Biology subjects

Call, C. L.

Publications and source records attributed to Call, C. L..

9 recordsLinked to original sources

bioRxiv2026

All-in-one, Cas13d-based cell-specific gene knockdown system for zebrafish

Cell type-specific genetic manipulation is essential for dissecting neural circuits and glial biology in vivo; however, methods for achieving and validating cell-specific gene knockdown in zebrafish remain limited. Here, we introduce a CRISPR-Cas13d-based mRNA knockdown platform for zebrafish that leverages RNA targeting, rather than DNA editing, to enable efficient and flexible perturbation of gene function in defined cell types of interest. We engineered an all-in-one vector system for cell type-specific RfxCas13d expression and ubiquitous expression of crispr RNAs (crRNAs) and validated its utility across the major classes of CNS glia: astrocytes, oligodendrocytes, and microglia. Cas13d-mediated knockdown consistently achieved target mRNA depletion and produced heritable reproducible morphological and functional phenotypes, demonstrating the robustness of the approach. This approach complements permanent genome-editing methods such as CRISPR-Cas9 by providing a reliable, efficient, and scalable strategy for RNA-level perturbation. Our Cas13d toolkit expands the repertoire of zebrafish genetic technologies, offering a powerful resource for in vivo cell type-specific studies to uncover cell-autonomous mechanisms and possibility for cell type-specific genetic screens in development and disease.

neuroscience↗

bioRxiv2026

PIEZOs regulate oligodendrocyte sheath formation, expansion, and myelination potential

Myelination requires precise integration of physical cues by oligodendrocyte lineage cells (OLCs), but the molecular sensors that detect these cues remain incompletely understood. Here, we demonstrate that oligodendrocyte progenitor cells (OPCs) are sensitive to sub-micron changes in membrane displacement. Based on channel properties, RNA expression, and protein abundance, we find that the mechanosensitive ion channel PIEZO1 contributes to OPC mechanosensitivity. In vivo, zebrafish with oligodendrocyte (OL)-specific disruption of piezo1 have fewer sheaths per OL. Zebrafish with OL-specific piezo2 disruption also have fewer sheaths as well as decreased total myelin capacity over time. OL-specific disruption of both piezo1 and piezo2 caused more severe phenotypes, with reduced OPC volume, and in myelinating OLs, reduced sheath number, sheath length, and total myelin output. Furthermore, piezo1/piezo2 disruption leads to sporadic sheath formation outside the normal developmental window. Our findings indicate that OLs use Piezo channels in vivo to influence sheath formation, expansion, and retractions.

neuroscience↗

bioRxiv2026

Pre-myelinating oligodendrocyte ADGRG1 is required for axon ensheathment and CNS myelin formation

Myelin is essential for axonal health and rapid propagation of action potentials. In the central nervous system (CNS), myelination is initiated with axon ensheathment by pre-myelinating oligodendrocytes (preOLs), followed by iterative membrane wrapping and longitudinal extension along the axon. The molecular mechanisms that govern preOL development remain largely unknown. In this study, we identify the adhesion G protein-coupled receptor ADGRG1 (also called GPR56) as a key, evolutionarily conserved regulator of this process. We show that ADGRG1 is highly expressed in preOLs and that its conditional deletion in mouse preOLs leads to simplified preOL morphology and defective axon ensheathment, resulting in CNS hypomyelination. Live imaging in zebrafish demonstrated conserved function of Adgrg1 in axon ensheathment. Mechanistically, we show that ADGRG1 promotes preOL maturation by RhoA activation. Collectively, these findings reveal an ADGRG1-mediated RhoA signaling pathway that governs axon ensheathment and myelin formation.

neuroscience↗

bioRxiv2025

P/Q-type voltage-gated calcium channels regulate calcium signaling and developmental myelination in oligodendrocyte lineage cells

Oligodendrocyte lineage cells (OLCs) are glia that arise as oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS) and may persist as progenitors or differentiate into myelin-producing oligodendrocytes (OLs). OLCs are sensitive to neuronal activity, which can influence myelin formation via activity-dependent myelination. Calcium influx in OLCs regulates many developmental processes, including stabilizing newly formed sheaths. OLCs possess P/Q-type voltage-gated calcium channels (VGCC) that contribute to calcium influx and mutations in these channels are implicated in a spectrum of neurological disorders, yet the functional significance of P/Q-type channels in OLCs is not well understood. In this study, we employ zebrafish to investigate the role of P/Q-type channels in OLCs in vivo during development. We use global and cell-type specific CRISPR/Cas9-mediated genome editing approaches in conjunction with live imaging and physiology to characterize the morphology and signaling properties of OLCs with mutated P/Q-type channel genes. P/Q-type channels are required for normal myelination in the developing CNS and mutants present with decreased amplitude sheath calcium transients, reduced myelin production, shorter myelin sheaths and blebbing membrane structures during development. These findings provide new insight into the role of P/Q-type calcium channels in regulating OLC development and myelination.

neuroscience↗

bioRxiv2025

Activation of cannabinoid receptor CB1 leads to aberrant myelination in development

The endocannabinoid system (ECS) has a widespread role in the development and function of the central nervous system (CNS). Cannabinoid receptors like CB1 and CB2 can be activated with exogenous cannabinoids most popularly known as tetrahydrocannabinol (THC) or cannabis and cannabidiol (CBD). The components of the ECS are expressed early in fetal development, and prenatal exposure to cannabis can lead to structural changes in white matter. White matter is composed of neuronal axons ensheathed in myelin, a lipid-rich insulation that facilitates saltatory conduction and maintains axon integrity. In the CNS, myelin is made by specialized glial cells called oligodendrocytes (OLs), which in addition to neurons also express components of the ECS. However, while several studies have focused on how the ECS regulates neuronal development, there is a limited understanding of its impact on OL development or myelin formation. Therefore, our current study set out to understand how pharmacological activation of the ECS alters OL differentiation and myelin formation in vivo. We administered WIN 55,212-2 (WIN 55), a CB1 and CB2 agonist, to larval zebrafish and longitudinally analyzed OL development and myelination in vivo. Interestingly, we observed an increase in non-axonal ensheathments in the spinal cord, which appeared to be surrounding neuronal cell bodies. These non-axonal ensheathments were dependent on CB1, as the addition of WIN 55 in a global CB1 mutant prevented this phenotype. Furthermore, this ectopic cell body ensheathment occurred independently from normal myelination processes, as individual OLs did not exhibit changes in the number of myelin sheaths, sheath length, or total myelin output. This study shows that activation of CB receptors in vivo leads to increased non-axonal ensheathment without significantly changing OL differentiation or normal myelin formation. Future studies can further investigate the pathways that drive this phenotype to better understand how exogenous cannabinoid activation can regulate the precision of oligodendrocyte ensheathment.

neuroscience↗

bioRxiv2022

Oligodendrocytes form paranodal bridges that generate chains of myelin sheaths vulnerable to degeneration with age

Myelin sheaths in the CNS are generated by the tips of oligodendrocyte processes, which wrap axons to accelerate action potential conduction, provide metabolic support and control excitability. Here we identify a distinct mode of myelination, conserved between zebrafish, mouse and human, in which oligodendrocytes extend myelin along individual axons by linking myelin sheaths across nodes of Ranvier (NoR). By forming thin extensions that cross NoR, which we term paranodal bridges, multiple sheaths can be connected to the soma by a single cytoplasmic process. Extensive in vivo live imaging-based analyses, complemented by serial electron microscopic reconstruction of paranodal bridges, revealed that many oligodendrocytes use this strategy to generate longer stretches of myelin along individual axons. In the mouse somatosensory cortex, paranodal bridges were particularly prevalent along the highly branched axons of parvalbumin expressing (PV) interneurons, which enabled oligodendrocytes to extend myelin sheaths around axon bifurcations. Sheaths at the distal ends of these chains of myelin degenerated more frequently in aged mice, suggesting that they may be more vulnerable to the aging brain environment. This previously undescribed and evolutionarily conserved feature of oligodendrocytes extends myelin coverage of individual axons without new oligodendrogenesis, which may reduce metabolic demand and preserve the fidelity of action potential propagation at axon branch points.

