Methylisothiazolinone toxicity and inhibition of wound healing and regeneration in planaria.

Methylisothiazolinone (MIT) is a common biocide used in cosmetic and industrial settings. Studies have demonstrated that MIT is a human sensitizer, to the extent that in 2013 MIT was named allergen of the year. Recently, we showed that MIT exposure in Xenopus laevis (the African clawed frog) inhibits wound healing and tail regeneration. However, it is unknown whether MIT affects these processes in other animals. Here, we investigated the effects of MIT exposure in planaria-non-parasitic freshwater flatworms able to regenerate all tissues after injury. Using a common research strain of Dugesia japonica, we determined that intact planarians exposed to 15µM MIT displayed both neuromuscular and epithelial-integrity defects. Furthermore, regenerating (head and tail) fragments exposed to 15µM MIT failed to close wounds or had significantly delayed wound healing. Planarian wounds normally close within 1h after injury. However, most MIT-exposed animals retained open wounds at 24h and subsequently died, and those few animals that were able to undergo delayed wound healing without dying exhibited abnormal regeneration. For instance, head regeneration was severely delayed or inhibited, with anterior structures such as eyes failing to form in newly produced tissues. These data suggest that MIT directly affects both wound healing and regeneration in planarians. Next, we investigated the ability of thiol-containing antioxidants to rescue planarian wound closure during MIT exposure. The data reveal both n-acetyl cysteine and glutathione were each able to fully rescue MIT inhibition of wound healing. Lastly, we established MIT toxicity levels by determining the LC50 of 5 different planarian species: D. japonica, Schmidtea mediterranea, Girardia tigrina, Girardia dorotocephala, and Phagocata gracilis. Our LC50 data revealed that concentrations as low as 39µM (4.5ppm) are lethal to planarians, with concentrations of just 5µM inhibiting wound healing, and suggest that phylogeny is predictive of species toxicity levels. Together these results indicate MIT may have broad wound healing effects on aquatic species in general and are not limited to X. laevis alone. Future studies should investigate the impact of MIT on wound healing in other organisms, including non-aquatic organisms and mammals.

Seeing the future: using Xenopus to understand eye regeneration.

Studies of Xenopus eye development have contributed considerably to the understanding of vertebrate neurogenesis, including eye field specification, cell fate determination and identification of genes critical for eye formation. This knowledge has served as a solid foundation for cellular and molecular examinations of the robust regenerative capacity of the Xenopus eye. The retina, lens, and the optic nerve are capable of regeneration after injury in both larval and adult stages. Here, we discuss the current models for studying eye regeneration in Xenopus and their potential applications for providing insights into human eye diseases. As Xenopus has many of the same tools that are available for other regeneration models, we thus highlight the distinct strengths and versatility of this organism that make it especially suited for extrapolating and testing strategies aimed at promoting regeneration and repair in eye tissues. Furthermore, we outline a promising future for the use of new techniques and approaches to address outstanding questions in understanding eye regeneration.

Effects of the biocide methylisothiazolinone on Xenopus laevis wound healing and tail regeneration.

The South African clawed frog, Xenopus laevis, has a strong history as a suitable model for environmental studies. Its embryos and transparent tadpoles are highly sensitive to the environment and their developmental processes are well described. It is also amenable for molecular studies. These characteristics enable its use for rapid identification and understanding of exposure-induced defects. To investigate the consequences of chemical exposure on aquatic animals, Xenopus laevis embryos and tadpoles were exposed to the biocide, methylisothiazolinone (MIT). Frog tadpoles exposed to MIT following tail amputation lost their natural regenerative ability. This inhibition of regeneration led to a failure to regrow tissues including the spinal cord, muscle, and notochord. This MIT-dependent regenerative defect is due to a failure to close the amputation wound. A wound healing assay revealed that while untreated embryos close their wounds within one day after injury, MIT-treated animals maintained open wounds that did not reduce in size and caused lethality. Concomitant exposure of MIT with chemicals containing thiol groups such as glutathione and N-acetyl cysteine restored normal wound healing and regeneration responses in tadpoles. Together these results indicate that exposure to MIT impairs developmental wound repair and tissue regeneration in Xenopus laevis. Thus, this study reveals new aspects of MIT activity and demonstrates that Xenopus laevis is a well-suited model for facilitating future research into chemical exposure effects on injury responses.

