Publications by authors named "Eva-Maria S Collins"

28 Publications

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Let it rip: The mechanics of self-bisection in asexual planarians determines their population reproductive strategies.

Phys Biol 2021 Oct 12. Epub 2021 Oct 12.

Biology Department, Swarthmore College, 500 College Ave, Swarthmore, Pennsylvania, 19081-1306, UNITED STATES.

Asexual freshwater planarians reproduce by transverse bisection (binary fission) into two pieces. This process produces a head and a tail, which fully regenerate within 1-2 weeks. How planarians split into two offspring - using only their musculature and substrate traction - is a challenging biomechanics problem. We found that three different species, Dugesia japonica, Girardia tigrina and Schmidtea mediterranea, have evolved three different mechanical solutions to self-bisect. Using time lapse imaging of the fission process, we quantitatively characterize the main steps of division in the three species and extract the distinct and shared key features. Across the three species, planarians actively alter their body shape, regulate substrate traction, and use their muscles to generate tensile stresses large enough to overcome the ultimate tensile strength of the tissue. Moreover, we show that how each planarian species divides dictates how resources are split among its offspring. This ultimately determines offspring survival and reproductive success. Thus, heterospecific differences in the mechanics of self-bisection of individual worms explain the observed differences in the population reproductive strategies of different planarian species.
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http://dx.doi.org/10.1088/1478-3975/ac2f29DOI Listing
October 2021

Planarian fragments behave as whole animals.

Curr Biol 2021 Oct 4. Epub 2021 Oct 4.

Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Electronic address:

Behavioral responses of freshwater planarians have been studied for over a century. In recent decades, behavior has been used as a readout to study planarian development and regeneration, wound healing, molecular evolution, neurotoxicology, and learning and memory.The planarian nervous system is among the simplest of the bilaterally symmetric animals, with an anterior brain attached to two ventral nerve cords interconnected by multiple commissures. We found that, in response to mechanical and near-UV stimulation, head stimulation produces turning, tail stimulation produces contraction, and trunk stimulation produces midbody elongation in the planarian Dugesia japonica. When cut into two or three pieces, the anterior end of each headless piece switched its behavior to turning instead of elongation; i.e., it responded as though it were the head. In addition, posterior ends of the head and midbody pieces sometimes produced contraction instead of elongation. Thus, each severed piece acts like an intact animal, with each midbody region having nearly complete behavioral capabilities. These observations show that each midbody region reads the global state of the organism and adapts its response to incoming signals from the remaining tissue. Selective lateral incisions showed that the changes in behavior are not due to nonselective pain responses and that the ventral nerve cords and cross-connectives are responsible for coordinating local behaviors. Our findings highlight a fast functional reorganization of the planarian nervous system that complements the slower repairs provided by regeneration. This reorganization provides needed behavioral responses for survival as regeneration proceeds.
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http://dx.doi.org/10.1016/j.cub.2021.09.056DOI Listing
October 2021

Wnt signaling determines body axis polarity in regenerating Hydra tissue fragments.

Dev Biol 2020 11 30;467(1-2):88-94. Epub 2020 Aug 30.

Department of Biology, Swarthmore College, Swarthmore, PA, 19081, USA; Department of Physics, University of California San Diego, La Jolla, CA, 92093, USA. Electronic address:

How an animal establishes its body axis is a fundamental question in developmental biology. The freshwater cnidarian Hydra is an attractive model for studying axis formation because it is radially symmetric, with a single oral-aboral axis. It was recently proposed that the orientation of the new body axis in a regenerating Hydra polyp is determined by the oral-aboral orientation of the actin-myosin contractile processes (myonemes) in the animal's outer epithelial layer. However, it remained unclear how the oral-aboral polarity of the body axis would be defined. As Wnt signaling is known to control axis polarity in Hydra and bilaterians, we hypothesized that it plays a role in axis formation during regeneration of Hydra tissue pieces. We tested this hypothesis using pharmacological perturbations and novel grafting experiments to set Wnt signaling and myoneme orientation perpendicular to each other to determine which controls axis formation. Our results demonstrate that Wnt signaling is the dominant encoder of axis orientation and polarity, in line with its conserved role in axial patterning.
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http://dx.doi.org/10.1016/j.ydbio.2020.08.012DOI Listing
November 2020

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing.

J Vis Exp 2020 07 30(161). Epub 2020 Jul 30.

Biology Department, Swarthmore College; Physics Department, UC San Diego;

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.
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http://dx.doi.org/10.3791/61549DOI Listing
July 2020

Dugesia japonica is the best suited of three planarian species for high-throughput toxicology screening.

Chemosphere 2020 Aug 8;253:126718. Epub 2020 Apr 8.

Department of Biology, Swarthmore College, Swarthmore, PA, USA; Department of Physics, University of California San Diego, La Jolla, CA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chemosphere.2020.126718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350771PMC
August 2020

A novel bilateral grafting technique for studying patterning in Hydra.

Dev Biol 2020 06 9;462(1):60-65. Epub 2020 Mar 9.

Department of Bioengineering, University of California San Diego, La Jolla, CA, 92093, USA; Department of Biology, Swarthmore College, Swarthmore, PA, 19081, USA; Department of Physics, University of California San Diego, La Jolla, CA, 92093, USA. Electronic address:

Control of patterning and the specification of body axes are fundamental aspects of animal development involving complex interactions between chemical, physical, and genetic signals. The freshwater polyp Hydra has long been recognized as a useful model system to address these questions due to its simple anatomy, optical transparency, and strong regenerative abilities, which enabled clever grafting experiments to alter and probe patterning. Reliable methods exist for the transplantation of small tissue pieces into the body column or the combination of sections cut perpendicular to the body axis, which can be used to examine oral-aboral gradients and axis induction potential of tissue fragments. However, existing methods do not allow researchers to probe questions of axis alignment and lateral information exchange. We therefore developed a technique to produce chimeric animals split longitudinally along the body axis of the animal by anesthetizing the animals with the terpene linalool and threading the donor pieces onto pairs of fine glass needles. Our novel approach can be applied to study questions in Hydra research that have thus far been inaccessible, including patterning processes acting perpendicular to the oral-aboral axis and the extent of lateral cell migration.
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http://dx.doi.org/10.1016/j.ydbio.2020.03.006DOI Listing
June 2020

Pharmacological or genetic targeting of Transient Receptor Potential (TRP) channels can disrupt the planarian escape response.

PLoS One 2019 5;14(12):e0226104. Epub 2019 Dec 5.

Department of Biology, Swarthmore College, Swarthmore, Pennsylvania, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226104PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894859PMC
March 2020

Linalool acts as a fast and reversible anesthetic in Hydra.

PLoS One 2019 24;14(10):e0224221. Epub 2019 Oct 24.

Department of Physics, University of California San Diego, La Jolla, CA, United States of America.

The ability to make transgenic Hydra lines has allowed for quantitative in vivo studies of Hydra regeneration and physiology. These studies commonly include excision, grafting and transplantation experiments along with high-resolution imaging of live animals, which can be challenging due to the animal's response to touch and light stimuli. While various anesthetics have been used in Hydra studies, they tend to be toxic over the course of a few hours or their long-term effects on animal health are unknown. Here, we show that the monoterpenoid alcohol linalool is a useful anesthetic for Hydra. Linalool is easy to use, non-toxic, fast acting, and reversible. It has no detectable long-term effects on cell viability or cell proliferation. We demonstrate that the same animal can be immobilized in linalool multiple times at intervals of several hours for repeated imaging over 2-3 days. This uniquely allows for in vivo imaging of dynamic processes such as head regeneration. We directly compare linalool to currently used anesthetics and show its superior performance. Linalool will be a useful tool for tissue manipulation and imaging in Hydra research in both research and teaching contexts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224221PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812832PMC
March 2020

Mouth Function Determines the Shape Oscillation Pattern in Regenerating Hydra Tissue Spheres.

Biophys J 2019 09 6;117(6):1145-1155. Epub 2019 Aug 6.

Department of Physics, University of California San Diego, La Jolla, California; Biology Department, Swarthmore College, Swarthmore, Pennsylvania. Electronic address:

Hydra is a small freshwater polyp capable of regeneration from small tissue pieces and from aggregates of cells. During regeneration, a hollow bilayered sphere is formed that undergoes osmotically driven shape oscillations of inflation and rupture. These oscillations are necessary for successful regeneration. Eventually, the oscillating sphere breaks rotational symmetry along the future head-foot axis of the animal. Notably, the shape oscillations show an abrupt shift from large-amplitude, long-period oscillations to small-amplitude, short-period oscillations. It has been widely accepted that this shift in oscillation pattern is linked to symmetry breaking and axis formation, and current theoretical models of Hydra symmetry breaking use this assumption as a model constraint. However, a mechanistic explanation for the shift in oscillation pattern is lacking. Using in vivo manipulation and imaging, we quantified the shape oscillation dynamics and dissected the timing and triggers of the pattern shift. Our experiments demonstrate that the shift in the shape oscillation pattern in regenerating Hydra tissue pieces is caused by the formation of a functional mouth and not by shape symmetry breaking as previously assumed. Thus, model assumptions must be revised in light of these new experimental data, which can be used to constrain and validate improved theoretical models of pattern formation in Hydra.
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http://dx.doi.org/10.1016/j.bpj.2019.07.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818176PMC
September 2019

Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Neurotoxicol Teratol 2019 May - Jun;73:54-66. Epub 2019 Mar 31.

Division of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA; Department of Physics, University of California San Diego, La Jolla, CA 92093, USA; Department of Biology, Swarthmore College Swarthmore, PA 19081, USA. Electronic address:

Asexual freshwater planarians are an attractive invertebrate model for high-throughput neurotoxicity screening, because they possess multiple quantifiable behaviors to assess distinct neuronal functions. Planarians uniquely allow direct comparisons between developing and adult animals to distinguish developmentally selective effects from general neurotoxicity. In this study, we used our automated planarian screening platform to compare the neurotoxicity of 15 flame retardants (FRs), consisting of representative phased-out brominated (BFRs) and replacement organophosphorus FRs (OPFRs). OPFRs have emerged as a proposed safer alternative to BFRs; however, limited information is available on their health effects. We found 11 of the 15 FRs (3/6 BFRs, 7/8 OPFRs, and Firemaster 550) caused adverse effects in both adult and developing planarians with similar nominal lowest-effect-levels for BFRs and OPFRs. This suggests that replacement OPFRs are comparably neurotoxic to the phased-out compounds. BFRs were primarily systemically toxic, whereas OPFRs, except Tris(2-chloroethyl) phosphate, shared a behavioral phenotype in response to noxious heat at sublethal concentrations, indicating specific neurotoxic effects. We found this behavioral phenotype was correlated with cholinesterase inhibition, thus linking behavioral outcomes to molecular targets. By directly comparing effects on adult and developing planarians, we further found that one BFR (3,3',5,5'-Tetrabromobisphenol A) caused a developmental selective defect. Together, these results demonstrate that our planarian screening platform yields high content data from various behavioral and morphological endpoints, allowing us to distinguish selective neurotoxic effects and effects specific to the developing nervous system. Ten of these 11 bioactive FRs were previously found to be bioactive in other models, including cell culture and alternative animal models (nematodes and zebrafish). This level of concordance across different platforms emphasizes the urgent need for further evaluation of OPFRs in mammalian systems.
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http://dx.doi.org/10.1016/j.ntt.2019.03.003DOI Listing
May 2020

SoxB1 Activity Regulates Sensory Neuron Regeneration, Maintenance, and Function in Planarians.

