CRISPR-Based Tools in Immunity.

CRISPR technology has opened a new era of genome interrogation and genome engineering. Discovered in bacteria, where it protects against bacteriophage by cleaving foreign nucleic acid sequences, the CRISPR system has been repurposed as an adaptable tool for genome editing and multiple other applications. CRISPR's ease of use, precision, and versatility have led to its widespread adoption, accelerating biomedical research and discovery in human cells and model organisms. Here we review CRISPR-based tools and discuss how they are being applied to decode the genetic circuits that control immune function in health and disease. Genetic variation in immune cells can affect autoimmune disease risk, infectious disease pathogenesis, and cancer immunotherapies. CRISPR provides unprecedented opportunities for functional mechanistic studies of coding and noncoding genome sequence function in immunity. Finally, we discuss the potential of CRISPR technology to engineer synthetic cellular immunotherapies for a wide range of human diseases.

Functional sensory circuits built from neurons of two species.

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.

A programmable reaction-diffusion system for spatiotemporal cell signaling circuit design.

Cells self-organize molecules in space and time to generate complex behaviors, but we lack synthetic strategies for engineering spatiotemporal signaling. We present a programmable reaction-diffusion platform for designing protein oscillations, patterns, and circuits in mammalian cells using two bacterial proteins, MinD and MinE (MinDE). MinDE circuits act like "single-cell radios," emitting frequency-barcoded fluorescence signals that can be spectrally isolated and analyzed using digital signal processing tools. We define how to genetically program these signals and connect their spatiotemporal dynamics to cell biology using engineerable protein-protein interactions. This enabled us to construct sensitive reporter circuits that broadcast endogenous cell signaling dynamics on a frequency-barcoded imaging channel and to build control signal circuits that synthetically pattern activities in the cell, such as protein condensate assembly and actin filamentation. Our work establishes a paradigm for visualizing, probing, and engineering cellular activities at length and timescales critical for biological function.

Brain-wide representations of behavior spanning multiple timescales and states in C. elegans.

Changes in an animal's behavior and internal state are accompanied by widespread changes in activity across its brain. However, how neurons across the brain encode behavior and how this is impacted by state is poorly understood. We recorded brain-wide activity and the diverse motor programs of freely moving C. elegans and built probabilistic models that explain how each neuron encodes quantitative behavioral features. By determining the identities of the recorded neurons, we created an atlas of how the defined neuron classes in the C. elegans connectome encode behavior. Many neuron classes have conjunctive representations of multiple behaviors. Moreover, although many neurons encode current motor actions, others integrate recent actions. Changes in behavioral state are accompanied by widespread changes in how neurons encode behavior, and we identify these flexible nodes in the connectome. Our results provide a global map of how the cell types across an animal's brain encode its behavior.

Generating parallel representations of position and identity in the olfactory system.

In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. Here, we show that separate cVA-processing streams extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, we identify 47 cell types with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts.

Structural and functional map for forelimb movement phases between cortex and medulla.

The cortex influences movement by widespread top-down projections to many nervous system regions. Skilled forelimb movements require brainstem circuitry in the medulla; however, the logic of cortical interactions with these neurons remains unexplored. Here, we reveal a fine-grained anatomical and functional map between anterior cortex (AC) and medulla in mice. Distinct cortical regions generate three-dimensional synaptic columns tiling the lateral medulla, topographically matching the dorso-ventral positions of postsynaptic neurons tuned to distinct forelimb action phases. Although medial AC (MAC) terminates ventrally and connects to forelimb-reaching-tuned neurons and its silencing impairs reaching, lateral AC (LAC) influences dorsally positioned neurons tuned to food handling, and its silencing impairs handling. Cortico-medullary neurons also extend collaterals to other subcortical structures through a segregated channel interaction logic. Our findings reveal a precise alignment between cortical location, its function, and specific forelimb-action-tuned medulla neurons, thereby clarifying interaction principles between these two key structures and beyond.

Parallel neural pathways control sodium consumption and taste valence.

The hedonic value of salt fundamentally changes depending on the internal state. High concentrations of salt induce innate aversion under sated states, whereas such aversive stimuli transform into appetitive ones under sodium depletion. Neural mechanisms underlying this state-dependent salt valence switch are poorly understood. Using transcriptomics state-to-cell-type mapping and neural manipulations, we show that positive and negative valences of salt are controlled by anatomically distinct neural circuits in the mammalian brain. The hindbrain interoceptive circuit regulates sodium-specific appetitive drive , whereas behavioral tolerance of aversive salts is encoded by a dedicated class of neurons in the forebrain lamina terminalis (LT) expressing prostaglandin E2 (PGE2) receptor, Ptger3. We show that these LT neurons regulate salt tolerance by selectively modulating aversive taste sensitivity, partly through a PGE2-Ptger3 axis. These results reveal the bimodal regulation of appetitive and tolerance signals toward salt, which together dictate the amount of sodium consumption under different internal states.

Cooperative assembly confers regulatory specificity and long-term genetic circuit stability.

A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits "wired" using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.

Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex.

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.5, they form a multi-layered circuit motif, composed of only embryonic near-projecting-type neurons. By E17.5, this transitions to a second motif involving all three embryonic types, analogous to the three adult layer 5 types. In vivo patch clamp recordings and two-photon calcium imaging of embryonic Rbp4-Cre neurons reveal active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses, from E14.5 onwards. Embryonic Rbp4-Cre neurons strongly express autism-associated genes and perturbing these genes interferes with the switch between the two motifs. Hence, pyramidal neurons form active, transient, multi-layered pyramidal-to-pyramidal circuits at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

A brainstem integrator for self-location memory and positional homeostasis in zebrafish.

To track and control self-location, animals integrate their movements through space. Representations of self-location are observed in the mammalian hippocampal formation, but it is unknown if positional representations exist in more ancient brain regions, how they arise from integrated self-motion, and by what pathways they control locomotion. Here, in a head-fixed, fictive-swimming, virtual-reality preparation, we exposed larval zebrafish to a variety of involuntary displacements. They tracked these displacements and, many seconds later, moved toward their earlier location through corrective swimming ("positional homeostasis"). Whole-brain functional imaging revealed a network in the medulla that stores a memory of location and induces an error signal in the inferior olive to drive future corrective swimming. Optogenetically manipulating medullary integrator cells evoked displacement-memory behavior. Ablating them, or downstream olivary neurons, abolished displacement corrections. These results reveal a multiregional hindbrain circuit in vertebrates that integrates self-motion and stores self-location to control locomotor behavior.

Synthetic mammalian signaling circuits for robust cell population control.

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum-sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed "Paradaux," in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, population control became more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system and demonstrate that paradoxical circuit architectures can provide robust population control.

Modularity and robustness of frontal cortical networks.

Neural activity underlying short-term memory is maintained by interconnected networks of brain regions. It remains unknown how brain regions interact to maintain persistent activity while exhibiting robustness to corrupt information in parts of the network. We simultaneously measured activity in large neuronal populations across mouse frontal hemispheres to probe interactions between brain regions. Activity across hemispheres was coordinated to maintain coherent short-term memory. Across mice, we uncovered individual variability in the organization of frontal cortical networks. A modular organization was required for the robustness of persistent activity to perturbations: each hemisphere retained persistent activity during perturbations of the other hemisphere, thus preventing local perturbations from spreading. A dynamic gating mechanism allowed hemispheres to coordinate coherent information while gating out corrupt information. Our results show that robust short-term memory is mediated by redundant modular representations across brain regions. Redundant modular representations naturally emerge in neural network models that learned robust dynamics.

Local Axonal Conduction Shapes the Spatiotemporal Properties of Neural Sequences.

Sequential activation of neurons has been observed during various behavioral and cognitive processes, but the underlying circuit mechanisms remain poorly understood. Here, we investigate premotor sequences in HVC (proper name) of the adult zebra finch forebrain that are central to the performance of the temporally precise courtship song. We use high-density silicon probes to measure song-related population activity, and we compare these observations with predictions from a range of network models. Our results support a circuit architecture in which heterogeneous delays between sequentially active neurons shape the spatiotemporal patterns of HVC premotor neuron activity. We gauge the impact of several delay sources, and we find the primary contributor to be slow conduction through axonal collaterals within HVC, which typically adds between 1 and 7.5 ms for each link within the sequence. Thus, local axonal "delay lines" can play an important role in determining the dynamical repertoire of neural circuits.

Defining the Teratoma as a Model for Multi-lineage Human Development.

We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.

