Biology subjects

Hammarlund, M.

Publications and source records attributed to Hammarlund, M..

At least 19 recordsLinked to original sources

bioRxiv2025

A gene expression atlas of a juvenile nervous system

Although the fundamental architecture of metazoan nervous systems is typically established in the embryo, substantial numbers of neurons are added during post-natal development while existing neurons expand in size, refine connectivity, and undergo additional differentiation. To reveal the underlying molecular determinants of postembryonic neurogenesis and maturation, we have produced gene expression profiles of all neuron types and their progenitors in the first larval stage (L1) of C. elegans. Comparisons of the L1 profile to the embryo and to the later L4 larval stage identified thousands of differentially expressed genes across individual neurons throughout the nervous system. Key neuropeptide signaling networks, for example, are remodeled during larval development. Gene regulatory network analysis revealed potential transcription factors driving the temporal changes in gene expression across the nervous system, including a broad role for the heterochronic transcription factor lin-14. We utilized available connectomic data of juvenile animals in combination with our neuron-specific atlas to identify potential molecular determinants of membrane contact and synaptic connectivity. These expression data are available through a user-friendly interface at CeNGEN.org for independent investigations of the maturation, connectivity and function of a developing nervous system.

neuroscience↗

bioRxiv2025

Tubulin autoregulation factors SCAPER and TTC5 recruit γ-tubulin to non-centrosomal MTOCs for neuronal microtubule nucleation and axon regeneration

Neuronal function and survival depend on highly stereotyped non-centrosomal microtubule (MT) arrays. How these arrays form remains poorly understood. Here we identified a role for SCAPER and TTC5, two factors previously implicated in tubulin mRNA autoregulation, in controlling neuronal MT content through {gamma}-tubulin-dependent nucleation. In C. elegans neurons, loss of scpr-1, ttc-5, or both reduced MT numbers to a similar degree as depletion of {gamma}-tubulin, the main MT nucleator. Using conditional single-cell degradation alleles and endogenous tagging, we find that {gamma}-tubulin nucleates MTs in the neuronal cell body from endosomal puncta, and that scpr-1 and ttc-5 are required to recruit {gamma}-tubulin to these structures. SCPR-1 is also instructive, as its overexpression drastically increases {gamma}-tubulin levels and enhances MT density. We propose that these mechanisms are conserved since human SCAPER rescues C. elegans mutants, and SCAPER knockdown in rat hippocampal neurons reduces both {gamma}-tubulin clustering at presynaptic sites and activity-dependent synaptic MT nucleation. Finally, while scpr-1, ttc-5, and{gamma} -tubulin are not required for developmental axon elongation, they are essential for regeneration, where SCPR-1 directs {gamma}-tubulin to the growth cone to facilitate regrowth following injury. These findings reveal mechanisms governing neuronal cytoskeleton assembly and function, and suggest potential crosstalk between tubulin autoregulation and microtubule nucleation.

cell biology↗

bioRxiv2025

Systematic identification of oscillatory gene expression in single cell types

Many biological cycles are driven by oscillatory gene expression coordinated across cell types. For example, larval development in Caenorhabditis elegans involves coordinated cyclic changes in cell division, behavior, and growth, the latter requiring production of a structured extracellular matrix called the cuticle. Here, we combine single-cell RNA sequencing and novel computational approaches to identify oscillatory gene expression in individual cell types. We find that many cell types exhibit looping structures in PCA and UMAP space that correspond to transcriptional oscillations at each larval stage. Oscillatory gene expression is found in all cuticle-producing cell types, including glia, but not detected in neurons or muscle. We develop rigorous statistical approaches for de novo identification of oscillatory genes and cell types, yielding >5,000 genes. While many oscillatory genes relate to cuticle production, each cell type expresses largely distinct genes, suggesting that cuticle production is a patchwork of cell-type-specific programs. Finally, we derive a potential set of regulatory transcription factors that can explain coordinated oscillatory gene expression and find that shared upstream factors likely control gene timing across cell types. Together, our results suggest that shared regulators control cell-type-specific oscillatory gene expression, including in previously overlooked cell types such as glia.

genomics↗

bioRxiv2025

CellSpliceNet: Interpretable Multimodal Modeling ofAlternative Splicing Across Neurons in C. elegans

Alternative splicing profoundly diversifies the transcriptome and proteome, but decoding its regulatory mechanisms remains a challenge. We introduce CellSpliceNet, an interpretable transformer-based multimodal deep learning framework designed to predict splicing outcomes across the neurons of C. elegans. By integrating four complementary data modalities, namely long-range genomic sequence, local regions of interest (ROIs) in the RNA sequence, secondary structure, and gene expression, CellSpliceNet captures the complex interplay of factors that influence splicing decisions within the cellular context. CellSpliceNet employs modality-specific transformer embeddings, incorporating structural representations guided by mutual information and scattering graph embeddings. To this end, a novel and carefully designed multimodal multi-head attention mechanism preserves the integrity of each modality while facilitating selective cross-modal interactions, notably allowing gene expression data to inform sequence and structural predictions. Attention-based pooling within each modality highlights biologically critical elements, such as canonical intron-exon splice boundaries and accessible single-stranded RNA loop structures within the exon. Quantitative comparisons with current state-of-the-art methods demonstrated CellSpliceNet s superior predictive accuracy (Spearman{rho} = 0.88) and high accuracy across diverse neuron subtypes. Furthermore, CellSpliceNet elucidates a hierarchical, neuron-specific splicing code by preferentially weighting upstream enhancer motifs (e.g., GGAAGAAC) and identifying neuron-class-specific splicing-factor signatures, including smu-1, unc-75, and hrp-1. Thus, CellSpliceNet not only advances the frontiers of alternative splicing predictive capabilities but also provides mechanistic insights into the multimodal regulation of alternative splicing.

genetics↗

bioRxiv2025

The neuron-intrinsic membrane skeleton is required for motor neuron integrity throughout lifespan

Axons experience physical stress throughout an organisms lifetime, and disruptions in axonal integrity are hallmarks of both neurodegenerative diseases and traumatic injuries. The spectrin-based membrane periodic skeleton (MPS) is proposed to have a crucial role in maintaining axonal strength, flexibility, and resilience. To investigate the importance of the intrinsic MPS for GABAergic motor neuron integrity in C. elegans, we employed the auxin-inducible degron system to degrade {beta}-spectrin/UNC-70 in a cell-type specific and time-dependent manner. Degradation of {beta}-spectrin from all neurons beginning at larval development resulted in widespread axon breakage and regeneration in VD/DD GABAergic motor neurons in both larval and adult animals. Similarly, targeted degradation of {beta}-spectrin in GABA neurons alone resulted in extensive breakage. Moreover, we found that depleting {beta}-spectrin from the mature nervous system also induced axon breaks. By contrast, epidermal {beta}-spectrin was not required for axon integrity of VD/DD neurons. These findings demonstrate the cell-intrinsic importance of neuronal {beta}-spectrin/UNC-70 for axon integrity both during development and in adulthood.

neuroscience↗

bioRxiv2025

Integrating bulk and single cell RNA-seq refines transcriptomic profiles of individual C. elegans neurons

Neuron-specific morphology and function are fundamentally tied to differences in gene expression across the nervous system. We previously generated a single cell RNA-seq (scRNA-Seq) dataset for every anatomical neuron class in the C. elegans hermaphrodite. Here we present a complementary set of bulk RNA-seq samples for 52 of the 118 canonical neuron classes in C. elegans. We show that the bulk RNA-seq dataset captures both lowly expressed and noncoding RNAs that are not detected in the scRNA-Seq profile, but also includes false positives due to contamination by other cell types. We present an analytical strategy that integrates the two datasets, preserving both the specificity of scRNA-Seq data and the sensitivity of bulk RNA-Seq. We show that this integrated dataset enhances the sensitivity and accuracy of transcript detection and differential gene analysis. In addition, we show that the bulk RNA-Seq data set detects differentially expressed non-coding RNAs across neuron types, including multiple families of non-polyadenylated transcripts. We propose that our approach provides a new strategy for interrogating gene expression by bridging the gap between bulk and single cell methodologies for transcriptomic studies. We suggest that these datasets advance the goal of delineating the mechanisms that define morphology and connectivity in the nervous system.