Dev Cell 2018 11;47(3):331-347.e5

Department of Biology, San Diego State University, San Diego, CA, USA. Electronic address:

SoxB1 genes play fundamental roles in neurodevelopmental processes and maintaining stem cell multipotency, but little is known about their function in regeneration. We addressed this question by analyzing the activity of the SoxB1 homolog soxB1-2 in the planarian Schmidtea mediterranea. Expression and functional analysis revealed that soxB1-2 marks ectodermal-lineage progenitors, and its activity is required for differentiation of subsets of ciliated epidermal and neuronal cells. Moreover, we show that inhibiting soxB1-2 or its candidate target genes leads to abnormal sensory neuron regeneration that causes planarians to display seizure-like movements or phenotypes associated with the loss of sensory modalities. Our analyses highlight soxB1-2-regulated genes that are expressed in sensory neurons and are homologous to factors implicated in epileptic disorders in humans and animal models of epilepsy, indicating that planarians can serve as a complementary model to investigate genetic causes of epilepsy.
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http://dx.doi.org/10.1016/j.devcel.2018.10.014DOI Listing
November 2018

Comparative Analysis of Zebrafish and Planarian Model Systems for Developmental Neurotoxicity Screens Using an 87-Compound Library.

Toxicol Sci 2019 01;167(1):15-25

Division of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093.

There is a clear need to establish and validate new methodologies to more quickly and efficiently screen chemicals for potential toxic effects, particularly on development. The emergence of alternative animal systems for rapid toxicology screens presents valuable opportunities to evaluate how systems complement each other. In this article, we compare a chemical library of 87-compounds in 2 such systems, developing zebrafish and freshwater planarians, by screening for developmental neurotoxic effects. We show that the systems' toxicological profiles are complementary to each other, with zebrafish yielding more detailed morphological endpoints and planarians more behavioral endpoints. Overall, zebrafish was more sensitive to this chemical library, yielding 86/87 hits, compared with 50/87 hits in planarians. The difference in sensitivity could not be attributed to molecular weight, log Kow, or the bioconcentration factor. Of the 87 chemicals, 28 had previously been evaluated in mammalian developmental neuro- (DNT), neuro-, or developmental toxicity studies. Of the 28, 20 were hits in the planarian, and 27 were hits in zebrafish. Eighteen of the 28 had previously been identified as DNT hits in mammals and were highly associated with activity in zebrafish and planarian behavioral assays in this study. Only 1 chemical (of 28) was a false negative in both zebrafish and planarian systems. The differences in endpoint coverage and system sensitivity illustrate the value of a dual systems approach to rapidly query a large chemical-bioactivity space and provide weight-of-evidence for prioritization of chemicals for further testing.
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http://dx.doi.org/10.1093/toxsci/kfy180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317421PMC
January 2019

Multi-Behavioral Endpoint Testing of an 87-Chemical Compound Library in Freshwater Planarians.

Toxicol Sci 2019 01;167(1):26-44

Division of Cell and Developmental Biology.

There is an increased recognition in the field of toxicology of the value of medium-to-high-throughput screening methods using in vitro and alternative animal models. We have previously introduced the asexual freshwater planarian Dugesia japonica as a new alternative animal model and proposed that it is particularly well-suited for the study of developmental neurotoxicology. In this article, we discuss how we have expanded and automated our screening methodology to allow for fast screening of multiple behavioral endpoints, developmental toxicity, and mortality. Using an 87-compound library provided by the National Toxicology Program, consisting of known and suspected neurotoxicants, including drugs, flame retardants, industrial chemicals, polycyclic aromatic hydrocarbons (PAHs), pesticides, and presumptive negative controls, we further evaluate the benefits and limitations of the system for medium-throughput screening, focusing on the technical aspects of the system. We show that, in the context of this library, planarians are the most sensitive to pesticides with 16/16 compounds causing toxicity and the least sensitive to PAHs, with only 5/17 causing toxicity. Furthermore, while none of the presumptive negative controls were bioactive in adult planarians, 2/5, acetaminophen and acetylsalicylic acid, were bioactive in regenerating worms. Notably, these compounds were previously reported as developmentally toxic in mammalian studies. Through parallel screening of adults and developing animals, planarians are thus a useful model to detect such developmental-specific effects, which was observed for 13 chemicals in this library. We use the data and experience gained from this screen to propose guidelines for best practices when using planarians for toxicology screens.
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http://dx.doi.org/10.1093/toxsci/kfy145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657585PMC
January 2019

Physical Mechanisms Driving Cell Sorting in Hydra.

