Eva-Maria S. Collins

Planarian brain regeneration as a model system for developmental neurotoxicology

Abstract 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 planarians particular amenability for neurotoxicology and neuroregeneration studies, owing to the planarians 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.

Dynamics of Mouth Opening in Hydra

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.

On-chip immobilization of planarians for in vivo imaging

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.

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

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.

Mechanics dictate where and how freshwater planarians fission

Significance How planarians reproduce by ripping themselves into a head and a tail piece, which subsequently regenerate into two new worms, is a centuries-old biomechanics problem. Michael Faraday contemplated how this feat can be achieved in the 1800s, but it remained unanswered because it is experimentally difficult to observe planarians “doing it.” We recorded Dugesia japonica planarians in the act and developed a physical model that captures pivotal steps of their reproduction dynamics. The model reproduces experimental time scales and rupture stresses without fit parameters. The key to rupture is a local reduction of the animal’s cross-sectional area, which greatly amplifies the stresses exerted by the planarian’s musculature and enables rupture at substrate stresses in the pascal range. 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 Dugesia japonica 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.

Physical Mechanisms Driving Cell Sorting in Hydra

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 Hydras 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 aggregates 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.

Forces driving cell sorting in Hydra

Cell sorting, whereby a heterogeneous cell mixture organizes into distinct tissues, is a fundamental patterning process in development. So far, most studies of cell sorting have relied either on 2-dimensional cellular aggregates, in vitro situations that do not have a direct counterpart in vivo, or were focused on the properties of single cells. Here, we report the first multiscale experimental study on 3-dimensional regenerating Hydra aggregates, capable of reforming a full animal. 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. Moreover, we measure the physical properties of separated tissues and determine their viscosities and surface tensions. Based on our experimental results and numerical simulations, we conclude that tissue interfacial tensions are sufficient to explain Hydra cell sorting. Doing so, we illustrate D’Arcy Thompson’s central idea that biological organization can be understood through physical principles, an idea which is currently re-shaping the field of developmental biology. Summary statement Hydra regenerates after dissociation into single cells. We show how physical mechanisms can explain the first step of regeneration, whereby ectodermal and endodermal cells sort out to form distinct tissue layers.

Why we need mechanics to understand animal regeneration

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.

Multi-Behavioral Endpoint Testing Of An 87-Chemical Compound Library In Freshwater Planarians

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.