Embryos assist morphogenesis of others through calcium and ATP signaling mechanisms in collective teratogen resistance.

Information for organismal patterning can come from a variety of sources. We investigate the possibility that instructive influences for normal embryonic development are provided not only at the level of cells within the embryo, but also via interactions between embryos. To explore this, we challenge groups of embryos with disruptors of normal development while varying group size. Here, we show that Xenopus laevis embryos are much more sensitive to a diverse set of chemical and molecular-biological perturbations when allowed to develop alone or in small groups, than in large groups. Keeping per-embryo exposure constant, we find that increasing the number of exposed embryos in a cohort increases the rate of survival while incidence of defects decreases. This inter-embryo assistance effect is mediated by short-range diffusible signals and involves the P2 ATP receptor. Our data and computational model emphasize that morphogenesis is a collective phenomenon not only at the level of cells, but also of whole bodies, and that cohort size is a crucial variable in studies of ecotoxicology, teratogenesis, and developmental plasticity.

Active inference, morphogenesis, and computational psychiatry.

Active inference is a leading theory in neuroscience that provides a simple and neuro-biologically plausible account of how action and perception are coupled in producing (Bayes) optimal behavior; and has been recently used to explain a variety of psychopathological conditions. In parallel, morphogenesis has been described as the behavior of a (non-neural) cellular collective intelligence solving problems in anatomical morphospace. In this article, we establish a link between the domains of cell biology and neuroscience, by analyzing disorders of morphogenesis as disorders of (active) inference. The aim of this article is three-fold. We want to: (i) reveal a connection between disorders of morphogenesis and disorders of active inference as apparent in psychopathological conditions; (ii) show how disorders of morphogenesis can be simulated using active inference; (iii) suggest that active inference can shed light on developmental defects or aberrant morphogenetic processes, seen as disorders of information processing, and perhaps suggesting novel intervention and repair strategies. We present four simulations illustrating application of these ideas to cellular behavior during morphogenesis. Three of the simulations show that the same forms of aberrant active inference (e.g., deficits of sensory attenuation and low sensory precision) that have been used to explain psychopathological conditions (e.g., schizophrenia and autism) also produce familiar disorders of development and morphogenesis when implemented at the level of the collective behavior of a group of cells. The fourth simulation involves two cells with too high precision, in which we show that the reduction of concentration signaling and sensitivity to the signals of other cells treats the development defect. Finally, we present the results of an experimental test of one of the model's predictions in early Xenopus laevis embryos: thioridazine (a dopamine antagonist that may reduce sensory precision in biological systems) induced developmental (anatomical) defects as predicted. The use of conceptual and empirical tools from neuroscience to understand the morphogenetic behavior of pre-neural agents offers the possibility of new approaches in regenerative medicine and evolutionary developmental biology.

Extra-genomic instructive influences in morphogenesis: A review of external signals that regulate growth and form.

Embryonic development and regeneration accomplish a remarkable feat: individual cells work together to create or repair complex anatomical structures. What is the source of the instructive signals that specify these invariant and robust organ-level outcomes? The most frequently studied source of morphogenetic control is the host genome and its transcriptional circuits. However, it is now apparent that significant information affecting patterning also arrives from outside of the body. Both biotic and physical factors, including temperature and various molecular signals emanating from pathogens, commensals, and conspecific organisms, affect developmental outcomes. Here, we review examples in which anatomical patterning decisions are strongly impacted by lateral signals that originate from outside of the zygotic genome. The endogenous pathways targeted by these influences often show transgenerational effects, enabling them to shape the evolution of anatomies even faster than traditional Baldwin-type assimilation. We also discuss recent advances in the biophysics of morphogenetic controls and speculate on additional sources of important patterning information which could be exploited to better understand the evolution of bodies and to design novel approaches for regenerative medicine.

Regenerative Adaptation to Electrochemical Perturbation in Planaria: A Molecular Analysis of Physiological Plasticity.

Anatomical homeostasis results from dynamic interactions between gene expression, physiology, and the external environment. Owing to its complexity, this cellular and organism-level phenotypic plasticity is still poorly understood. We establish planarian regeneration as a model for acquired tolerance to environments that alter endogenous physiology. Exposure to barium chloride (BaCl2) results in a rapid degeneration of anterior tissue in Dugesia japonica. Remarkably, continued exposure to fresh solution of BaCl2 results in regeneration of heads that are insensitive to BaCl2. RNA-seq revealed transcriptional changes in BaCl2-adapted heads that suggests a model of adaptation to excitotoxicity. Loss-of-function experiments confirmed several predictions: blockage of chloride and calcium channels allowed heads to survive initial BaCl2 exposure, inducing adaptation without prior exposure, whereas blockade of TRPM channels reversed adaptation. Such highly adaptive plasticity may represent an attractive target for biomedical strategies in a wide range of applications beyond its immediate relevance to excitotoxicity preconditioning.

Meta-omics analysis of elite athletes identifies a performance-enhancing microbe that functions via lactate metabolism.

The human gut microbiome is linked to many states of human health and disease1. The metabolic repertoire of the gut microbiome is vast, but the health implications of these bacterial pathways are poorly understood. In this study, we identify a link between members of the genus Veillonella and exercise performance. We observed an increase in Veillonella relative abundance in marathon runners postmarathon and isolated a strain of Veillonella atypica from stool samples. Inoculation of this strain into mice significantly increased exhaustive treadmill run time. Veillonella utilize lactate as their sole carbon source, which prompted us to perform a shotgun metagenomic analysis in a cohort of elite athletes, finding that every gene in a major pathway metabolizing lactate to propionate is at higher relative abundance postexercise. Using 13C3-labeled lactate in mice, we demonstrate that serum lactate crosses the epithelial barrier into the lumen of the gut. We also show that intrarectal instillation of propionate is sufficient to reproduce the increased treadmill run time performance observed with V. atypica gavage. Taken together, these studies reveal that V. atypica improves run time via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance.

CRISPR Guide RNA Cloning for Mammalian Systems.

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.