Wendy S. Beane

Induction of Vertebrate Regeneration by a Transient Sodium Current

Amphibians such as frogs can restore lost organs during development, including the lens and tail. To design biomedical therapies for organ repair, it is necessary to develop a detailed understanding of natural regeneration. Recently, ion transport has been implicated as a functional regulator of regeneration. Whereas voltage-gated sodium channels play a well known and important role in propagating action potentials in excitable cells, we have identified a novel role in regeneration for the ion transport function mediated by the voltage-gated sodium channel, NaV1.2. A local, early increase in intracellular sodium is required for initiating regeneration following Xenopus laevis tail amputation, and molecular and pharmacological inhibition of sodium transport causes regenerative failure. NaV1.2 is absent under nonregenerative conditions, but misexpression of human NaV1.5 can rescue regeneration during these states. Remarkably, pharmacological induction of a transient sodium current is capable of restoring regeneration even after the formation of a nonregenerative wound epithelium, confirming that it is the regulation of sodium transport that is critical for regeneration. Our studies reveal a previously undetected competency window in which cells retain their intrinsic regenerative program, identify a novel endogenous role for NaV in regeneration, and show that modulation of sodium transport represents an exciting new approach to organ repair.

Bioelectric signaling regulates head and organ size during planarian regeneration.

A main goal of regenerative medicine is to replace lost or damaged tissues and organs with functional parts of the correct size and shape. But the proliferation of new cells is not sufficient; we will also need to understand how the scale and ultimate form of newly produced tissues are determined. Using the planarian model system, we report that membrane voltage-dependent bioelectric signaling determines both head size and organ scaling during regeneration. RNA interference of the H+,K+-ATPase ion pump results in membrane hyperpolarization, which has no effect on the amount of new tissue (blastema) that is regenerated yet produces regenerates with tiny ‘shrunken’ heads and proportionally oversized pharynges. Our data show that this disproportionality results from a lack of the apoptosis required to adjust head and organ size and placement, highlighting apoptotic remodeling as the link between bioelectric signaling and the establishment of organ size during regeneration.

Regeneration: The Origin of Cancer or a Possible Cure?

A better understanding of the forces controlling cell growth will be essential for developing effective therapies in regenerative medicine and cancer. Historically, the literature has linked cancer and tissue regeneration-proposing regeneration as both the source of cancer and a method to inhibit tumorigenesis. This review discusses two powerful regeneration models, the vertebrate urodele amphibians and invertebrate planarians, in light of cancer regulation. Urodele limb and eye lens regeneration is described, as well as the planarians emergence as a molecular and genetic model system in which recent insights begin to molecularly dissect cancer and regeneration in adult tissues.

Modeling planarian regeneration: a primer for reverse-engineering the worm.

A mechanistic understanding of robust self-assembly and repair capabilities of complex systems would have enormous implications for basic evolutionary developmental biology as well as for transformative applications in regenerative biomedicine and the engineering of highly fault-tolerant cybernetic systems. Molecular biologists are working to identify the pathways underlying the remarkable regenerative abilities of model species that perfectly regenerate limbs, brains, and other complex body parts. However, a profound disconnect remains between the deluge of high-resolution genetic and protein data on pathways required for regeneration, and the desired spatial, algorithmic models that show how self-monitoring and growth control arise from the synthesis of cellular activities. This barrier to progress in the understanding of morphogenetic controls may be breached by powerful techniques from the computational sciences—using non-traditional modeling approaches to reverse-engineer systems such as planaria: flatworms with a complex bodyplan and nervous system that are able to regenerate any body part after traumatic injury. Currently, the involvement of experts from outside of molecular genetics is hampered by the specialist literature of molecular developmental biology: impactful collaborations across such different fields require that review literature be available that presents the key functional capabilities of important biological model systems while abstracting away from the often irrelevant and confusing details of specific genes and proteins. To facilitate modeling efforts by computer scientists, physicists, engineers, and mathematicians, we present a different kind of review of planarian regeneration. Focusing on the main patterning properties of this system, we review what is known about the signal exchanges that occur during regenerative repair in planaria and the cellular mechanisms that are thought to underlie them. By establishing an engineering-like style for reviews of the molecular developmental biology of biomedically important model systems, significant fresh insights and quantitative computational models will be developed by new collaborations between biology and the information sciences.

Inhibition of planar cell polarity extends neural growth during regeneration, homeostasis, and development

The ability to stop producing or replacing cells at the appropriate time is essential, as uncontrolled growth can lead to loss of function and even cancer. Tightly regulated mechanisms coordinate the growth of stem cell progeny with the patterning needs of the host organism. Despite the importance of proper termination during regeneration, cell turnover, and embryonic development, very little is known about how tissues determine when patterning is complete during these processes. Using planarian flatworms, we show that the planar cell polarity (PCP) pathway is required to stop the growth of neural tissue. Although traditionally studied as regulators of tissue polarity, we found that loss of the PCP genes Vangl2, DAAM1, and ROCK by RNA interference (individually or together) resulted in supernumerary eyes and excess optical neurons in intact planarians, while regenerating planarians had continued hyperplasia throughout the nervous system long after controls ceased new growth. This failure to terminate growth suggests that neural tissues use PCP as a readout of patterning, highlighting a potential role for intact PCP as a signal to stem and progenitor cells to halt neuronal growth when patterning is finished. Moreover, we found this mechanism to be conserved in vertebrates. Loss of Vangl2 during normal development, as well as during Xenopus tadpole tail regeneration, also leads to the production of excess neural tissue. This evolutionarily conserved function of PCP represents a tractable new approach for controlling the growth of nerves.