genomics↗

bioRxiv2024

Cytoplasmic ribosomes hitchhike on mitochondria to dendrites

Neurons rely on local protein synthesis to rapidly modify the proteome of neurites distant from the cell body. A prerequisite for local protein synthesis is the presence of ribosomes in the neurite, but the mechanisms of ribosome transport in neurons remain poorly defined. Here, we find that ribosomes hitchhike on mitochondria for their delivery to the dendrite of a sensory neuron in C. elegans. Ribosomes co-transport with dendritic mitochondria, and their association requires the atypical Rho GTPase MIRO-1. Disrupting mitochondrial transport prevents ribosomes from reaching the dendrite, whereas ectopic re-localization of mitochondria results in a concomitant re-localization of ribosomes, demonstrating that mitochondria are required and sufficient for instructing ribosome distribution in dendrites. Endolysosomal organelles that are involved in mRNA transport and translation can associate with mitochondria and ribosomes but do not play a significant role in ribosome transport. These results reveal a mechanism for dendritic ribosome delivery, which is a critical upstream requirement for local protein synthesis.

cell biology↗

bioRxiv2024

Alternative splicing across the C. elegans nervous system

Alternative splicing is a key mechanism that shapes neuronal transcriptomes, helping to define neuronal identity and modulate function. Here, we present an atlas of alternative splicing across the nervous system of Caenorhabditis elegans. Our analysis identifies novel alternative splicing in key neuronal genes such as unc-40/DCC and sax-3/ROBO. Globally, we delineate patterns of differential alternative splicing in almost 2,000 genes, and estimate that a quarter of neuronal genes undergo differential splicing. We introduce a web interface for examination of splicing patterns across neuron types. We explore the relationship between neuron type and splicing patterns, and between splicing patterns and differential gene expression. We identify RNA features that correlate with differential alternative splicing, and describe the enrichment of microexons. Finally, we compute a splicing regulatory network that can be used to generate hypotheses on the regulation and targets of alternative splicing in neurons.

neuroscience↗

bioRxiv2023

Programmed Cell Death Modifies Neural Circuits and Tunes Intrinsic Behavior

Programmed cell death (PCD) is a common feature of animal development. During development of the C. elegans hermaphrodite, programmed cell death eliminates 131 cells in stereotyped positions in the cell lineage, mostly in neuronal lineages. Blocking cell death results in supernumerary "undead" neurons. We find that undead neurons can be wired into circuits, can display activity, and can modify specific behaviors. The two undead RIM-like neurons participate in the RIM-containing circuit that computes movement. The presence of these two extra neurons results in animals that initiate fewer reversals and lengthens the duration of those reversals that do occur. We describe additional behavioral alterations of cell-death mutants, including in locomotory turning angle and pharyngeal pumping. These findings indicate that physiological or evolutionary variations in PCD might reveal latent neuronal elements that the nervous system can incorporate to modify nervous system function and animal behavior.

neuroscience↗

bioRxiv2023

Glycolytic Metabolon Assembly on Mitochondria via Hexokinase O-GlcNAcylation Promotes Metabolic Efficiency

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1s regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1s mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1s O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.

cell biology↗

bioRxiv2023

Local and dynamic regulation of neuronal glycolysis in vivo

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use in C. elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapes in vivo. Significance statementWhile it is generally accepted that energy metabolism underpins neuronal function, how it is distributed and dynamically regulated in different tissues of the brain to meet varying energy demands is not well understood. Here we utilized a fluorescent biosensor, HYlight, to observe glycolytic metabolism at cellular and subcellular scales in vivo. By leveraging both the stereotyped identities of individual neurons in C. elegans, and genetic tools for manipulating glycolytic metabolism, we determined that neurons perform and dynamically regulate glycolysis to match changing cellular demands for energy. Our findings support a model whereby glycolytic states should be considered distinct and related to individual neuron identities in vivo, and introduce new questions about the interconnected nature of metabolism and neuronal function.