Light-activation of the Archaerhodopsin H(+)-pump reverses age-dependent loss of vertebrate regeneration: sparking system-level controls in vivo.

Optogenetics, the regulation of proteins by light, has revolutionized the study of excitable cells, and generated strong interest in the therapeutic potential of this technology for regulating action potentials in neural and muscle cells. However, it is currently unknown whether light-activated channels and pumps will allow control of resting potential in embryonic or regenerating cells in vivo. Abnormalities in ion currents of non-excitable cells are known to play key roles in the etiology of birth defects and cancer. Moreover, changes in transmembrane resting potential initiate Xenopus tadpole tail regeneration, including regrowth of a functioning spinal cord, in tails that have been inhibited by natural inactivity of the endogenous H(+)-V-ATPase pump. However, existing pharmacological and genetic methods allow neither non-invasive control of bioelectric parameters in vivo nor the ability to abrogate signaling at defined time points. Here, we show that light activation of a H(+)-pump can prevent developmental defects and induce regeneration by hyperpolarizing transmembrane potentials. Specifically, light-dependent, Archaerhodopsin-based, H(+)-flux hyperpolarized cells in vivo and thus rescued Xenopus embryos from the craniofacial and patterning abnormalities caused by molecular blockade of endogenous H(+)-flux. Furthermore, light stimulation of Arch for only 2 days after amputation restored regenerative capacity to inhibited tails, inducing cell proliferation, tissue innervation, and upregulation of notch1 and msx1, essential genes in two well-known endogenous regenerative pathways. Electroneutral pH change, induced by expression of the sodium proton exchanger, NHE3, did not rescue regeneration, implicating the hyperpolarizing activity of Archaerhodopsin as the causal factor. The data reveal that hyperpolarization is required only during the first 48 hours post-injury, and that expression in the spinal cord is not necessary for the effect to occur. Our study shows that complex, coordinated sets of stable bioelectric events that alter body patterning-prevention of birth defects and induction of regeneration-can be elicited by the temporal modulation of a single ion current. Furthermore, as optogenetic reagents can be used to achieve that manipulation, the potential for this technology to impact clinical approaches for preventive, therapeutic, and regenerative medicine is extraordinary. We expect this first critical step will lead to an unprecedented expansion of optogenetics in biomedical research and in the probing of novel and fundamental biophysical determinants of growth and form.

Transducing bioelectric signals into epigenetic pathways during tadpole tail regeneration.

One important component of the cell-cell communication that occurs during regenerative patterning is bioelectrical signaling. In particular, the regeneration of the tail in Xenopus laevis tadpoles both requires, and can be initiated at non-regenerative stages by, specific regulation of bioelectrical signaling (alteration in resting membrane potential and a subsequent change in sodium content of blastemal cells). Although standing gradients of transmembrane voltage and ion concentration can provide positional guidance and other morphogenetic cues, these biophysical parameters must be transduced into transcriptional responses within cells. A number of mechanisms have been described for linking slow voltage changes to gene expression, but recent data on the importance of epigenetic regulation for regeneration suggest a novel hypothesis: that sodium/butyrate transporters link ion flows to influx of small molecules needed to modify chromatin state. Here, we briefly review the data on bioelectricity in tadpole tail regeneration, present a technique for convenient alteration of transmembrane potential in vivo that does not require transgenes, show augmentation of regeneration in vivo by manipulation of voltage, and present new data in the Xenopus tail consistent with the hypothesis that the monocarboxlyate transporter SLC5A8 may link regeneration-relevant epigenetic modification with upstream changes in ion content.

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.

HDAC activity is required during Xenopus tail regeneration.

The ability to fully restore damaged or lost organs is present in only a subset of animals. The Xenopus tadpole tail is a complex appendage, containing epidermis, muscle, nerves, spinal cord, and vasculature, which regenerates after amputation. Understanding the mechanisms of tail regeneration may lead to new insights to promote biomedical regeneration in non-regenerative tissues. Although chromatin remodeling is known to be critical for stem cell pluripotency, its role in complex organ regeneration in vivo remains largely uncharacterized. Here we show that histone deacetylase (HDAC) activity is required for the early stages of tail regeneration. HDAC1 is expressed during the 1(st) two days of regeneration. Pharmacological blockade of HDACs using Trichostatin A (TSA) increased histone acetylation levels in the amputated tail. Furthermore, treatment with TSA or another HDAC inhibitor, valproic acid, specifically inhibited regeneration. Over-expression of wild-type Mad3, a transcriptional repressor known to associate in a complex with HDACs via Sin3, inhibited regeneration. Similarly, expression of a Mad3 mutant lacking the Sin3-interacting domain that is required for HDAC binding also blocks regeneration, suggesting that HDAC and Mad3 may act together to regulate regeneration. Inhibition of HDAC function resulted in aberrant expression of Notch1 and BMP2, two genes known to be required for tail regeneration. Our results identify a novel early role for HDAC in appendage regeneration and suggest that modulation of histone acetylation is important in regenerative repair of complex appendages.

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, Na(V)1.2. 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. Na(V)1.2 is absent under nonregenerative conditions, but misexpression of human Na(V)1.5 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 Na(V) in regeneration, and show that modulation of sodium transport represents an exciting new approach to organ repair.

Capicua regulates cell proliferation downstream of the receptor tyrosine kinase/ras signaling pathway.

Signaling via the receptor tyrosine kinase (RTK)/Ras pathway promotes tissue growth during organismal development and is increased in many cancers [1]. It is still not understood precisely how this pathway promotes cell growth (mass accumulation). In addition, the RTK/Ras pathway also functions in cell survival, cell-fate specification, terminal differentiation, and progression through mitosis [2-7]. An important question is how the same canonical pathway can elicit strikingly different responses in different cell types. Here, we show that the HMG-box protein Capicua (Cic) restricts cell growth in Drosophila imaginal discs, and its levels are, in turn, downregulated by Ras signaling. Moreover, unlike normal cells, the growth of cic mutant cells is undiminished in the complete absence of a Ras signal. In addition to a general role in growth regulation, the importance of cic in regulating cell-fate determination downstream of Ras appears to vary from tissue to tissue. In the developing eye, the analysis of cic mutants shows that the functions of Ras in regulating growth and cell-fate determination are separable. Thus, the DNA-binding protein Cic is a key downstream component in the pathway by which Ras regulates growth in imaginal discs.

Apoptosis is required during early stages of tail regeneration in Xenopus laevis.

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.

The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes.

The maintenance of self-renewal in stem cells appears to be distinct from the induction of proliferation of the terminally differentiated mammalian cardiomyocytes because it is believed that the latter are unable to divide. However, proliferation is a necessary step in both processes. Interestingly, the small molecule 6-bromoindirubin-3'-oxime (BIO) is the first pharmacological agent shown to maintain self-renewal in human and mouse embryonic stem cells. To determine whether a molecule that can maintain stem cell properties can also participate in controlling the proliferative capability of the highly differentiated cardiomyocytes, we examine the effect of BIO in postmitotic cardiac cells. Here, we show that BIO promotes proliferation in mammalian cardiomyocytes. Our demonstration of a second role for BIO suggests that the maintenance of stem cell self-renewal and the induction of proliferation in differentiated cardiomyocytes may share common molecular pathways.

An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye.

We screened for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, we identified 46 lines, corresponding to insertions in 32 different loci, that elicited a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation.