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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.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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.

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.

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.

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.

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.

Ion transporters, and the resulting voltage gradients and electric fields, have been implicated in embryonic development and regeneration. These biophysical signals are key physiological aspects of the microenvironment that epigenetically regulate stem and tumor cell behavior. Here, we identify a previously unrecognized function for KCNQ1, a potassium channel known to be involved in human Romano-Ward and Jervell-Lange-Nielsen syndromes when mutated. Misexpression of its modulatory wild-type beta-subunit XKCNE1 in the Xenopus embryo resulted in a striking alteration of the behavior of one type of embryonic stem cell: the pigment cell lineage of the neural crest. Depolarization of embryonic cells by misexpression of KCNE1 non-cell-autonomously induced melanocytes to overproliferate, spread out, and become highly invasive of blood vessels, liver, gut, and neural tube, leading to a deeply hyperpigmented phenotype. This effect is mediated by the up-regulation of Sox10 and Slug genes, thus linking alterations in ion channel function to the control of migration, shape, and mitosis rates during embryonic morphogenesis. Taken together, these data identify a role for the KCNQ1 channel in regulating key cell behaviors and reveal the molecular identity of a biophysical switch, by means of which neoplastic-like properties can be conferred upon a specific embryonic stem cell subpopulation.

Several ion transporters have been implicated in left-right (LR) patterning. Here, we characterize a new component of the early bioelectrical circuit: the potassium channel KCNQ1 and its accessory subunit KCNE1. Having cloned the native Xenopus versions of both genes, we show that both are asymmetrically localized as maternal proteins during the first few cleavages of frog embryo development in a process dependent on microtubule and actin organization. Molecular loss-of-function using dominant negative constructs demonstrates that both gene products are required for normal LR asymmetry. We propose a model whereby these channels provide an exit path for K(+) ions brought in by the H(+),K(+)-ATPase. This physiological module thus allows the obligate but electroneutral H(+),K(+)-ATPase to generate an asymmetric voltage gradient on the left and right sides. Our data reveal a new, bioelectrical component of the mechanisms patterning a large-scale axis in vertebrate embryogenesis., ((c) 2008 S. Karger AG, Basel.)