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During infection, animals exhibit adaptive changes in physiology and behavior aimed at increasing survival. Although many causes of infection exist, they trigger similar stereotyped symptoms such as fever, warmth seeking, loss of appetite and fatigue(1,2). Yet exactly how the nervous system alters body temperature and triggers sickness behaviors to coordinate responses to infection remains unknown. Here we identify a previously uncharacterized population of neurons in the ventral medial preoptic area (VMPO) of the hypothalamus that are activated after lipopolysaccharide (LPS)- and Poly (I:C)- induced sickness and are critical for generating a fever response and other sickness symptoms such as warmth-seeking and loss of appetite. Single-nucleus RNA-sequencing and multiplexed error-robust fluorescent in situ hybridization (MERFISH) uncovered the identity and distribution of LPS-activated VMPO (VMPO(LPS)) neurons and non-neuronal cells. Gene expression and electrophysiological measurements suggest a paracrine mechanism in which the release of immune signals by non-neuronal cells during infection activates nearby VMPO(LPS) neurons. Finally, we show that VMPO(LPS) neurons exert a broad influence on the activity of brain areas associated with behavioral and homeostatic functions and are synaptically and functionally connected to circuit nodes controlling body temperature and appetite. Together these results uncover VMPO(LPS) neurons as a control hub that integrates immune signals to orchestrate multiple sickness symptoms in response to infection.

Elucidation of the mechanisms of T-cell–mediated antitumor responses will provide information for the rational design and development of cancer immunotherapies. Here, we found that calnexin, an endoplasmic reticulum (ER) chaperone protein, is significantly upregulated in oral squamous cell carcinoma (OSCC). Upregulation of its membranous expression on OSCC cells is associated with inhibited T-cell infiltration in tumor tissues and correlates with poor survival of patients with OSCC. We found that calnexin inhibits the proliferation of CD4+ and CD8+ T cells isolated from the whole blood of healthy donors and patients with OSCC and inhibits the secretion of IFNγ, TNFα, and IL2 from these cells. Furthermore, in a melanoma model, knockdown of calnexin enhanced the infiltration and effector functions of T cells in the tumor microenvironment and conferred better control of tumor growth, whereas treatment with a recombinant calnexin protein impaired the infiltration and effector functions of T cells and promoted tumor growth. We also found that calnexin enhanced the expression of PD-1 on CD4+ and CD8+ T cells by restraining the DNA methylation status of a CpG island in the PD-1 promoter. Thus, this work uncovers a mechanism by which T-cell antitumor responses are regulated by calnexin in tumor cells and suggests that calnexin might serve as a potential target for the improvement of antitumor immunotherapy.

How does wiring specificity of neural maps emerge during development? Formation of the adultDrosophilaolfactory glomerular map begins with patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type–specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon– and adaptive optical lattice light-sheet microscope–based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.

Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. Here, we present the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole agingDrosophila. We characterize 163 distinct cell types and perform an in-depth analysis of changes in tissue cell composition, gene expression, and cell identities. We further develop aging clock models to predict the fly age and show that ribosomal gene expression is a conserved predictive factor for age. Combining all aging features, we find unique cell type-specific aging patterns. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.

The function of serotonin in the mammalian brain has been challenging to unravel. In this issue of Neuron, Paquelet et al. (2022) employ microendoscopy to record over 2,000 dorsal raphe serotonin neurons, yielding new insights into their activity from the single neuron to the population level.

Selective separation of pyrite and galena using 2-mercaptobenzimidazole (MBI) as a chelating collector, was investigated by flotation, scanning electron microscope (SEM), Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) calculations. Single-mineral flotation showed a significant flotability of pyrite at pH = 2–4 and galena at pH = 8–10 in the presence of MBI. The depressant effect of pyrite was observed to be more pronounced under alkaline conditions. Artificially mixed minerals flotation suggested the galena-pyrite could be separated under alkaline conditions, especially at pH = 10. SEM analysis indicates that the adsorption of MBI on galena surface is more intense than pyrite. However, the interaction between MBI and pyrite is stronger than that of MBI-galena. FT-IR analysis shows the iron and lead ions may chelate with -N = and -SH groups of MBI. Pyrite contains more iron hydroxide (or sulfate forms) in solution from XPS analysis, only half of the metallic ions on the surface function as active sites, thus reducing the amount of metal that can be chelated with MBI. DFT calculations revealed that MBI was often in the molecular form under acidic mediums and the ionic form under alkaline condition, while the ionic form of MBI was more active.

Iron in refractory iron ore composed of 47.04% Fe, 0.39% Pb and 0.30% Zn was enriched through reduction roasting followed by magnetic separation in this work. The phase transformations occurring in iron ore during reduction roasting were investigated through XRD, VSM, and SEM-EDS. Results show reduction process of hematite follows Fe2O3 → Fe3O4 → FeO → Fe. The quartz in iron ore reacts with FeO to form fayalite. At elevated temperatures, fayalite decomposes into metallic iron and cristobalite solid solution. The reductively roasted product (RRP) with main phases of magnetite and metallic iron has higher magnetization intensity than that with main phases of wustite and fayalite. Iron in RRP-800 °C and RRP-1150 °C is extracted by magnetic separation, and obtained concentrates have main minerals of magnetite and metallic iron, respectively. Elevated temperature decreases zinc and lead contents in concentrate. Thus, deep reduction-magnetic separation is more suitable for enriching iron from Zn- and Pb-bearing refractory iron ore.

Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in diverse tissue contexts, however, their spatial resolution is limited by array density. We present Expansion Spatial Transcriptomics (Ex-ST) to overcome this limitation by clearing and expanding tissue prior to capturing the entire transcriptome. This approach allows us to achieve near cellular resolution and higher capture efficiency of lowly-expressed genes, which we demonstrate using mouse brain samples.

Hyperexcitability of brain circuits is a common feature of autism spectrum disorders (ASDs). Genetic deletion of a chromatin-binding protein, retinoic acid induced 1 ( RAI1 ), causes Smith–Magenis syndrome (SMS). SMS is a syndromic ASD associated with intellectual disability, autistic features, maladaptive behaviors, overt seizures, and abnormal electroencephalogram (EEG) patterns. The molecular and neural mechanisms underlying abnormal brain activity in SMS remain unclear. Here we show that panneural Rai1 deletions in mice result in increased seizure susceptibility and prolonged hippocampal seizure duration in vivo and increased dentate gyrus population spikes ex vivo. Brain-wide mapping of neuronal activity pinpointed selective cell types within the limbic system, including the hippocampal dentate gyrus granule cells (dGCs) that are hyperactivated by chemoconvulsant administration or sensory experience in Rai1 -deficient brains. Deletion of Rai1 from glutamatergic neurons, but not from gamma-aminobutyric acidergic (GABAergic) neurons, was responsible for increased seizure susceptibility. Deleting Rai1 from the Emx1 Cre -lineage glutamatergic neurons resulted in abnormal dGC properties, including increased excitatory synaptic transmission and increased intrinsic excitability. Our work uncovers the mechanism of neuronal hyperexcitability in SMS by identifying Rai1 as a negative regulator of dGC intrinsic and synaptic excitability.

The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2’s tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2’s role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.

Mosquitoes are strongly attracted to humans, and their bites not only cause intense itch but can beget severe diseases. In this issue of Cell, Herre et al. reveal that non-canonical olfactory circuit organization and coding likely endow mosquitoes with a robust ability to locate human hosts.

Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless ( fru ) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 ( Hnf4 ) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C ( RhoGEF64c ), Frequenin 2 ( Frq2 ), Prip , and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.

Social interactions require awareness and understanding of the behavior of others. Mirror neurons, cells representing an action by self and others, have been proposed to be integral to the cognitive substrates that enable such awareness and understanding. Mirror neurons of the primate neocortex represent skilled motor tasks, but it is unclear if they are critical for the actions they embody, enable social behaviors, or exist in non-cortical regions. We demonstrate that the activity of individual VMHvlPR neurons in the mouse hypothalamus represents aggression performed by self and others. We used a genetically encoded mirror-TRAP strategy to functionally interrogate these aggression-mirroring neurons. We find that their activity is essential for fighting and that forced activation of these cells triggers aggressive displays by mice, even toward their mirror image. Together, we have discovered a mirroring center in an evolutionarily ancient region that provides a subcortical cognitive substrate essential for a social behavior.

Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

Variations in neuronal connectivity occur widely in nervous systems from invertebrates to mammals. Yet, it is unclear how neuronal variability originates, to what extent and at what time scales it exists, and what functional consequences it might carry. To assess inter- and intraindividual neuronal variability, it would be ideal to analyze the same identified neuron across different brain hemispheres and individuals. Here, using genetic labeling and electron microscopy connectomics, we show that an identified inhibitory olfactory local interneuron, TC-LN, exhibits extraordinary variability in its glomerular innervation patterns. Moreover, TC-LN’s innervation of the VL2a glomerulus, which processes food signals and modulates mating behavior, is sexually dimorphic, is influenced by female’s courtship experience, and correlates with food intake in mated females. Mating also affects output connectivity of TC-LN to specific local interneurons. We propose that mating-associated variability of TC-LNs regulates how food odor is interpreted by an inhibitory network to modulate feeding.

The ability to obtain single cell transcriptomes for stable cell types and dynamic cell states is ushering in a new era for biology. We created the Tabula Drosophilae, a single cell atlas of the adult fruit fly which includes 580k cells from 15 individually dissected sexed tissues as well as the entire head and body. Over 100 researchers from the fly community contributed annotations to >250 distinct cell types across all tissues. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types that are shared between tissues, such as blood and muscle cells, allowed the discovery of rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the entire Drosophila community and serves as a comprehensive reference to study genetic perturbations and disease models at single cell resolution.

Transcription factors are central commanders specifying cell fate, morphology, and physiology while cell-surface proteins execute these commands through interaction with cellular environment. In developing neurons, it is presumed that transcription factors control wiring specificity through regulation of cell-surface protein expression. However, the number and identity of cell-surface protein(s) a transcription factor regulates remain largely unclear1,2. Also unknown is whether a transcription factor regulates the same or different cell-surface proteins in different neuron types to specify their connectivity. Here we use a lineage-defining transcription factor, Acj6 (ref. 3), to investigate how it controls precise dendrite targeting of Drosophila olfactory projection neurons (PNs). Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion proteins and proteins previously not associated with wiring, such as the mechanosensitive ion channel Piezo—whose channel activity is dispensable for its wiring function. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combinatorial expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, a key transcription factor controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.

SUMMARYTranscription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combinatorial expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell- surface executors.

Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.