Biophys J 2017 Dec;113(12):2827-2841

Department of Physics, University of California, San Diego, La Jolla, California; Division of Biological Sciences, University of California, San Diego, La Jolla, California. Electronic address:

Cell sorting, whereby a heterogeneous cell mixture organizes into distinct tissues, is a fundamental patterning process in development. Hydra is a powerful model system for carrying out studies of cell sorting in three dimensions, because of its unique ability to regenerate after complete dissociation into individual cells. The physicists Alfred Gierer and Hans Meinhardt recognized Hydra's self-organizing properties more than 40 years ago. However, what drives cell sorting during regeneration of Hydra from cell aggregates is still debated. Differential motility and differential adhesion have been proposed as driving mechanisms, but the available experimental data are insufficient to distinguish between these two. Here, we answer this longstanding question by using transgenic Hydra expressing fluorescent proteins and a multiscale experimental and numerical approach. By quantifying the kinematics of single cell and whole aggregate behaviors, we show that no differences in cell motility exist among cell types and that sorting dynamics follow a power law with an exponent of ∼0.5. Additionally, we measure the physical properties of separated tissues and quantify their viscosities and surface tensions. Based on our experimental results and numerical simulations, we conclude that tissue interfacial tensions are sufficient to explain cell sorting in aggregates of Hydra cells. Furthermore, we demonstrate that the aggregate's geometry during sorting is key to understanding the sorting dynamics and explains the exponent of the power law behavior. Our results answer the long standing question of the physical mechanisms driving cell sorting in Hydra cell aggregates. In addition, they demonstrate how powerful this organism is for biophysical studies of self-organization and pattern formation.
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http://dx.doi.org/10.1016/j.bpj.2017.10.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771031PMC
December 2017

Why we need mechanics to understand animal regeneration.

Dev Biol 2018 01 24;433(2):155-165. Epub 2017 Nov 24.

Physics Department, UC San Diego, La Jolla, CA 92093, USA; Section of Cell&Developmental Biology, UC San Diego, La Jolla, CA 92093, USA. Electronic address:

Mechanical forces are an important contributor to cell fate specification and cell migration during embryonic development in animals. Similarities between embryogenesis and regeneration, particularly with regards to pattern formation and large-scale tissue movements, suggest similarly important roles for physical forces during regeneration. While the influence of the mechanical environment on stem cell differentiation in vitro is being actively exploited in the fields of tissue engineering and regenerative medicine, comparatively little is known about the role of stresses and strains acting during animal regeneration. In this review, we summarize published work on the role of physical principles and mechanical forces in animal regeneration. Novel experimental techniques aimed at addressing the role of mechanics in embryogenesis have greatly enhanced our understanding at scales from the subcellular to the macroscopic - we believe the time is ripe for the field of regeneration to similarly leverage the tools of the mechanobiological research community.
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http://dx.doi.org/10.1016/j.ydbio.2017.09.021DOI Listing
January 2018

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.

Arch Toxicol 2018 Mar 22;92(3):1161-1176. Epub 2017 Nov 22.

Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.

The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 µM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.
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http://dx.doi.org/10.1007/s00204-017-2130-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413736PMC
March 2018

Mechanics dictate where and how freshwater planarians fission.

Proc Natl Acad Sci U S A 2017 10 25;114(41):10888-10893. Epub 2017 Sep 25.

Physics Department, University of California, San Diego, La Jolla, CA 92093;

Asexual freshwater planarians reproduce by tearing themselves into two pieces by a process called binary fission. The resulting head and tail pieces regenerate within about a week, forming two new worms. Understanding this process of ripping oneself into two parts poses a challenging biomechanical problem. Because planarians stop "doing it" at the slightest disturbance, this remained a centuries-old puzzle. We focus on fission and show that it proceeds in three stages: a local constriction ("waist formation"), pulsation-which increases waist longitudinal stresses-and transverse rupture. We developed a linear mechanical model with a planarian represented by a thin shell. The model fully captures the pulsation dynamics leading to rupture and reproduces empirical time scales and stresses. It asserts that fission execution is a mechanical process. Furthermore, we show that the location of waist formation, and thus fission, is determined by physical constraints. Together, our results demonstrate that where and how a planarian rips itself apart during asexual reproduction can be fully explained through biomechanics.
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http://dx.doi.org/10.1073/pnas.1700762114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642676PMC
October 2017

Generation and Long-term Maintenance of Nerve-free Hydra.

J Vis Exp 2017 07 7(125). Epub 2017 Jul 7.

Division of Biological Sciences, UC San Diego; Physics Department, UC San Diego;

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.
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http://dx.doi.org/10.3791/56115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609432PMC
July 2017

Coordination of size-control, reproduction and generational memory in freshwater planarians.

Phys Biol 2017 05 23;14(3):036003. Epub 2017 May 23.

Department of Physics and Astronomy, Northwestern University, Evanston, IL, United States of America. These authors contributed equally to this work.

Uncovering the mechanisms that control size, growth, and division rates of organisms reproducing through binary division means understanding basic principles of their life cycle. Recent work has focused on how division rates are regulated in bacteria and yeast, but this question has not yet been addressed in more complex, multicellular organisms. We have, over the course of several years, assembled a unique large-scale data set on the growth and asexual reproduction of two freshwater planarian species, Dugesia japonica and Girardia tigrina, which reproduce by transverse fission and succeeding regeneration of head and tail pieces into new planarians. We show that generation-dependent memory effects in planarian reproduction need to be taken into account to accurately capture the experimental data. To achieve this, we developed a new additive model that mixes multiple size control strategies based on planarian size, growth, and time between divisions. Our model quantifies the proportions of each strategy in the mixed dynamics, revealing the ability of the two planarian species to utilize different strategies in a coordinated manner for size control. Additionally, we found that head and tail offspring of both species employ different mechanisms to monitor and trigger their reproduction cycles. Thus, we find a diversity of strategies not only between species but between heads and tails within species. Our additive model provides two advantages over existing 2D models that fit a multivariable splitting rate function to the data for size control: firstly, it can be fit to relatively small data sets and can thus be applied to systems where available data is limited. Secondly, it enables new biological insights because it explicitly shows the contributions of different size control strategies for each offspring type.
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http://dx.doi.org/10.1088/1478-3975/aa70c4DOI Listing
May 2017

Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation.

Arch Toxicol 2017 Aug 18;91(8):2837-2847. Epub 2016 Dec 18.

Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093, USA.

The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.
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http://dx.doi.org/10.1007/s00204-016-1908-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485937PMC
August 2017

Slo1 regulates ethanol-induced scrunching in freshwater planarians.

Phys Biol 2016 09 9;13(5):055001. Epub 2016 Sep 9.

Department of Physics, University of California San Diego, La Jolla, CA, USA.

When freshwater planarians are exposed to a low-percentage (0.5%-1%) alcohol solution, they display a characteristic 'drunken' phenotype. Here we show that this drunken phenotype is a mixture of cilia-mediated gliding and scrunching, a muscular-based planarian gait which we recently demonstrated to be triggered by adverse environmental stimuli. At exogenous ethanol concentrations ≥2% (v/v), planarians become gradually immobilized and ultimately die. Using RNA interference (RNAi) for targeted gene knockdown, we elucidate the molecular basis for ethanol sensing and show that the big potassium ion channel SLO1 is necessary for ethanol sensitivity in planarians. Because slo1(RNAi) animals maintain their ability to scrunch in response to other adverse triggers, these results suggest that slo1 specifically regulates ethanol sensitivity and not the scrunching gait per se. Furthermore, this study demonstrates the ease of performing pharmacological studies in planarians. Combined with the worms' amenability to quantitative behavioral assays and targeted gene knockdown, planarians are a valuable model organism for studying the effect of neuroactive compounds on brain function and behavior.
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http://dx.doi.org/10.1088/1478-3975/13/5/055001DOI Listing
September 2016

Planarian brain regeneration as a model system for developmental neurotoxicology.