From Cell Types to an Integrated Understanding of Brain Evolution: The Case of the Cerebral Cortex.

With the discovery of the incredible diversity of neurons, Cajal and coworkers laid the foundation of modern neuroscience. Neuron types are not only structural units of nervous systems but also evolutionary units, because their identities are encoded in the genome. With the advent of high-throughput cellular transcriptomics, neuronal identities can be characterized and compared systematically across species. The comparison of neurons in mammals, reptiles, and birds indicates that the mammalian cerebral cortex is a mosaic of deeply conserved and recently evolved neuron types. Using the cerebral cortex as a case study, this review illustrates how comparing neuron types across species is key to reconciling observations on neural development, neuroanatomy, circuit wiring, and physiology for an integrated understanding of brain evolution.

Sour Sensing from the Tongue to the Brain.

The ability to sense sour provides an important sensory signal to prevent the ingestion of unripe, spoiled, or fermented foods. Taste and somatosensory receptors in the oral cavity trigger aversive behaviors in response to acid stimuli. Here, we show that the ion channel Otopetrin-1, a proton-selective channel normally involved in the sensation of gravity in the vestibular system, is essential for sour sensing in the taste system. We demonstrate that knockout of Otop1 eliminates acid responses from sour-sensing taste receptor cells (TRCs). In addition, we show that mice engineered to express otopetrin-1 in sweet TRCs have sweet cells that also respond to sour stimuli. Next, we genetically identified the taste ganglion neurons mediating each of the five basic taste qualities and demonstrate that sour taste uses its own dedicated labeled line from TRCs in the tongue to finely tuned taste neurons in the brain to trigger aversive behaviors.

ASICs Mediate Food Responses in an Enteric Serotonergic Neuron that Controls Foraging Behaviors.

Animals must respond to the ingestion of food by generating adaptive behaviors, but the role of gut-brain signaling in behavioral regulation is poorly understood. Here, we identify conserved ion channels in an enteric serotonergic neuron that mediate its responses to food ingestion and decipher how these responses drive changes in foraging behavior. We show that the C. elegans serotonergic neuron NSM acts as an enteric sensory neuron that acutely detects food ingestion. We identify the novel and conserved acid-sensing ion channels (ASICs) DEL-7 and DEL-3 as NSM-enriched channels required for feeding-dependent NSM activity, which in turn drives slow locomotion while animals feed. Point mutations that alter the DEL-7 channel change NSM dynamics and associated behavioral dynamics of the organism. This study provides causal links between food ingestion, molecular and physiological properties of an enteric serotonergic neuron, and adaptive feeding behaviors, yielding a new view of how enteric neurons control behavior.

Synthetic Developmental Biology: Understanding Through Reconstitution.

Reconstitution is an experimental strategy that seeks to recapitulate biological events outside their natural contexts using a reduced set of components. Classically, biochemical reconstitution has been extensively applied to identify the minimal set of molecules sufficient for recreating the basic chemistry of life. By analogy, reconstitution approaches to developmental biology recapitulate aspects of developmental events outside an embryo, with the goal of revealing the basic genetic circuits or physical cues sufficient for recreating developmental decisions. The rapidly growing repertoire of genetic, molecular, microscopic, and bioengineering tools is expanding the complexity and precision of reconstitution experiments. We review the emerging field of synthetic developmental biology, with a focus on the ways in which reconstitution strategies and new biological tools have enhanced our modern understanding of fundamental questions in developmental biology.

A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster.

Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. VIDEO ABSTRACT.

Circuit Design Features of a Stable Two-Cell System.

Cell communication within tissues is mediated by multiple paracrine signals including growth factors, which control cell survival and proliferation. Cells and the growth factors they produce and receive constitute a circuit with specific properties that ensure homeostasis. Here, we used computational and experimental approaches to characterize the features of cell circuits based on growth factor exchange between macrophages and fibroblasts, two cell types found in most mammalian tissues. We found that the macrophage-fibroblast cell circuit is stable and robust to perturbations. Analytical screening of all possible two-cell circuit topologies revealed the circuit features sufficient for stability, including environmental constraint and negative-feedback regulation. Moreover, we found that cell-cell contact is essential for the stability of the macrophage-fibroblast circuit. These findings illustrate principles of cell circuit design and provide a quantitative perspective on cell interactions.