A low percent ethanol method for immobilizing planarians.

Planarians have recently become a popular model system for the study of adult stem cells, regeneration and polarity. The system is attractive for both undergraduate and graduate research labs, since planarian colonies are low cost and easy to maintain. Also in situ hybridization, immunofluorescence and RNA-interference (RNAi) gene knockdown techniques have been developed for planarian studies. However, imaging of live worms (particularly at high magnifications) is difficult because animals are strongly photophobic; they quickly move away from light sources and out of frame. The current methods available to inhibit movement in planarians include RNAi injection and exposure to cold temperatures. The former is labor and time intensive, while the latter precludes the use of many fluorescent reporter dyes. Here, we report a simple, inexpensive and reversible method to immobilize planarians for live imaging. Our data show that a short 1 hour treatment with 3% ethanol (EtOH) is sufficient to inhibit both the fine and gross movements of Schmidtea mediterranea planarians, of the typical size used (4–6 mm), with full recovery of movement within 3–4 hours. Importantly, EtOH treatment did not interfere with regeneration, even after repeated exposure, nor lyse epithelial cells (as assayed by H&E staining). We demonstrate that a short exposure to a low concentration of EtOH is a quick and effective method of immobilizing planarians, one that is easily adaptable to planarians of all sizes and will increase the accessibility of live imaging assays to planarian researchers.

Planarian phototactic assay reveals differential behavioral responses based on wavelength

Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli), planarians are an emerging model for regenerative eye research. However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths (red, blue, green), as well as ultraviolet (UV) and infrared (IR) which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths. These responses occur in a hierarchy, with the shortest wavelengths (UV) causing the most intense photophobic responses while longer wavelengths produce no effect (red) or an apparent attraction (IR). In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response (that drives planarians to escape the light source regardless of wavelength) and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths. Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment.

The planarian TRPA1 homolog mediates extraocular behavioral responses to near-ultraviolet light

ABSTRACT Although light is most commonly thought of as a visual cue, many animals possess mechanisms to detect light outside of the eye for various functions, including predator avoidance, circadian rhythms, phototaxis and migration. Here we confirm that planarians (like Caenorhabditis elegans, leeches and Drosophila larvae) are capable of detecting and responding to light using extraocular photoreception. We found that, when either eyeless or decapitated worms were exposed to near-ultraviolet (near-UV) light, intense wild-type photophobic behaviors were still observed. Our data also revealed that behavioral responses to green wavelengths were mediated by ocular mechanisms, whereas near-UV responses were driven by extraocular mechanisms. As part of a candidate screen to uncover the genetic basis of extraocular photoreception in the planarian species Schmidtea mediterranea, we identified a potential role for a homolog of the transient receptor potential channel A1 (TRPA1) in mediating behavioral responses to extraocular light cues. RNA interference (RNAi) to Smed-TrpA resulted in worms that lacked extraocular photophobic responses to near-UV light, a mechanism previously only identified in Drosophila. These data show that the planarian TRPA1 homolog is required for planarian extraocular-light avoidance and may represent a potential ancestral function of this gene. TRPA1 is an evolutionarily conserved detector of temperature and chemical irritants, including reactive oxygen species that are byproducts of UV-light exposure. Our results suggest that planarians possess extraocular photoreception and display an unconventional TRPA1-mediated photophobic response to near-UV light. Summary: Planarians use a non-traditional TRPA1-mediated mechanism for extraocular photoresponses to near-UV light, a response previously identified only in Drosophila.

H+/K+ ATPase activity is required for biomineralization in sea urchin embryos

The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H(+)/K(+) ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles.

Temporal regulation of planarian eye regeneration

Abstract While tissue regeneration is typically studied using standard injury models, in nature injuries vary greatly in the amount and location of tissues lost. Planarians have the unique ability to regenerate from many different injuries (including from tiny fragments with no brain), allowing us to study the effects of different injuries on regeneration timelines. We followed the timing of regeneration for one organ, the eye, after multiple injury types that involved tissue loss (single‐ and double‐eye ablation, and decapitation) in Schmidtea mediterranea. Our data reveal that the timing of regeneration remained constant despite changing injury parameters. Optic tissue regrowth, nerve re‐innervation, and functional recovery were similar between injury types (even when the animal was simultaneously regrowing its brain). Changes in metabolic rate (i.e., starving vs. fed regenerates) also had no effect on regeneration timelines. In addition, our data suggest there may exist a role for optic nerve degeneration following eye ablation. Our results suggest that the temporal regulation of planarian eye regeneration is tightly controlled and resistant to variations in injury type.