Regeneration (Oxf) 2016 04 15;3(2):65-77. Epub 2016 Mar 15.

Division of Biological Sciences University of California San Diego, La Jolla California 92093 USA; Department of Physics University of California San Diego, La Jolla California 92093 USA.

Freshwater planarians, famous for their regenerative prowess, have long been recognized as a valuable in vivo animal model to study the effects of chemical exposure. In this review, we summarize the current techniques and tools used in the literature to assess toxicity in the planarian system. We focus on the planarian's particular amenability for neurotoxicology and neuroregeneration studies, owing to the planarian's unique ability to regenerate a centralized nervous system. Zooming in from the organismal to the molecular level, we show that planarians offer a repertoire of morphological and behavioral readouts while also being amenable to mechanistic studies of compound toxicity. Finally, we discuss the open challenges and opportunities for planarian brain regeneration to become an important model system for modern toxicology.
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http://dx.doi.org/10.1002/reg2.52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895328PMC
April 2016

Dynamics of Mouth Opening in Hydra.

Biophys J 2016 Mar;110(5):1191-201

Division of Biological Sciences, University of California San Diego, La Jolla, California; Department of Physics, University of California San Diego, La Jolla, California. Electronic address:

Hydra, a simple freshwater animal famous for its regenerative capabilities, must tear a hole through its epithelial tissue each time it opens its mouth. The feeding response of Hydra has been well-characterized physiologically and is regarded as a classical model system for environmental chemical biology. However, due to a lack of in vivo labeling and imaging tools, the biomechanics of mouth opening have remained completely unexplored. We take advantage of the availability of transgenic Hydra lines to perform the first dynamical analysis, to our knowledge, of Hydra mouth opening and test existing hypotheses regarding the underlying cellular mechanisms. Through cell position and shape tracking, we show that mouth opening is accompanied by changes in cell shape, but not cellular rearrangements as previously suggested. Treatment with a muscle relaxant impairs mouth opening, supporting the hypothesis that mouth opening is an active process driven by radial contractile processes (myonemes) in the ectoderm. Furthermore, we find that all events exhibit the same relative rate of opening. Because one individual can open consecutively to different amounts, this suggests that the degree of mouth opening is controlled through neuronal signaling. Finally, from the opening dynamics and independent measurements of the elastic properties of the tissues, we estimate the forces exerted by the myonemes to be on the order of a few nanoNewtons. Our study provides the first dynamical framework, to our knowledge, for understanding the remarkable plasticity of the Hydra mouth and illustrates that Hydra is a powerful system for quantitative biomechanical studies of cell and tissue behaviors in vivo.
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http://dx.doi.org/10.1016/j.bpj.2016.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788721PMC
March 2016

Scrunching: a novel escape gait in planarians.

Phys Biol 2015 Sep 10;12(5):056010. Epub 2015 Sep 10.

Physics Department, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

The ability to escape a predator or other life-threatening situations is central to animal survival. Different species have evolved unique strategies under anatomical and environmental constraints. In this study, we describe a novel musculature-driven escape gait in planarians, 'scrunching', which is quantitatively different from other planarian gaits, such as gliding and peristalsis. We show that scrunching is a conserved gait among different flatworm species, underlying its importance as an escape mechanism. We further demonstrate that it can be induced by a variety of physical stimuli, including amputation, high temperature, electric shock and low pH. We discuss the functional basis for scrunching as the preferential gait when gliding is impaired due to a disruption of mucus production. Finally, we show that the key mechanical features of scrunching are adequately captured by a simple biomechanical model that is solely based on experimental data from traction force microscopy and tissue rheology without fit parameters. Together, our results form a complete description of this novel form of planarian locomotion. Because scrunching has distinct dynamics, this gait can serve as a robust behavioral readout for studies of motor neuron and muscular functions in planarians and in particular the restoration of these functions during regeneration.
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http://dx.doi.org/10.1088/1478-3975/12/5/056010DOI Listing
September 2015

Freshwater Planarians as an Alternative Animal Model for Neurotoxicology.

