TIM29 is required for enhanced stem cell activity during regeneration in the flatworm Macrostomum lignano

TIM29 is a mitochondrial inner membrane protein that interacts with the protein import complex TIM22. TIM29 was shown to stabilize the TIM22 complex but its biological function remains largely unknown. Until recently, it was classified as one of the Domain of Unknown Function (DUF) genes, with a conserved protein domain DUF2366 of unclear function. Since characterizing DUF genes can provide novel biological insight, we used previously established transcriptional profiles of the germline and stem cells of the flatworm Macrostomum lignano to probe conserved DUFs for their potential role in germline biology, stem cell function, regeneration, and development. Here, we demonstrate that DUF2366/TIM29 knockdown in M. lignano has very limited effect during the normal homeostatic condition but prevents worms from adapting to a highly proliferative state required for regeneration

Pharmacological Or Genetic Targeting Of Transient Receptor Potential (TRP) Channels Can Disrupt The Planarian Escape Response

In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit an oscillatory type of locomotion called scrunching. We have previously characterized the biomechanics of scrunching and shown that it is induced by specific stimuli, such as amputation, noxious heat, and extreme pH. Because these specific inducers are known to activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other systems induce scrunching in the planarian species Dugesia japonica and, except for anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. We further confirmed that TRPA1 in both planarian species is necessary for AITC-induced scrunching using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit capsaicin-induced scrunching. RNAi of DjTRPVa/DjTRPVb attenuated scrunching induced by the endocannabinoid and TRPV agonist, anandamide. Overall, our results show that although scrunching induction can involve different initial pathways for sensing stimuli, this behavior’s signature dynamical features are independent of the inducer, implying that scrunching is a stereotypical planarian escape behavior in response to various noxious stimuli that converge on a single downstream pathway. Understanding which aspects of nociception are conserved or not across different organisms can provide insight into the underlying regulatory mechanisms to better understand pain sensation

The pioneer factor Smed-gata456-1 is required for gut cell differentiation and maintenance in planarians

How adult stem cells differentiate into different cell types remains one of the most intriguing questions in regenerative medicine. Pioneer factors are transcription factors that can bind to and open chromatin, and are among the first elements involved in cell differentiation. We used the freshwater planarian Schmidtea mediterranea as a model system to study the role of the gata456 family of pioneer factors in gut cell differentiation during both regeneration and maintenance of the digestive system. Our findings reveal the presence of two members of the gata456 family in the Schmidtea mediterranea genome; Smed-gata456-1 and Smed-gata456-2. Our results show that Smed-gata456-1 is the only ortholog with a gut cell-related function. Smed-gata456-1 is essential for the differentiation of precursors into intestinal cells and for the survival of these differentiated cells, indicating a key role in gut regeneration and maintenance. Furthermore, tissues other than the gut appear normal following Smed-gata456-1 RNA interference (RNAi), indicating a gut-specific function. Importantly, different neoblast subtypes are unaffected by Smed-gata456-1(RNAi), suggesting that 1) Smed-gata456-1 is involved in the differentiation and maintenance, but not in the early determination, of gut cells; and 2) that the stem cell compartment is not dependent on a functional gut

Developmental scRNAseq Trajectories in Gene- and Cell-State Space—The Flatworm Example

Single-cell RNA sequencing has become a standard technique to characterize tissue development. Hereby, cross-sectional snapshots of the diversity of cell transcriptomes were transformed into (pseudo-) longitudinal trajectories of cell differentiation using computational methods, which are based on similarity measures distinguishing cell phenotypes. Cell development is driven by alterations of transcriptional programs e.g., by differentiation from stem cells into various tissues or by adapting to micro-environmental requirements. We here complement developmental trajectories in cell-state space by trajectories in gene-state space to more clearly address this latter aspect. Such trajectories can be generated using self-organizing maps machine learning. The method transforms multidimensional gene expression patterns into two dimensional data landscapes, which resemble the metaphoric Waddington epigenetic landscape. Trajectories in this landscape visualize transcriptional programs passed by cells along their developmental paths from stem cells to differentiated tissues. In addition, we generated developmental “vector fields” using RNA-velocities to forecast changes of RNA abundance in the expression landscapes. We applied the method to tissue development of planarian as an illustrative example. Gene-state space trajectories complement our data portrayal approach by (pseudo-)temporal information about changing transcriptional programs of the cells. Future applications can be seen in the fields of tissue and cell differentiation, ageing and tumor progression and also, using other data types such as genome, methylome, and also clinical and epidemiological phenotype data

Cellular and Molecular Responses Unique to Major Injury Are Dispensable for Planarian Regeneration

The fundamental requirements for regeneration are poorly understood. Planarians can robustly regenerate all tissues after injury, involving stem cells, positional information, and a set of cellular and molecular responses collectively called the “missing tissue” or “regenerative” response. follistatin, which encodes an extracellular Activin inhibitor, is required for the missing tissue response after head amputation and for subsequent regeneration. We found that follistatin is required for the missing tissue response regardless of the wound context, but causes regeneration failure only after head amputation. This head regeneration failure involves follistatin-mediated regulation of Wnt signaling at wounds and is not a consequence of a diminished missing tissue response. All tested contexts of regeneration, including head regeneration, could occur with a defective missing tissue response, but at a slower pace. Our findings suggest that major cellular and molecular programs induced specifically by large injuries function to accelerate regeneration but are dispensable for regeneration itself. In regenerative organisms, a large array of cellular responses are triggered at major injuries. However, which of these responses are fundamentally required for regeneration to occur remains unknown. Tewari et al. find that hallmark cellular and molecular responses induced uniquely at large injuries are dispensable for planarian regeneration. Keywords: regeneration; wound response; WnT signaling; TGF-β signaling; planarians; follistatinNational Institutes of Health (U.S.) (Grant R01GM080639

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system

Outstanding intraindividual genetic diversity in fissiparous planarians (Dugesia, Platyhelminthes) with facultative sex.

Predicted genetic consequences of asexuality include high intraindividual genetic diversity (i.e., the Meselson effect) and accumulation of deleterious mutations (i.e., Muller’s Ratchet), among others. These consequences have been largely studied in parthenogenetic organisms, but studies on fissiparous species are scarce. Differing from parthenogens, fissiparous organisms inherit part of the soma of the progenitor, including somatic mutations. Thus, in the long term, fissiparous reproduction may also result in genetic mosaicism, besides the presence of the Meselson effect and Muller’s Ratchet. Dugesiidae planarians show outstanding regeneration capabilities, allowing them to naturally reproduce by fission, either strictly or combined with sex (facultative). Therefore, they are an ideal model to analyze the genetic footprint of fissiparous reproduction, both when it is alternated with sex and when it is the only mode of reproduction

Transcriptional signatures of somatic neoblasts and germline cells in <i>Macrostomum lignano</i>

The regeneration-capable flatworm <i>Macrostomum lignano</i> is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as <i>Schmidtea mediterranea</i>. However, information on the transcriptome and markers of stem cells in <i>M. lignano</i> is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of <i>M. lignano</i>, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including <i>Mlig-ddx39</i>, <i>Mlig-rrm1</i>, <i>Mlig-rpa3</i>, <i>Mlig-cdk1</i>, and <i>Mlig-h2a</i>, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated <i>M. lignano</i> transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in <i>M. lignano</i>

Bioelectrical model of head-tail patterning based on cell ion channels and intercellular gap junctions

Robust control of anterior-posterior axial patterning during regeneration is mediated by bioelectric signaling. However, a number of systems-level properties of bioelectrochemical circuits, including stochastic outcomes such as seen in permanently de-stabilized "cryptic" flatworms, are not completely understood. We present a bioelectrical model for head-tail patterning that combines single-cell characteristics such as membrane ion channels with multicellular community effects via voltage-gated gap junctions. It complements the biochemically-focused models by describing the effects of intercellular electrochemical coupling, cutting plane, and gap junction blocking of the multicellular ensemble. We provide qualitative insights into recent experiments concerning planarian anterior/posterior polarity by showing that: (i) bioelectrical signals can help separated cell domains to know their relative position after injury and contribute to the transitions between the abnormal double-head state and the normal head-tail state; (ii) the bioelectrical phase-space of the system shows a bi-stability region that can be interpreted as the cryptic system state; and (iii) context-dependent responses are obtained depending on the cutting plane position, the initial bioelectrical state of the multicellular system, and the intercellular connectivity. The model reveals how simple bioelectric circuits can exhibit complex tissue-level patterning and suggests strategies for regenerative control in vivo and in synthetic biology contexts

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts