Biological Robots: Perspectives on an Emerging Interdisciplinary Field.

Advances in science and engineering often reveal the limitations of classical approaches initially used to understand, predict, and control phenomena. With progress, conceptual categories must often be re-evaluated to better track recently discovered invariants across disciplines. It is essential to refine frameworks and resolve conflicting boundaries between disciplines such that they better facilitate, not restrict, experimental approaches and capabilities. In this essay, we address specific questions and critiques which have arisen in response to our research program, which lies at the intersection of developmental biology, computer science, and robotics. In the context of biological machines and robots, we explore changes across concepts and previously distinct fields that are driven by recent advances in materials, information, and life sciences. Herein, each author provides their own perspective on the subject, framed by their own disciplinary training. We argue that as with computation, certain aspects of developmental biology and robotics are not tied to specific materials; rather, the consilience of these fields can help to shed light on issues of multiscale control, self-assembly, and relationships between form and function. We hope new fields can emerge as boundaries arising from technological limitations are overcome, furthering practical applications from regenerative medicine to useful synthetic living machines.

Kinematic self-replication in reconfigurable organisms.

All living systems perpetuate themselves via growth in or on the body, followed by splitting, budding, or birth. We find that synthetic multicellular assemblies can also replicate kinematically by moving and compressing dissociated cells in their environment into functional self-copies. This form of perpetuation, previously unseen in any organism, arises spontaneously over days rather than evolving over millennia. We also show how artificial intelligence methods can design assemblies that postpone loss of replicative ability and perform useful work as a side effect of replication. This suggests other unique and useful phenotypes can be rapidly reached from wild-type organisms without selection or genetic engineering, thereby broadening our understanding of the conditions under which replication arises, phenotypic plasticity, and how useful replicative machines may be realized.

A cellular platform for the development of synthetic living machines.

Robot swarms have, to date, been constructed from artificial materials. Motile biological constructs have been created from muscle cells grown on precisely shaped scaffolds. However, the exploitation of emergent self-organization and functional plasticity into a self-directed living machine has remained a major challenge. We report here a method for generation of in vitro biological robots from frog (Xenopus laevis) cells. These xenobots exhibit coordinated locomotion via cilia present on their surface. These cilia arise through normal tissue patterning and do not require complicated construction methods or genomic editing, making production amenable to high-throughput projects. The biological robots arise by cellular self-organization and do not require scaffolds or microprinting; the amphibian cells are highly amenable to surgical, genetic, chemical, and optical stimulation during the self-assembly process. We show that the xenobots can navigate aqueous environments in diverse ways, heal after damage, and show emergent group behaviors. We constructed a computational model to predict useful collective behaviors that can be elicited from a xenobot swarm. In addition, we provide proof of principle for a writable molecular memory using a photoconvertible protein that can record exposure to a specific wavelength of light. Together, these results introduce a platform that can be used to study many aspects of self-assembly, swarm behavior, and synthetic bioengineering, as well as provide versatile, soft-body living machines for numerous practical applications in biomedicine and the environment.

A scalable pipeline for designing reconfigurable organisms.

Living systems are more robust, diverse, complex, and supportive of human life than any technology yet created. However, our ability to create novel lifeforms is currently limited to varying existing organisms or bioengineering organoids in vitro. Here we show a scalable pipeline for creating functional novel lifeforms: AI methods automatically design diverse candidate lifeforms in silico to perform some desired function, and transferable designs are then created using a cell-based construction toolkit to realize living systems with the predicted behaviors. Although some steps in this pipeline still require manual intervention, complete automation in future would pave the way to designing and deploying unique, bespoke living systems for a wide range of functions.

Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel.

Regeneration is regulated not only by chemical signals but also by physical processes, such as bioelectric gradients. How these may change in the absence of the normal gravitational and geomagnetic fields is largely unknown. Planarian flatworms were moved to the International Space Station for 5 weeks, immediately after removing their heads and tails. A control group in spring water remained on Earth. No manipulation of the planaria occurred while they were in orbit, and space-exposed worms were returned to our laboratory for analysis. One animal out of 15 regenerated into a double-headed phenotype-normally an extremely rare event. Remarkably, amputating this double-headed worm again, in plain water, resulted again in the double-headed phenotype. Moreover, even when tested 20 months after return to Earth, the space-exposed worms displayed significant quantitative differences in behavior and microbiome composition. These observations may have implications for human and animal space travelers, but could also elucidate how microgravity and hypomagnetic environments could be used to trigger desired morphological, neurological, physiological, and bacteriomic changes for various regenerative and bioengineering applications.

Serotonergic stimulation induces nerve growth and promotes visual learning via posterior eye grafts in a vertebrate model of induced sensory plasticity.

The major goal of regenerative medicine is to repair damaged tissues and organ systems, thereby restoring their native functions in the host. Control of innervation by re-grown or implanted structures, and integration of the nascent nerves into behavioral/cognitive programs of the host, remains a critical barrier. In the case of sensory organs, this is particularly true, as afferent neurons must form connections with the host to communicate auditory, visual, and tactile information. Xenopus embryos and tadpoles are powerful models for such studies, as grafting techniques allow for the creation of eyes and other sensory structures along the body axis, and the behavior of the resulting organism can be quantitatively analyzed. Previous work has demonstrated that ectopic eyes could be grafted in blinded tadpoles, allowing some of the animals to learn in a simple light-preference assay. Here, we show that it is possible to improve the efficiency of the process in the context of a novel image-forming vision assay, using a drug already approved for human use. Innervation of the host by ectopic eyes can be increased by targeting a serotonergic signaling mechanism: grafts treated with a 5-HT1B/D agonist strongly innervate the recipient compared with untreated grafts, without large-scale disruption of the host nervous system. Blind animals possessing eye grafts with the augmented innervation demonstrate increased performance over untreated siblings in wavelength-based learning assays. Furthermore, treated animals also exhibit enhanced visual pattern recognition, suggesting that the increased innervation in response to 5-HT1B/D activation leads to enhanced functional integration of the ectopic organ with the host central nervous system and behavioral programs. These data establish a model system and reveal a new roadmap using small molecule neurotransmitter drugs to augment innervation, integration, and function of transplanted heterologous organs in regenerative medicine.

Color and intensity discrimination in Xenopus laevis tadpoles.

Investigations into the physiology of Xenopus laevis have the potential to greatly accelerate biomedical research, especially concerning neural plasticity and sensory systems, but are limited by the lack of available information on behavioral learning in this species. Here, we attempt to lay the foundations for a behavioral assay in Xenopus that can be used in conjunction with biological manipulations. We tested cohorts of Xenopus tadpoles across four light-mediated active-avoidance experiments, using either wavelength or intensity as the salient discriminative cue. In the wavelength task, we determine a baseline learning rate and characterize retention of learning, identifying active extinction effects as far more potent than the passage of time in the loss of behavior. In the intensity task, we examine the effects of varying differences between the discriminative stimuli on acquisition and extinction and identify a critical range of intensity differences where learning changes. The results of our experiments demonstrate that Xenopus is a tractable model organism for cognitive research and can learn a variety of associative tasks in the laboratory settings. These data reveal new aspects of the Xenopus larval visual processing system and facilitate future research between cognitive methods and biological/chemical manipulations to study mechanisms of brain structure and function.

Serotonergic regulation of melanocyte conversion: A bioelectrically regulated network for stochastic all-or-none hyperpigmentation.

Experimentally induced depolarization of resting membrane potential in "instructor cells" in Xenopus laevis embryos causes hyperpigmentation in an all-or-none fashion in some tadpoles due to excess proliferation and migration of melanocytes. We showed that this stochastic process involved serotonin signaling, adenosine 3',5'-monophosphate (cAMP), and the transcription factors cAMP response element-binding protein (CREB), Sox10, and Slug. Transcriptional microarray analysis of embryos taken at stage 15 (early neurula) and stage 45 (free-swimming tadpole) revealed changes in the abundance of 45 and 517 transcripts, respectively, between control embryos and embryos exposed to the instructor cell-depolarizing agent ivermectin. Bioinformatic analysis revealed that the human homologs of some of the differentially regulated genes were associated with cancer, consistent with the induced arborization and invasive behavior of converted melanocytes. We identified a physiological circuit that uses serotonergic signaling between instructor cells, melanotrope cells of the pituitary, and melanocytes to control the proliferation, cell shape, and migration properties of the pigment cell pool. To understand the stochasticity and properties of this multiscale signaling system, we applied a computational machine-learning method that iteratively explored network models to reverse-engineer a stochastic dynamic model that recapitulated the frequency of the all-or-none hyperpigmentation phenotype produced in response to various pharmacological and molecular genetic manipulations. This computational approach may provide insight into stochastic cellular decision-making that occurs during normal development and pathological conditions, such as cancer.

The stability of memories during brain remodeling: A perspective.

One of the most important features of the nervous system is memory: the ability to represent and store experiences, in a manner that alters behavior and cognition at future times when the original stimulus is no longer present. However, the brain is not always an anatomically stable structure: many animal species regenerate all or part of the brain after severe injury, or remodel their CNS toward a new configuration as part of their life cycle. This raises a fascinating question: what are the dynamics of memories during brain regeneration? Can stable memories remain intact when cellular turnover and spatial rearrangement modify the biological hardware within which experiences are stored? What can we learn from model species that exhibit both, regeneration and memory, with respect to robustness and stability requirements for long-term memories encoded in living tissues? In this Perspective, we discuss relevant data in regenerating planaria, metamorphosing insects, and hibernating ground squirrels. While much remains to be done to understand this remarkable process, molecular-level insight will have important implications for cognitive science, regenerative medicine of the brain, and the development of non-traditional computational media in synthetic bioengineering.

A novel method for inducing nerve growth via modulation of host resting potential: gap junction-mediated and serotonergic signaling mechanisms.

A major goal of regenerative medicine is to restore the function of damaged or missing organs through the implantation of bioengineered or donor-derived components. It is necessary to understand the signals and cues necessary for implanted structures to innervate the host, as organs devoid of neural connections provide little benefit to the patient. While developmental studies have identified neuronal pathfinding molecules required for proper patterning during embryogenesis, strategies to initiate innervation in structures transplanted at later times or alternate locations remain limited. Recent work has identified membrane resting potential of nerves as a key regulator of growth cone extension or arrest. Here, we identify a novel role of bioelectricity in the generation of axon guidance cues, showing that neurons read the electric topography of surrounding cells, and demonstrate these cues can be leveraged to initiate sensory organ transplant innervation. Grafts of fluorescently labeled embryological eye primordia were used to produce ectopic eyes in Xenopus laevis tadpoles. Depolarization of host tissues through anion channel activation or other means led to a striking hyperinnervation of the body by these ectopic eyes. A screen of possible transduction mechanisms identified serotonergic signaling to be essential for hyperinnervation to occur, and our molecular data suggest a possible model of bioelectrical control of the distribution of neurotransmitters that guides nerve growth. Together, these results identify the molecular components of bioelectrical signaling among cells that regulates axon guidance, and suggest novel biomedical and bioengineering strategies for triggering neuronal outgrowth using ion channel drugs already approved for human use.

Left-right patterning in Xenopus conjoined twin embryos requires serotonin signaling and gap junctions.

A number of processes operating during the first cell cleavages enable the left-right (LR) axis to be consistently oriented during Xenopus laevis development. Prior work showed that secondary organizers induced in frog embryos after cleavage stages (i.e. conjoined twins arising from ectopic induced primary axes) correctly pattern their own LR axis only when a primary (early) organizer is also present. This instructive effect confirms the unique LR patterning functions that occur during early embryogenesis, but leaves open the question: which mechanisms that operate during early stages are also involved in the orientation of later-induced organizers? We sought to distinguish the two phases of LR patterning in secondary organizers (LR patterning of the primary twin and the later transfer of this information to the secondary twin) by perturbing only the latter process. Here, we used reagents that do not affect primary LR patterning at the time secondary organizers form to inhibit each of 4 mechanisms in the induced twin. Using pharmacological, molecular-genetic, and photo-chemical tools, we show that serotonergic and gap-junctional signaling, but not proton or potassium flows, are required for the secondary organizer to appropriately pattern its LR axis in a multicellular context. We also show that consistently-asymmetric gene expression begins prior to ciliary flow. Together, our data highlight the importance of physiological signaling in the propagation of cleavage-derived LR orientation to multicellular cell fields.

Inversion of left-right asymmetry alters performance of Xenopus tadpoles in nonlateralized cognitive tasks.

Left-right behavioural biases are well documented across the animal kingdom, and handedness has long been associated with cognitive performance. However, the relationship between body laterality and cognitive ability is poorly understood. The embryonic pathways dictating normal left-right patterning have been molecularly dissected in model vertebrates, and numerous genetic and pharmacological treatments now facilitate experimental randomization or reversal of the left-right axis in these animals. Several recent studies showed a link between brain asymmetry and strongly lateralized behaviours such as eye use preference. However, links between laterality of the body and performance on cognitive tasks utilizing nonlateralized cues remain unknown. Xenopus tadpoles are an established model for the study of early left-right patterning, and protocols were recently developed to quantitatively evaluate learning and memory in these animals. Using an automated testing and training platform, we tested wild-type, left-right-randomized and left-right-reversed tadpoles for their ability to learn colour cues in an automated assay. Our results indicate that animals with either randomization or reversal of somatic left-right patterning learned more slowly than wild-type siblings, although all groups were able to reach the same performance optimum given enough training sessions. These results are the first analysis of the link between body laterality and learning of nonlateralized cues, and they position the Xenopus tadpole as an attractive and tractable model for future studies of the links between asymmetry of the body, lateralization of the brain and behaviour.

Ectopic eyes outside the head in Xenopus tadpoles provide sensory data for light-mediated learning.

A major roadblock in the biomedical treatment of human sensory disorders, including blindness, has been an incomplete understanding of the nervous system and its ability to adapt to changes in sensory modality. Likewise, fundamental insight into the evolvability of complex functional anatomies requires understanding brain plasticity and the interaction between the nervous system and body architecture. While advances have been made in the generation of artificial and biological replacement components, the brain's ability to interpret sensory information arising from ectopic locations is not well understood. We report the use of eye primordia grafts to create ectopic eyes along the body axis of Xenopus tadpoles. These eyes are morphologically identical to native eyes and can be induced at caudal locations. Cell labeling studies reveal that eyes created in the tail send projections to the stomach and trunk. To assess function we performed light-mediated learning assays using an automated machine vision and environmental control system. The results demonstrate that ectopic eyes in the tail of Xenopus tadpoles could confer vision to the host. Thus ectopic visual organs were functional even when present at posterior locations. These data and protocols demonstrate the ability of vertebrate brains to interpret sensory input from ectopic structures and incorporate them into adaptive behavioral programs. This tractable new model for understanding the robust plasticity of the central nervous system has significant implications for regenerative medicine and sensory augmentation technology.

Aversive training methods in Xenopus laevis: general principles.

Xenopus laevis is an ideal organism in which to study the mechanisms linking genetics, the embryogenesis of the central nervous system, and the generation of cognitive behavior. Frog embryos facilitate the targeting of many pathways of importance to neuroscience via pharmacological, genetic, and surgical manipulations. A limiting factor for investigations of memory and learning has been the difficulty of eliciting learning in Xenopus. Here, we outline a simple strategy for aversive conditioning (associative learning) in Xenopus tadpoles, and present sample data using a quantitative automated analysis system. We also discuss the factors and variables that must be considered to ensure optimal learning and recall performance, for use as behavioral endpoints in any experiment.

Neurally Derived Tissues in Xenopus laevis Embryos Exhibit a Consistent Bioelectrical Left-Right Asymmetry.

Consistent left-right asymmetry in organ morphogenesis is a fascinating aspect of bilaterian development. Although embryonic patterning of asymmetric viscera, heart, and brain is beginning to be understood, less is known about possible subtle asymmetries present in anatomically identical paired structures. We investigated two important developmental events: physiological controls of eye development and specification of neural crest derivatives, in Xenopus laevis embryos. We found that the striking hyperpolarization of transmembrane potential (V(mem)) demarcating eye induction usually occurs in the right eye field first. This asymmetry is randomized by perturbing visceral left-right patterning, suggesting that eye asymmetry is linked to mechanisms establishing primary laterality. Bilateral misexpression of a depolarizing channel mRNA affects primarily the right eye, revealing an additional functional asymmetry in the control of eye patterning by V(mem). The ATP-sensitive K(+) channel subunit transcript, SUR1, is asymmetrically expressed in the eye primordia, thus being a good candidate for the observed physiological asymmetries. Such subtle asymmetries are not only seen in the eye: consistent asymmetry was also observed in the migration of differentiated melanocytes on the left and right sides. These data suggest that even anatomically symmetrical structures may possess subtle but consistent laterality and interact with other developmental left-right patterning pathways.

Color vision and learning in the monarch butterfly, Danaus plexippus (Nymphalidae).

The monarch butterfly, Danaus plexippus, is well known for its intimate association with milkweed plants and its incredible multi-generational trans-continental migrations. However, little is known about monarch butterflies' color perception or learning ability, despite the importance of visual information to butterfly behavior in the contexts of nectar foraging, host-plant location and mate recognition. We used both theoretical and experimental approaches to address basic questions about monarch color vision and learning ability. Color space modeling based on the three known spectral classes of photoreceptors present in the eye suggests that monarchs should not be able to discriminate between long wavelength colors without making use of a dark orange lateral filtering pigment distributed heterogeneously in the eye. In the context of nectar foraging, monarchs show strong innate preferences, rapidly learn to associate colors with sugar rewards and learn non-innately preferred colors as quickly and proficiently as they do innately preferred colors. Butterflies also demonstrate asymmetric confusion between specific pairs of colors, which is likely a function of stimulus brightness. Monarchs readily learn to associate a second color with reward, and in general, learning parameters do not vary with temporal sequence of training. In addition, monarchs have true color vision; that is, they can discriminate colors on the basis of wavelength, independent of intensity. Finally, behavioral trials confirm that monarchs do make use of lateral filtering pigments to enhance long-wavelength discrimination. Our results demonstrate that monarchs are proficient and flexible color learners; these capabilities should allow them to respond rapidly to changing nectar availabilities as they travel over migratory routes, across both space and time.

Transmembrane potential of GlyCl-expressing instructor cells induces a neoplastic-like conversion of melanocytes via a serotonergic pathway.

Understanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest's environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl). Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT). These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

High-throughput Xenopus laevis immunohistochemistry using agarose sections.

Characterizing protein localization in Xenopus laevis embryos is an important aspect of developmental and regenerative studies that use this advantageous model system. Although whole-mount immunohistochemistry is an efficient and powerful way to visualize surface and ectodermal expression, its ability to localize proteins in internal tissues and cells is limited by the incomplete penetration of antibodies through outer layers of the embryo. Microtome sections of paraffin-embedded embryos provide good internal resolution, but precise orientation of embryos can be difficult, and sectioning many samples is time intensive. Further, care must be taken with sections to minimize tissue damage, because heat and organic solvents used during the process can render some proteins invisible to antibody detection. The method described here is a short protocol for generating robust sections for use in immunoreactions with as little as two days from collection to visualization, making it useful as a rapid screening process. Advantages of this method include: (1) the durability of the sections produced (which can be treated as if they were wholemounts and processed by fluid aspiration in vials rather than mounted onto slides); (2) the ability to examine multiple antibody targets in tandem, in tissue that is never heated or extracted with harsh reagents; (3) the lack of autofluorescence as occurs in glutaraldehyde-containing media; and (4) the ease of orientation of embryos in a fully transparent block.

A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science.

Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle.

All cells possess long-term, steady-state voltage gradients across the plasma membrane. These transmembrane potentials arise from the combined activity of numerous ion channels, pumps and gap junction complexes. Increasing data from molecular physiology now reveal that the role of changes in membrane voltage controls, and is in turn controlled by, progression through the cell cycle. We review recent functional data on the regulation of mitosis by bioelectric signals, and the function of membrane voltage and specific potassium, sodium and chloride ion channels in the proliferation of embryonic, somatic and neoplastic cells. Its unique properties place this powerful, well-conserved, but still poorly-understood signaling system at the center of the coordinated cellular interactions required for complex pattern formation. Moreover, disregulation of ion channel expression and function is increasingly observed to be not only a useful marker but likely a functional element in oncogenesis. New advances in genomics and the development of in vivo biophysical techniques suggest exciting opportunities for molecular medicine, bioengineering and regenerative approaches to human health.