Toxicol Sci 2015 Sep 26;147(1):270-85. Epub 2015 Jun 26.

*Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093; Physics Department, University of California, San Diego, La Jolla, California 92093; and

Traditional toxicology testing has relied on low-throughput, expensive mammalian studies; however, timely testing of the large number of environmental toxicants requires new in vitro and in vivo platforms for inexpensive medium- to high-throughput screening. Herein, we describe the suitability of the asexual freshwater planarian Dugesia japonica as a new animal model for the study of developmental neurotoxicology. As these asexual animals reproduce by binary fission, followed by regeneration of missing body structures within approximately 1 week, development and regeneration occur through similar processes allowing us to induce neurodevelopment "at will" through amputation. This short time scale and the comparable sizes of full and regenerating animals enable parallel experiments in adults and developing worms to determine development-specific aspects of toxicity. Because the planarian brain, despite its simplicity, is structurally and molecularly similar to the mammalian brain, we are able to ascertain neurodevelopmental toxicity that is relevant to humans. As a proof of concept, we developed a 5-step semiautomatic screening platform to characterize the toxicity of 9 known neurotoxicants (consisting of common solvents, pesticides, and detergents) and a neutral agent, glucose, and quantified effects on viability, stimulated and unstimulated behavior, regeneration, and brain structure. Comparisons of our findings with other alternative toxicology animal models, such as zebrafish larvae and nematodes, demonstrated that planarians are comparably sensitive to the tested chemicals. In addition, we found that certain compounds induced adverse effects specifically in developing animals. We thus conclude that planarians offer new complementary opportunities for developmental neurotoxicology animal models.
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http://dx.doi.org/10.1093/toxsci/kfv129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838007PMC
September 2015

Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea.

PLoS One 2015 27;10(5):e0127074. Epub 2015 May 27.

Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127074PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446096PMC
April 2016

On-chip immobilization of planarians for in vivo imaging.

Sci Rep 2014 Sep 17;4:6388. Epub 2014 Sep 17.

1] Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544 USA [2] Department of Physics, University of California, San Diego, La Jolla, CA 92093 USA [3] Division of Biology, University of California, San Diego, La Jolla, CA 92093.

Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra.
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http://dx.doi.org/10.1038/srep06388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4165980PMC
September 2014

Smed-dynA-1 is a planarian nervous system specific dynamin 1 homolog required for normal locomotion.

Biol Open 2014 Jun 20;3(7):627-34. Epub 2014 Jun 20.

Lewis-Sigler Institute for Integrative Genomics, Carl C. Icahn Laboratory, Princeton University, Princeton, NJ 08544, USA Physics Department, University of California at San Diego, La Jolla, CA 92093, USA Division of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA 92093, USA

Dynamins are GTPases that are required for separation of vesicles from the plasma membrane and thus are key regulators of endocytosis in eukaryotic cells. This role for dynamin proteins is especially crucial for the proper function of neurons, where they ensure that synaptic vesicles and their neurotransmitter cargo are recycled in the presynaptic cell. Here we have characterized the dynamin protein family in the freshwater planarian Schmidtea mediterranea and showed that it possesses six dynamins with tissue specific expression profiles. Of these six planarian homologs, two are necessary for normal tissue homeostasis, and the loss of another, Smed-dynA-1, leads to an abnormal behavioral phenotype, which we have quantified using automated center of mass tracking. Smed-dynA-1 is primarily expressed in the planarian nervous system and is a functional homolog of the mammalian Dynamin I. The distinct expression profiles of the six dynamin genes makes planarians an interesting new system to reveal novel dynamin functions, which may be determined by their differential tissue localization. The observed complexity of neurotransmitter regulation combined with the tools of quantitative behavioral assays as a functional readout for neuronal activity, renders planarians an ideal system for studying how the nervous system controls behavior.
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http://dx.doi.org/10.1242/bio.20147583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154299PMC
June 2014
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