neuroscience↗

bioRxiv2023

Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport

Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport. Summary statementAnterograde transport of axonal mitochondria is critical for maintenance of the mitochondria pool and neuronal health. Wu et al. show that the endogenous kinesin-1 and RIC-7 localize at the leading end of mitochondria to drive axonal anterograde transport in vivo.

cell biology↗

bioRxiv2022

The neuropeptidergic connectome of C. elegans

SummaryEfforts are currently ongoing to map synaptic wiring diagrams or connectomes in order to understand the neural basis of brain function. However, chemical synapses represent only one type of functionally important neuronal connection; in particular, extrasynaptic, "wireless" signaling by neuropeptides is widespread and plays essential roles in all nervous systems. By integrating single-cell anatomical and gene expression datasets with a biochemical analysis of receptor-ligand interactions, we have generated a draft connectome of neuropeptide signaling in the C. elegans nervous system. This connectome is characterized by a high connection density, extended signaling cascades, autocrine foci, and a decentralized topology, with a large, highly interconnected core containing three constituent communities sharing similar patterns of input connectivity. Intriguingly, several of the most important nodes in this connectome are little-studied neurons that are specialized for peptidergic neuromodulation. We anticipate that the C. elegans neuropeptidergic connectome will serve as a prototype to understand basic organizational principles of neuroendocrine signaling networks.

neuroscience↗

bioRxiv2022

Scaled-expansion of the membrane associated cytoskeleton requires conserved kinesin adaptors

A periodic lattice of actin rings and spectrin tetramers scaffolds the axonal membrane. How spectrin is delivered to this structure to scale its size to that of the growing axon is unknown. We found that endogenous spectrin, visualized with singe axon resolution in vivo, is delivered to hotspots in the lattice that support its expansion at rates set by axon stretch-growth. Unlike other cytoskeletal proteins, whose apparent slow movement consists of intermittent bouts of fast movements, spectrin moves slowly and processively. We identified a pair of coiled coil proteins that mediate this slow movement and the expansion of the lattice by linking spectrin to kinesin-1. Thus, processive slow transport and local lattice incorporation support scaled cytoskeletal expansion during axon stretch-growth. One-Sentence SummaryKinesin adaptors control spectrin transport and expansion of the membrane periodic skeleton.

cell biology↗

bioRxiv2022

Integrating bulk and single cell RNA-seq refines transcriptomic profiles of specific C. elegans neurons.

genetics↗

bioRxiv2021

Activation of the CaMKII-Sarm1-ASK1 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria

Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In C. elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 protects against degeneration through the conserved Sarm1/TIR-1-ASK1/NSY-1 MAPK pathway. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated in this context, and uncover a surprising neuroprotective role for the Sarm1-ASK1 pathway.

neuroscience↗

bioRxiv2021

An optimized ribodepletion approach for C. elegans RNA-sequencing libraries

A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in C. elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Further, some common resources, such as sequence specific kits for removing ribosomal RNA, are not optimized for non-mammalian samples. To optimize library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.

genomics↗

bioRxiv2020

Molecular topography of an entire nervous system

Nervous systems are constructed from a deep repertoire of neuron types but the underlying gene expression programs that specify individual neuron identities are poorly understood. To address this deficit, we have produced an expression profile of all 302 neurons of the C. elegans nervous system that matches the single cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses unique codes of [~]23 neuropeptide-encoding genes and [~]36 neuropeptide receptors thus pointing to an expansive "wireless" signaling network. To demonstrate the utility of this uniquely comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression across the nervous system and (2) reveal adhesion proteins with potential roles in synaptic specificity and process placement. These data are available at cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity and function throughout the C. elegans nervous system.

neuroscience↗