neuroscience↗

bioRxiv2021

Remyelination restores myelin content on distinct neuronal subtypes in the cerebral cortex

Axons in the cerebral cortex exhibit diverse patterns of myelination, with some axons devoid of myelin, some exhibiting discontinuous patches of myelin, and others continuous myelin that is interrupted only by nodes of Ranvier. Oligodendrocytes establish this pattern by sorting through a high density of potential targets to select a small cohort of axons for myelination; however, the myelination patterns established on distinct excitatory and inhibitory neurons within the cortex remain to be fully defined and little is known about the extent to which these patterns are restored after oligodendrocyte regeneration. Here we show that axons in layer I of the somatosensory cortex, a key region for integration of input from local and distant sources, exhibit an extraordinarily diverse range of myelination patterns, even among distinct neuronal subtypes. Although larger axons were more often selected for myelination, neuronal identity profoundly influenced the probability of myelination. The relative differences in myelination among neuron subtypes were preserved between cortical areas with widely varying myelin density, suggesting that regional differences in myelin abundance arises through local control of oligodendrogenesis, rather than selective reduction of myelin on distinct neuron subtypes. By following the loss and regeneration of myelin sheaths along defined neurons in vivo we show that even though the distribution of myelin on individual PV and VM neuron axons was altered following remyelination, the overall myelin content on these neurons was restored. The findings suggest that local changes in myelin can be tolerated, allowing opportunistic selection of available targets by newly formed oligodendrocytes to restore relative differences in myelin content between functionally distinct neurons.

neuroscience↗

bioRxiv2021

Automated in vivo tracking of cortical oligodendrocytes

Oligodendrocytes exert a profound influence on neural circuits by accelerating axon potential conduction, altering excitability and providing metabolic support. As oligodendrogenesis continues in the adult brain and is essential for myelin repair, uncovering the factors that control their dynamics is necessary to understand the consequences of adaptive myelination and develop new strategies to enhance remyelination in diseases such as multiple sclerosis. Unfortunately, few methods exist for analysis of oligodendrocyte dynamics, and even fewer are suitable for in vivo investigation. Here, we describe the development of a fully automated cell tracking pipeline using convolutional neural networks (Oligo-Track) that provides rapid volumetric segmentation and tracking of thousands of cells over weeks in vivo. This system reliably replicated human analysis, outperformed traditional analytic approaches, and extracted injury and repair dynamics at multiple cortical depths, establishing that oligodendrogenesis after cuprizone-mediated demyelination is suppressed in deeper cortical layers. Volumetric data provided by this analysis revealed that oligodendrocyte soma size progressively decreases after their generation, and declines further prior to death, providing a means to predict cell age and eventual cell death from individual time points. This new CNN-based analysis pipeline offers a rapid, robust method to quantitatively analyze oligodendrocyte dynamics in vivo, which will aid in understanding how changes in these myelinating cells influence circuit function and recovery from injury and disease.

neuroscience↗

bioRxiv2020

Remyelination alters the pattern of myelin in the cerebral cortex

Destruction of oligodendrocytes and myelin sheaths in cortical gray matter profoundly alters neural activity and is associated with cognitive disability in multiple sclerosis (MS). Myelin can be restored by regenerating oligodendrocytes from resident progenitors; however, it is not known whether regeneration restores the complex myelination patterns in cortical circuits. Here we performed time lapse in vivo two photon imaging in somatosensory cortex of adult mice to define the kinetics and specificity of myelin regeneration after acute oligodendrocyte ablation. These longitudinal studies revealed that the pattern of myelination in cortex changed dramatically after regeneration, as new oligodendrocytes were formed in different locations and new sheaths were often established along axon segments previously lacking myelin. Despite the dramatic increase in axonal territory available, oligodendrogenesis was persistently impaired in deeper cortical layers that experienced higher gliosis. The repeated reorganization of myelin patterns in MS may alter circuit function and contribute to cognitive decline.

neuroscience↗