create a detailed catalog of cells, their differences and the roles they perform. Forsthoefel et al. have now used LCM to study cells from the planarian digestive system. This approach found 1,800 genes that have high activity in cells from the gut and showed many similarities between planaria and humans. LCM made it possible to study these cells in a new level of detail, revealing several hundred new genes as well as new cell types. The study showed that regeneration and survival of cells known as goblet cells particularly depended on two genes, gli-1 and RREB2. Irreversible gut damage in humans can result from surgeries and conditions such as acid reflux. Other animals are able to repair and regenerate the gut more successfully. Techniques like LCM can help researchers to understand the differences between humans and other species. In time, these insights may lead to technologies and therapies that can improve our own abilities to heal following injuries.

Fixation, Processing, and Immunofluorescent Labeling of Whole Mount Planarians.

Efforts to elucidate mechanisms of regeneration in the planarian Schmidtea mediterranea have included the application of immunocytochemical methods to detect specific molecules and label cells and tissues in situ. Here we describe methods for immunofluorescent labeling of whole mount planarians. We outline protocols for fixation and steps for processing animals prior to immunolabeling, incorporating commonly utilized reagents for mucus removal, pigment bleaching, tissue permeabilization, and antigen retrieval. Because processing steps can mask or degrade antigens, we also recommend protocol parameters that can be tested simultaneously to optimize sample preparation for novel antibodies.

Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.

BACKGROUND: Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling. RESULTS: To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. CONCLUSIONS: We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. Our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms.

RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics.

BACKGROUND: The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. RESULTS: We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. CONCLUSIONS: This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems.

A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors.

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-ß. Indeed, whereas many biological responses elicited by Gq depend on the transient activation of second-messenger systems, Gq utilizes a hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.

An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians.

Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.

Stem cell-based growth, regeneration, and remodeling of the planarian intestine.

Although some animals are capable of regenerating organs, the mechanisms by which this is achieved are poorly understood. In planarians, pluripotent somatic stem cells called neoblasts supply new cells for growth, replenish tissues in response to cellular turnover, and regenerate tissues after injury. For most tissues and organs, however, the spatiotemporal dynamics of stem cell differentiation and the fate of tissue that existed prior to injury have not been characterized systematically. Utilizing in vivo imaging and bromodeoxyuridine pulse-chase experiments, we have analyzed growth and regeneration of the planarian intestine, the organ responsible for digestion and nutrient distribution. During growth, we observe that new gut branches are added along the entire anteroposterior axis. We find that new enterocytes differentiate throughout the intestine rather than in specific growth zones, suggesting that branching morphogenesis is achieved primarily by remodeling of differentiated intestinal tissues. During regeneration, we also demonstrate a previously unappreciated degree of intestinal remodeling, in which pre-existing posterior gut tissue contributes extensively to the newly formed anterior gut, and vice versa. By contrast to growing animals, differentiation of new intestinal cells occurs at preferential locations, including within newly generated tissue (the blastema), and along pre-existing intestinal branches undergoing remodeling. Our results indicate that growth and regeneration of the planarian intestine are achieved by co-ordinated differentiation of stem cells and the remodeling of pre-existing tissues. Elucidation of the mechanisms by which these processes are integrated will be critical for understanding organogenesis in a post-embryonic context.

Emerging patterns in planarian regeneration.

In the past decade, the planarian has become an increasingly tractable invertebrate model for the investigation of regeneration and stem cell biology. Application of a variety of techniques and development of genomic reagents in this system have enabled exploration of the molecular mechanisms by which pluripotent somatic stem cells called neoblasts replenish, repair, and regenerate planarian tissues and organs. Recent investigations have implicated evolutionarily conserved signaling pathways in the re-establishment of anterior-posterior (A-P), dorsal-ventral (D-V), and medial-lateral (M-L) polarity after injury. These studies have significantly advanced our understanding of early events during planarian regeneration and have raised new questions about the mechanisms of stem cell-based tissue repair and renewal.

The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila.

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. We detect genetic and/or physical interactions between Fra and these effector molecules that suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline.

Interactions between the secreted protein Amalgam, its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding.

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase (Abl) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. We have identified an unusual missense ama allele, ama(M109), which dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the Ama(M109) mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, we have found that all five mutations originally attributed to disabled are in fact alleles of nrt. These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone.

The activity of the Drosophila morphogenetic protein Bicoid is inhibited by a domain located outside its homeodomain.

The Drosophila morphogenetic protein Bicoid (Bcd) is a homeodomain-containing activator that stimulates the expression of target genes during early embryonic development. We demonstrate that a small domain of Bcd located immediately N-terminally of the homeodomain represses its own activity in Drosophila cells. This domain, referred to as a self-inhibitory domain, works as an independent module that does not rely on any other sequences of Bcd and can repress the activity of heterologous activators. We further show that this domain of Bcd does not affect its properties of DNA binding or subcellular distribution. A Bcd derivative with point mutations in the self-inhibitory domain severely affects pattern formation and target gene expression in Drosophila embryos. We also provide evidence to suggest that the action of the self-inhibitory domain requires a Drosophila co-factor(s), other than CtBP or dSAP18. Our results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen during embryonic development is dependent on the downregulation of its own activity through an interaction with a novel co-repressor(s) or complex(es).