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.
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. However, recent work showed that regenerating tissue pieces inherit the parent animal's body axis and thus are asymmetric from the beginning. Thus, there is no mechanistic explanation for the observed shift in oscillation pattern and no clear understanding of its significance for Hydra regeneration. 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, thereby linking morphological readouts to physiologically relevant events during regeneration. This study shows the power of using modern experimental techniques to revisit old questions in pattern formation and development.
Answers to mechanistic questions about biological phenomena require fluency in a variety of molecular biology techniques and physical concepts. Here, we present an interdisciplinary approach to introducing undergraduate students to an important problem in the areas of animal behavior and neuroscience—the neuronal control of animal behavior. In this lab module, students explore planarian behavior by quantitative image and data analysis with freely available software and low-cost resources. Planarians are ∼1–2-cm-long aquatic free-living flatworms famous for their regeneration abilities. They are inexpensive and easy to maintain, handle, and perturb, and their fairly large size allows for image acquisition with a webcam, which makes this lab module accessible and scalable. Our lab module integrates basic physical concepts such as center of mass, velocity and speed, periodic signals, and time series analysis in the context of a biological system. The module is designed to attract students with diverse disciplinary backgrounds. It challenges the students to form hypotheses about behavior and equips them with a basic but broadly applicable toolkit to achieve this quantitatively. We give a detailed description of the necessary resources and show how to implement the module. We also provide suggestions for advanced exercises and possible extensions. Finally, we provide student feedback from a pilot implementation.
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.
15The ability to make transgenic Hydra lines has opened the door for quantitative in vivo studies of 16 Hydra regeneration and physiology. These studies commonly include excision, grafting and 17 transplantation experiments along with high-resolution imaging of live animals, which can be 18 challenging due to the animal's response to touch and light stimuli. While various anesthetics 19 have been used in Hydra studies over the years, they tend to be toxic over the course of a few 20 hours or their long-term effects on animal health have not been studied. Here we show that the 21 monoterpenoid linalool is a useful anesthetic for Hydra. Linalool is easy to use, non-toxic, fast 22 acting, and reversible. It has no detectable long-term effects on cell viability or cell proliferation. 23 We demonstrate that the same animal can be immobilized in linalool multiple times at intervals 24 of several hours for repeated imaging over 2-3 days. This uniquely allows for in vivo imaging of 25 dynamic processes such as head regeneration. We further directly compare linalool to currently 26 used anesthetics and show its superior performance. Because linalool, which is frequently 27 utilized in perfumes and cosmetic products, is also non-hazardous to humans, it will be a useful 28 tool for Hydra research in both research and teaching contexts. 29 30 31 112
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. However, recent work showed that regenerating tissue pieces inherit the parent animal's body axis and thus are asymmetric from the beginning. Thus, there is no mechanistic explanation for the observed shift in oscillation pattern and no clear understanding of its significance for Hydra regeneration. 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, thereby linking morphological readouts to physiologically relevant events during regeneration. This study shows the power of using modern experimental techniques to revisit old questions in pattern formation and development. Hydra strains and cultureHydra vulgaris strain AEP, Hydra vulgaris (formerly Hydra magnipapillata) strain sf-1 (temperature sensitive interstitial stem cells), Hydra vulgaris strain A10 (chimera consisting of Hydra vulgaris (formerly Hydra magnipapillata strain 105) epithelial cells and sf-1 interstitial cells) (20) and Hydra vulgaris "watermelon" (AEP expressing GFP in the ectoderm and DsRed2 in the endoderm) (7) were used for experiments. Polyps were kept in Hydra medium (HM) composed of 1 mM CaCl 2 (Spectrum Chemical, New Brunswick, NJ), 0.1 mM MgCl 2 (Sigma-Aldrich, St. Louis, MO), 0.03 mM KNO 3 (Fisher Scientific, Waltham, MA), 0.5 mM NaHCO 3 (Fisher Scientific), and 0.08 mM MgSO 4 (Fisher Scientific) prepared with MilliQ water, with pH between 7 and 7.3, at 18°C in a Panasonic incubator (Panasonic MIR-554) in the dark. The Hydra were fed three times per week with Artemia nauplii (Brine Shrimp Direct, Ogden, UT). Animals were cleaned daily using published procedures (21). Generation of nerve-free HydraNerve-free Hydra were generated using either of two methods. Watermelon animals were made nerve-free as described by Tran et al. (22). Briefly, the animals were incubated in 0.4% colchicine (Acros Organics, Thermo-Fisher Scientific, Waltham, MA) in HM for 8 h in the dark. This 8 h incubation was then repeated 3 weeks following the first treatment. Colchicine-treated Hydra are susceptible to bacterial infection, so the animals were kept in HM supplemented with 50 μg/mL rifampicin (EMD Millipore, Burlington, MA) at 18°C in t...
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