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

To Be or Not to Be a Tapeworm Parasite: That Is the Post-Genomic Question in <em>Taenia solium</em> Cysticercosis

Cestode parasites rely on their host to obtain their nutrients. Elucidation of tapeworm genomes has shown a remarkable reduction in the coding of multiple enzymes, particularly those of anabolic pathways. Previous findings showed that 10–13% of the proteins found in the vesicular fluid of Taenia solium cysticerci are of host origin. Further proteomic characterization allowed identification of 4,259 different proteins including 891 of host origin in the parasite’s protein lysates. One explanation for this high abundance and diversity of host proteins in the parasite lysates is related to the functional exploitation of host proteins by cysticerci. Supporting this concept is the uptake of host haptoglobin and hemoglobin by the parasite, as a way to acquire iron. Surprisingly, internalized host proteins are minimally degraded by the parasite physiological machinery. Additional proteomic analysis demonstrated that these host proteins become part of the organic matrix of calcareous corpuscles; as 60–70% of the protein content are host proteins. In this review, a collection of available genomic and proteomic data for taeniid cestodes is assembled, the subject of the use and processing of host proteins is particularly addressed; a sketchy and unique cell physiological profile starts to emerge for these parasitic organisms

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

Analysis of Fox genes in Schmidtea mediterranea reveals new families and a conserved role of Smed‑foxO in controlling cell death

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa clade, and specifically from planarians, which are a unique model for understanding development, due to the striking plasticity of the adult. The aim of this study was to identify and perform evolutionary and functional studies of the Fox genes of lophotrochozoan species and, specifically, of the planarian Schmidtea mediterranea. Generating a pipeline for identifying Forkhead domains and using phylogenetics allowed us the phylogenetic reconstruction of Fox genes. We corrected the annotation for misannotated genes and uncovered a new family, the QD, present in all metazoans. According to the new phylogeny, the 27 Fox genes found in Schmidtea mediterranea were classified into 12 families. In Platyhelminthes, family losses were accompanied by extensive gene diversification and the appearance of specific families, the A(P) and N(P). Among the newly identified planarian Fox genes, we found a single copy of foxO, which shows an evolutionary conserved role in controlling cell death

Dugesia Japonica Is The Best Suited Of Three Planarian Species For High-Throughput Toxicology Screening

High-throughput screening (HTS) using new approach methods is revolutionizing toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct comparison with D. japonica. Through quantitative assessments of morphology and multiple behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 concentrations at two time points over a twelve-day period. We obtained two main results: First, G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to D. japonica under HTS conditions, due to decreased health over time and lack of movement under red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. Second, we observed species differences in sensitivity to the solvents, suggesting that care must be taken when extrapolating chemical effects across planarian species. Overall, our data show that D. japonica is best suited for behavioral HTS given the limitations of the other species. Standardizing which planarian species is used in neurotoxicity screening will facilitate data comparisons across research groups and accelerate the application of this promising invertebrate system for first-tier chemical HTS, helping streamline toxicology testing

An investigation into mechanisms of regeneration specificity in planarian flatworms.

Many animals have the extraordinary ability to replace lost body parts, even so we humans do not. One critical but poorly understood aspect of this phenomenon is how wounds tailor the regeneration response to the particular target structure that needs to be regrown. In my thesis work I have attempted to address this problem in the champions of regeneration, the planarian flatworms. If one of these animals is cut into tiny pieces, each of the pieces will regenerate a head at the anterior end and tail at the posterior end. For over a century investigators have searched for the intrinsic polarity cue underlying this regeneration polarity, but until now its mechanistic basis is not known. The explicit goal of my thesis work was to identify this cue. The general approach that I have taken toward identification of the intrinsic polarity is to systematically compare two different planarian species with subtle variations in the establishment of regeneration polarity, Schmidtea mediterranea and Girardia tigrina. First, I demonstrate through systematic comparison of different amputation paradigms that regeneration polarity is dependent not only on species, but also on piece length, body size and anteroposterior axis position. Second, given that these findings are consistent with a gradient- based intrinsic polarity cue as prevalent hypothesis in the field, I tested whether the recently identified tail-to-head gradient of canonical Wnt (cWnt) signalling could be mechanistic basis of regeneration polarity. As precondition for doing so, I developed new approaches to measure and manipulate cWnt signalling in planaria. The data acquired with these tools suggest that the cWnt gradient may contribute to the observed position-dependence of regeneration polarity but is overall not the (only) intrinsic polarity cue. Third, I present my initial efforts to test whether the longitudinal muscle fibres (LMFs) in which notum is exclusively activated are an intrinsic polarity cue. My results suggest that “bundles” of short, intrinsically polarised LMFs running along the AP axis may express notum when they are cut anterior to their nucleus and moreover that misregulation of such a mechanism may underlie the species-dependence of regeneration polarity. Overall, the work presented in this thesis offers new insight into the cellular and conceptual basis of planarian regeneration polarity and, in doing so, the more general question of how regenerative organisms “sense” precisely what body part is missing and therefore needs to be regrown. Furthermore, it puts forward new hypotheses that through additional experimentation may explain lead to elucidation of the underlying molecular mechanisms

Genomic analyses reveal FoxG as an upstream regulator of wnt1 required for posterior identity specification in planarians

Embryonic specification of the first body axis requires the formation of an Organizer, a group of cells with the ability to instruct fates in the surrounding tissue. The existence of organizing regions in adults, i.e. during regeneration, which also requires patterning of new tissues, remains unstudied. To that aim, we study regeneration in planarians, flatworms that can regenerate any missing structure, even the head, in a few days. In planarians, as described in embryonic models, the cWNT pathway specifies the anterior-posterior axis. During the first 12-24h after amputation both wnt1 and notum (a Wnt inhibitor) are expressed in any wound, but 48 hours later they become restricted to posterior or anterior facing wounds, forming the anterior and the posterior organizers, respectively. In this study we undertook a genomic approach to further understand the mechanism that triggers the early expression of wnt1 and the specification of the posterior identity. Through ATAC-sequencing and CHIPmentation techniques we uncovered Cis-Regulatory Elements of Schmidtea mediterranea genome and analyzed them in notum and wnt1 (RNAi) animals. The result shows that already at 12 hours after amputation the chromatin structure of the wounds has changed its conformation according to the polarity of the pre-existing tissue. Analysing the DNA binding motives present in the proximal regulatory regions of genes down-regulated after wnt1 (RNAi) we found a few genes containing a TCF binding site, which include posterior Homeobox genes and chromatin remodelling proteins, suggesting that those are direct targets of the cWNT pathway and the responsible to trigger the expression of the posterior effectors. Furthermore, we have identified FoxG as an up-stream regulator of wnt1 transcription, probably though binding to an enhancer found in its first intron. Silencing of foxG inhibits the early phase of wnt1 expression and phenocopies the wnt1 (RNAi) phenotype, indicating its early role in specifying posterior versus anterior identity. Moreover, we have created a new open platform to interpret all transcriptomic and genomic results obtained (https://compgen.bio.ub.edu/PlanNET/planexp)

PlanMine 3.0-improvements to a mineable resource of flatworm biology and biodiversity.

Flatworms (Platyhelminthes) are a basally branching phylum that harbours a wealth of fascinating biology, including planarians with their astonishing regenerative abilities and the parasitic tape worms and blood flukes that exert a massive impact on human health. PlanMine (http://planmine.mpi-cbg.de/) has the mission objective of providing both a mineable sequence repository for planarians and also a resource for the comparative analysis of flatworm biology. While the original PlanMine release was entirely based on transcriptomes, the current release transitions to a more genomic perspective. Building on the recent availability of a high quality genome assembly of the planarian model species Schmidtea mediterranea, we provide a gene prediction set that now assign existing transcripts to defined genomic coordinates. The addition of recent single cell and bulk RNA-seq datasets greatly expands the available gene expression information. Further, we add transcriptomes from a broad range of other flatworms and provide a phylogeny-aware interface that makes evolutionary species comparisons accessible to non-experts. At its core, PlanMine continues to utilize the powerful InterMine framework and consistent data annotations to enable meaningful inter-species comparisons. Overall, PlanMine 3.0 thus provides a host of new features that makes the fascinating biology of flatworms accessible to the wider research community