Biology subjects

Lemire, A. L.

Publications and source records attributed to Lemire, A. L..

12 recordsLinked to original sources

bioRxiv2025

Imaging cellular activity simultaneously across all organs of a vertebrate reveals body-wide circuits

All cells in an animal collectively ensure, moment-to-moment, the survival of the whole organism in the face of environmental stressors1,2. Physiology seeks to elucidate the intricate network of interactions that sustain life, which often span multiple organs, cell types, and timescales, but a major challenge lies in the inability to simultaneously record time-varying cellular activity throughout the entire body. We developed WHOLISTIC (WHole Organism Live Imaging System for recording Tissue and IntraCellular activity), a method to image second-timescale, time-varying intracellular dynamics across cell-types of the vertebrate body. By advancing and integrating volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors in transparent young Danio rerio (zebrafish) and with proof of concept in adult Danionella, the method enables real-time recording of cellular dynamics across the organism. Calcium is a universal intracellular messenger, with a large array of cellular processes depending on changes in calcium concentration across varying time-scales, making it an ideal proxy of cellular activity3. Using this platform to screen the dynamics of most cells in the body, we discovered unexpected responses of specific cell types to stimuli, such as chondrocyte reactions to cold, meningeal responses to ketamine, and state-dependent activity, such as oscillatory ependymal-cell activity during periods of extended motor quiescence. At the organ scale, the method uncovered pulsating traveling waves along the kidney nephron. At the multi-organ scale, we uncovered muscle synergies and independencies, as well as muscle-organ interactions. Integration with optogenetics allowed us to all-optically determine the causal direction of brain-body interactions. At the whole-organism scale, the method captured the rapid brainstem-controlled redistribution of blood flow across the body. Finally, we advanced Whole-Body Expansion Microscopy4 to provide ground-truth molecular and ultrastructural anatomical context, explaining the spatiotemporal structure of activity captured by WHOLISTIC. Together, these innovations establish a new paradigm for systems biology, bridging cellular and organismal physiology, with broad implications for both fundamental research and drug discovery.

neuroscience↗

bioRxiv2023

Programmed withdrawal of cilia maintenance followed by centriole capping leads to permanent cilia loss during cerebellar granule cell neurogenesis

Primary cilia in brain neurons provide a privileged compartment for binding and responding to extracellular ligands such as sonic hedgehog. Paradoxically, cilia in differentiating cerebellar granule cells are deconstructed during neurogenesis. To identify mechanisms underlying this newly defined cilia deconstruction pathway, we used single cell transcriptomic and immunocytological analyses to compare the transcript and protein signatures of differentiating and progenitor granule cells. We found that differentiating granule cells lacked transcripts for key regulators of pre-mitotic cilia resorption, suggesting cilia disassembly in differentiating cells was distinct from pre-mitotic cilia resorption. Further analysis revealed that during differentiation, transcription of genes required for cilia maintenance decreased. Specifically, protein components of intraflagellar transport complexes, pericentrosomal material and centriolar satellites all decreased as granule cells matured. The changes in transcription and translation correlated with the downregulation of sonic hedgehog signaling at the onset of differentiation. We also found binding of centriolar cap proteins to the mother centrioles as granule cell neurons matured. These data indicate that global, developmentally programmed, diminution of cilium maintenance caused cilia deconstruction in differentiating granule cells. Furthermore, the capping of docked mother centrioles prevents cilia regrowth likely blocking dysregulated sonic hedgehog signaling and tumorigenesis.

cell biology↗

bioRxiv2022

Spatial organization of the 3D genome encodes gene co-expression programs in single cells

Deconstructing the mechanism by which the 3D genome encodes genetic information to generate diverse cell types during animal development is a major challenge in biology. The contrast between the elimination of chromatin loops and domains upon Cohesin loss and the lack of downstream gene expression changes at the cell population level instigates intense debates regarding the structure-function relationship between genome organization and gene regulation. Here, by analyzing single cells after acute Cohesin removal with sequencing and spatial genome imaging techniques, we discover that, instead of dictating population-wide gene expression levels, 3D genome topology mediated by Cohesin safeguards long-range gene co-expression correlations in single cells. Notably, Cohesin loss induces gene co-activation and chromatin co-opening between active domains in cis up to tens of megabase apart, far beyond the typical length scale of enhancer-promoter communication. In addition, Cohesin separates Mediator protein hubs, prevents active genes in cis from localizing into shared hubs and blocks intersegment transfer of diverse transcriptional regulators. Together, these results support that spatial organization of the 3D genome orchestrates dynamic long-range gene and chromatin co-regulation in single living cells.

genomics↗

bioRxiv2022

Multimodal mapping of cell types and projections in the central nucleus of the amygdala

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-Seq) to classify molecularly defined cell types in the CEA and identified marker-genes to map the location of these neuronal types using expansion assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of [~]30,000 molecularly defined CEA neurons. Our study revealed spatio-molecular organization of the CEA, with medial and lateral CEA associated with distinct cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker-gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

neuroscience↗

bioRxiv2022

Sharp cell-type-identity changes differentiate the retrosplenial cortex from the neocortex

The laminae of the neocortex are fundamental processing layers of the mammalian brain. Notably, such laminae are believed to be relatively stereotyped across short spatial scales, such that shared laminae between nearby brain regions exhibit similar constituent cells. Here, we considered a potential exception to this rule by studying the retrosplenial cortex (RSC), a brain region known for sharp cytoarchitectonic differences across its granular-dysgranular border. Using a variety of transcriptomics techniques, we identified, spatially mapped, and interpreted the excitatory cell-type landscape of the mouse RSC. In doing so, we surprisingly uncovered that RSC gene expression and cell types change sharply at the granular-dysgranular border. Additionally, supposedly homologous laminae between the RSC and neocortex are effectively wholly distinct in their cell-type composition. In collection, the RSC exhibits a variety of intrinsic cell-type specializations, and embodies a new neocortical organizational principle wherein cell-type identities vary sharply within and between brain regions.

neuroscience↗

bioRxiv2021

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased {Delta}F/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

neuroscience↗

bioRxiv2021

A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility

Chemical synapses between axons and dendrites mediate much of the brains intercellular communication. Here we describe a new kind of synapse - the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam - scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

cell biology↗

bioRxiv2021

Spatially patterned excitatory neuron subtypes and circuits within the claustrum

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow region that primarily projects to the neocortex, whereas circuit-based approaches suggest a broader region embedding neocortical and other neural circuits. Here, we took a bottom-up, cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum is comprised of two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential projection targets. Thus, the claustrum is comprised of excitatory neuron subtypes with distinct molecular and circuit properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.

neuroscience↗

bioRxiv2021

Expansion-Assisted Iterative-FISH defines lateral hypothalamus spatio-molecular organization

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 {micro}m) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization. Highlights- EASI-FISH enables robust gene expression profiling in thick brain slices - A turnkey analysis pipeline for facile analysis of large EASI-FISH image datasets - EASI-FISH reveals novel subregions of the lateral hypothalamus - Identification of rare cell types based on morphological and spatial heterogeneity

neuroscience↗

bioRxiv2020

A novel family of secreted proteins linked to plant gall development

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls, and that this results in differential expression of a small number of plant genes. Dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. Bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. Bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants. One Sentence SummaryAphid bicycle genes, which encode diverse secreted proteins, contribute to plant gall development.

genetics↗

bioRxiv2020

The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson's disease

Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinsons disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remains largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes, and we identify the abnormal accumulation of key PD-related proteins within multi vesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

neuroscience↗

bioRxiv2019

Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics

Animals employ multiple and distributed neuronal networks with diverse learning rules and synaptic plasticity dynamics to record distinct temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso & Rubin 2016). Here we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NOs effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

neuroscience↗