Normal Table of Xenopus development: a new graphical resource.

Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

Bioelectricity: Reasons to Be Amazed and Hopeful-My Personal Favorites.

The field of bioelectricity is growing so rapidly, any attempt on my part to summarize its extraordinary breadth and depth would be a shallow report, a list really. Instead, I've zoomed in to introduce two marvelous examples of new understanding and application of bioelectricity. The signaling Venus flytraps use to capture and digest insects is a marvel of electrical engineering. The application of magnetic fields to treat conditions with brain involvement can offer relief from debilitating symptoms that have been refractory to other treatments. These, and a multitude of other fascinating stories, are helping to spread the word that bioelectricity can and should be seen as a critical approach that can change both science and medicine. This important journal will continue to carry that message.

Live imaging of intracellular pH in planarians using the ratiometric fluorescent dye SNARF-5F-AM.

Physiological parameters such as resting potential and pH are increasingly recognized as important regulators of cell activity and tissue-level events in regeneration, development, and cancer. The availability of fluorescent reporter dyes has greatly increased the ability to track these properties in vivo. The planarian flatworm is an important and highly tractable model system for regeneration, stem cell biology, and neuroscience; however, no protocols have been published for investigating pH in this system. Here, we report a simple and effective protocol for imaging pH gradients in living planaria suitable for intact and regenerating flatworms.

Preventing Ethanol-Induced Brain and Eye Morphology Defects Using Optogenetics.

Background: Embryonic exposure to the teratogen ethanol leads to dysmorphias, including eye and brain morphology defects associated with fetal alcohol spectrum disorder (FASD). Exposure of Xenopus laevis embryos to ethanol leads to similar developmental defects, including brain and eye dysmorphism, confirming our work and the work of others showing Xenopus as a useful system for studies of the brain and eye birth defects associated with FASD. Several targets of ethanol action have been hypothesized, one being regulation of Kir2.1 potassium channel. Endogenous ion fluxes and membrane voltage variation (bioelectric signals) have been shown to be powerful regulators of embryonic cell behaviors that are required for correct brain and eye morphology. Disruptions to these voltage patterns lead to spatially correlated disruptions in gene expression patterns and corresponding morphology. Materials and Methods: Here, we use controlled membrane voltage modulation to determine when and where voltage modulation is sufficient to rescue ethanol-induced brain and eye defects in Xenopus embryos. Results: We found (1) that modulating membrane voltage using light activation of the channelrhodopsin-2 variant D156A rescues ethanol exposed embryos, resulting in normal brain and eye morphologies; (2) hyperpolarization is required for the full duration of ethanol exposure; (3) hyperpolarization of only superficial ectoderm is sufficient for this effect; and(4) the rescue effect acts at a distance. Conclusions: These results, particularly the last, raise the exciting possibility of using bioelectric modulation to treat ethanol-induced brain and eye birth defects, possibly with extant ion channel drugs already prescribed to pregnant women. This may prove to be a simple and cost-effective strategy for reducing the impact of FASD.

Synthesis of Water-Soluble Far-Red-Emitting Amphiphilic BODIPY Dyes.

We report the synthesis of two water-soluble BODIPY dyes with far-red absorption and near-infrared fluorescence following cell membrane insertion. Introduction of dicationic or dianionic groups imparts water solubility and prevents translocation of the dye through the plasma membrane for highly effective labeling. The dicationic form is particularly well localized to the plasma membrane and resists quenching even after >8 min of continuous light exposure. The dyes are almost completely nonemissive in water and other highly polar solvents, but display high-fluorescence yields in chloroform and upon insertion into the extracellular leaflet.

The Zahn drawings: new illustrations of Xenopus embryo and tadpole stages for studies of craniofacial development.

The embryos and tadpoles of the frog Xenopus are increasingly important subjects for studies of the development of the head and face - studies that are providing novel and crucial insight into the causes and prevention of a suite of devastating birth defects, as well as basic evolutionary and developmental biology. However, many studies are conducted on a range of embryonic stages that are not fully represented in the beloved Xenopus resource, Nieuwkoop and Faber's classic Normal Table of Xenopus laevis (Daudin) The lack of standardized images at these stages acts as a barrier to the efficient and accurate representation and communication of experimental methodology and expression data. To fill this gap, we have created 27 new high-quality illustrations. Like their oft-used predecessors from Nieuwkoop and Faber, these drawings can be freely downloaded and used, and will, we hope, serve as an essential resource for this important model system.

Long-Term, Stochastic Editing of Regenerative Anatomy via Targeting Endogenous Bioelectric Gradients.

We show that regenerating planarians' normal anterior-posterior pattern can be permanently rewritten by a brief perturbation of endogenous bioelectrical networks. Temporary modulation of regenerative bioelectric dynamics in amputated trunk fragments of planaria stochastically results in a constant ratio of regenerates with two heads to regenerates with normal morphology. Remarkably, this is shown to be due not to partial penetrance of treatment, but a profound yet hidden alteration to the animals' patterning circuitry. Subsequent amputations of the morphologically normal regenerates in water result in the same ratio of double-headed to normal morphology, revealing a cryptic phenotype that is not apparent unless the animals are cut. These animals do not differ from wild-type worms in histology, expression of key polarity genes, or neoblast distribution. Instead, the altered regenerative bodyplan is stored in seemingly normal planaria via global patterns of cellular resting potential. This gradient is functionally instructive, and represents a multistable, epigenetic anatomical switch: experimental reversals of bioelectric state reset subsequent regenerative morphology back to wild-type. Hence, bioelectric properties can stably override genome-default target morphology, and provide a tractable control point for investigating cryptic phenotypes and the stochasticity of large-scale epigenetic controls.

Use of genetically encoded, light-gated ion translocators to control tumorigenesis.

It has long been known that the resting potential of tumor cells is depolarized relative to their normal counterparts. More recent work has provided evidence that resting potential is not just a readout of cell state: it regulates cell behavior as well. Thus, the ability to control resting potential in vivo would provide a powerful new tool for the study and treatment of tumors, a tool capable of revealing living-state physiological information impossible to obtain using molecular tools applied to isolated cell components. Here we describe the first use of optogenetics to manipulate ion-flux mediated regulation of membrane potential specifically to prevent and cause regression of oncogene-induced tumors. Injection of mutant-KRAS mRNA induces tumor-like structures with many documented similarities to tumors, in Xenopus tadpoles. We show that expression and activation of either ChR2D156A, a blue-light activated cation channel, or Arch, a green-light activated proton pump, both of which hyperpolarize cells, significantly lowers the incidence of KRAS tumor formation. Excitingly, we also demonstrate that activation of co-expressed light-activated ion translocators after tumor formation significantly increases the frequency with which the tumors regress in a process called normalization. These data demonstrate an optogenetic approach to dissect the biophysics of cancer. Moreover, they provide proof-of-principle for a novel class of interventions, directed at regulating cell state by targeting physiological regulators that can over-ride the presence of mutations.

Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome.

KEY POINTS: Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT: Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms.

The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together, these data and analyses shed light on important physiological modifiers of morphological information in dictating species-specific shape, and reveal them to be a novel instructive input into head patterning in regenerating planaria.

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry.

Optogenetics in Developmental Biology: using light to control ion flux-dependent signals in Xenopus embryos.

Developmental bioelectricity, electrical signaling among non-excitable cells, is now known to regulate proliferation, apoptosis, gene expression, and patterning during development. The extraordinary temporal and spatial resolution offered by optogenetics could revolutionize the study of bioelectricity the same way it has revolutionized neuroscience. There is, however, no guide to adapting optogenetics to patterning systems. To fill this gap, we used optogenetic reagents, both proteins and photochemical switches, to vary steady-state bioelectrical properties of non-spiking embryonic cells in Xenopus laevis. We injected mRNA for various proteins, including Channelrhodopsins and Archaerhodopsin, into 1-8 cell embryos, or soaked embryos in media containing photochemical switches, then examined the effect of light and dark on membrane voltage (Vmem) using both electrodes and fluorescent membrane voltage reporters. We also scored tadpoles for known effects of varying Vmem, including left-right asymmetry disruption, hyperpigmentation, and craniofacial phenotypes. The majority of reagents we tested caused a significant increase in the percentage of light-exposed tadpoles showing relevant phenotypes; however, the majority of reagents also induced phenotypes in controls kept in the dark. Experiments on this "dark phenotype" yielded evidence that the direction of ion flux via common optogenetic reagents may be reversed, or unpredictable in non-neural cells. When used in combination with rigorous controls, optogenetics can be a powerful tool for investigating ion-flux based signaling in non-excitable systems. Nonetheless, it is crucial that new reagents be designed with these non-neural cell types in mind.

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.

Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation.

Alongside the well-known chemical modes of cell-cell communication, we find an important and powerful system of bioelectrical signaling: changes in the resting voltage potential (Vmem) of the plasma membrane driven by ion channels, pumps and gap junctions. Slow Vmem changes in all cells serve as a highly conserved, information-bearing pathway that regulates cell proliferation, migration and differentiation. In embryonic and regenerative pattern formation and in the disorganization of neoplasia, bioelectrical cues serve as mediators of large-scale anatomical polarity, organ identity and positional information. Recent developments have resulted in tools that enable a high-resolution analysis of these biophysical signals and their linkage with upstream and downstream canonical genetic pathways. Here, we provide an overview for the study of bioelectric signaling, focusing on state-of-the-art approaches that use molecular physiology and developmental genetics to probe the roles of bioelectric events functionally. We highlight the logic, strategies and well-developed technologies that any group of researchers can employ to identify and dissect ionic signaling components in their own work and thus to help crack the bioelectric code. The dissection of bioelectric events as instructive signals enabling the orchestration of cell behaviors into large-scale coherent patterning programs will enrich on-going work in diverse areas of biology, as biophysical factors become incorporated into our systems-level understanding of cell interactions.

Photoconversion for tracking the dynamics of cell movement in Xenopus laevis embryos.

Xenopus laevis is an ideal model system for investigating dynamic morphogenetic processes during embryogenesis, regeneration, and homeostasis. Our understanding of these events has been greatly facilitated by lineage labeling, that is, marking a cell or a group of cells and all their descendants using vital dyes, fluorescent molecules, or transplantation techniques. Unfortunately, these strategies are limited in their spatiotemporal resolution: They do not allow long-term dynamic in vivo imaging, are generally invasive, and labeling is restricted to cells on the surface. Genetically encoded fluorescent proteins (FPs), on the other hand, provide excellent alternative methods to traditional lineage labeling, enabling labeling with high spatiotemporal resolution and tracking of cellular and subcellular structures to study patterning events. Over the past decade, FPs have evolved to allow fine control of their spectral properties (in a defined region of interest) for greater labeling specificity. One example is EosFP, which is a protein cloned from the scleractinian coral Lobophyllia hemprichii that can be photoconverted from green to red fluorescence state with near-ultraviolet (UV) light irradiation. Here, we describe EosFP-photoconversion of Xenopus embryos to track cells during developmental and regenerative processes using a metal-halide- or xenon-arc-based fluorescent microscope system, which provides a simpler, less expensive alternative to photoconversion using laser microscopy.

Measuring resting membrane potential using the fluorescent voltage reporters DiBAC4(3) and CC2-DMPE.

Slow changes in steady-state (resting) transmembrane potential (V(mem)) of non-excitable cells often encode important instructive signals controlling differentiation, proliferation, and cell:cell communication. Probing the function of such bioelectric gradients in vivo or in culture requires the ability to track V(mem), to characterize endogenous patterns of differential potential, map out isopotential cell groups (compartments or cell fields), and confirm the results of functional perturbation of V(mem). The use of fluorescent bioelectricity reporters (FBRs) has become more common as continuing research and innovation have produced better and more options. These dyes are now used routinely for cell sorting and for studies of cultured cells. Important advantages over single cell electrode measurements are offered by dyes, including: (1) subcellular resolution, (2) the ability to monitor multicellular areas and volumes in vivo, (3) simplicity of use, (4) ability to measure moving targets, and (5) ability to measure over long time periods. Thus, FBRs are suitable for longitudinal studies of systems that change and move over time, for example, embryos. Existing protocols focus on measurements of rapid action potentials in cultured cells or neurons. This article describes a dye pair that can be used to measure resting V(mem) in cultured cells and in vivo in Xenopus laevis embryos and tadpoles (and is readily applied to other model systems, such as zebrafish, for studies of developmental bioelectricity). It is assumed that the reader is fully familiar with the process and terminology of fluorescence microscopy.