Biology subjects

Monk, K. R.

Publications and source records attributed to Monk, K. R..

8 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↗

bioRxiv2025

Developmental axon diameter growth of central nervous system axons does not depend on ensheathment or myelination by oligodendrocytes

Myelination facilitates the rapid conduction of action potentials along axons. In the central nervous system (CNS), myelinated axons vary over 100-fold in diameter, with conduction speed scaling linearly with increasing diameter. Axon diameter and myelination are closely interlinked, with axon diameter exerting a strong influence on myelination. Conversely, myelinating Schwann cells in the peripheral nervous system can both positively and negatively affect axon diameter. However, whether axon diameter is regulated by CNS oligodendrocytes is less clear. Here, we investigated CNS axon diameter growth in the absence of myelin using mouse (Mbpshi/shi and Myrf conditional knockout) and zebrafish (olig2 morpholino) models. We find that neither the ensheathment of axons, nor the formation of compact myelin are required for CNS axons to achieve appropriate and diverse diameters. This indicates that developmental CNS axon diameter growth is independent of myelination, and shows that myelinating cells of CNS and PNS differentially influence axonal morphology.

neuroscience↗

bioRxiv2024

FBXW7 regulates MYRF levels to control myelin capacity and homeostasis in the adult CNS

Myelin, along with the oligodendrocytes (OLs) that produce it, is essential for proper central nervous system (CNS) function in vertebrates. Although the accurate targeting of myelin to axons and its maintenance are critical for CNS performance, the molecular pathways that regulate these processes remain poorly understood. Through a combination of zebrafish genetics, mouse models, and primary OL cultures, we found FBXW7, a recognition subunit of an E3 ubiquitin ligase complex, is a regulator of adult myelination in the CNS. Loss of Fbxw7 in myelinating OLs resulted in increased myelin sheath lengths with no change in myelin thickness. As the animals aged, they developed progressive abnormalities including myelin outfolds, disrupted paranodal organization, and ectopic ensheathment of neuronal cell bodies with myelin. Through biochemical studies we found that FBXW7 directly binds and degrades the N-terminal of Myelin Regulatory Factor (N-MYRF), to control the balance between oligodendrocyte myelin growth and homeostasis.

neuroscience↗

bioRxiv2023

Dock1 acts cell-autonomously in Schwann cells to regulate the development, maintenance, and repair of peripheral myelin

Schwann cells, the myelinating glia of the peripheral nervous system (PNS), are critical for myelin development, maintenance, and repair. Rac1 is a known regulator of radial sorting, a key step in developmental myelination, and we previously showed in zebrafish that loss of Dock1, a Rac1-specific guanine nucleotide exchange factor, results in delayed peripheral myelination in development. We demonstrate here that Dock1 is necessary for myelin maintenance and remyelination after injury in adult zebrafish. Furthermore, it performs an evolutionary conserved role in mice, acting cell-autonomously in Schwann cells to regulate peripheral myelin development, maintenance, and repair. Additionally, manipulating Rac1 levels in larval zebrafish reveals that dock1 mutants are sensitized to inhibition of Rac1, suggesting an interaction between the two proteins during PNS development. We propose that the interplay between Dock1 and Rac1 signaling in Schwann cells is required to establish, maintain, and facilitate repair and remyelination within the peripheral nervous system.

neuroscience↗

bioRxiv2022

Synapses in oligodendrocyte precursor cells are dynamic and contribute to Ca2+ activity

In the nervous system, only one type of neuron-glial synapse is known to exist: that between neurons and oligodendrocyte precursor cells (OPCs). Neuron-OPC synapses are thought to bridge neuronal activity to OPCs. However, their composition, assembly, downstream signaling, and in vivo functions remain largely unclear. Here, we use zebrafish to address these questions and identify postsynaptic molecules PSD-95 and Gephyrin in OPCs. They increase during early development and decrease upon OPC differentiation. PSD-95 and Gephyrin in OPCs are highly dynamic and frequently assemble at "hotspots." Gephyrin hotspots and synapse-associated Ca2+ activity in OPCs predict where a subset of myelin sheaths form in oligodendrocytes. Further analyses reveal that spontaneous synaptic release is integral to OPC Ca2+ activity, while evoked synaptic release contributes only in early development. Finally, disruption of the synaptic genes dlg4a&b, gephyrinb, and nlgn3b impairs OPC differentiation and myelination. Together, we propose that neuron-OPC synapses are dynamically assembled and can predetermine myelination patterns through Ca2+ signaling.

neuroscience↗

bioRxiv2020

Postembryonic screen for mutations affecting spine development in zebrafish

The spinal vertebral column gives structural support for the adult body plan, protects the spinal cord, and provides muscle attachment and stability, which allows the animal to move within its environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10{degrees} around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant mutant zebrafish, displaying overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1,229 F1 animals, and subsequently bred to homozygosity in F3 families, from these, 314 haploid genomes were screened for recessive phenotypes. We cumulatively found 39 adult-viable (3 dominant and 36 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing within this phenotypic group revealed at least 16 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report several recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Many of the alleles characterized thus far are non-synonymous mutations in known essential scospondin and adamts9 genes. Our results provide evidence of monogenic traits that are critical for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

developmental biology↗

bioRxiv2018

Fbxw7 is a critical regulator of Schwann cell myelinating potential

Myelin insulates and protects axons in vertebrate nervous systems. In the central nervous system (CNS), oligodendrocytes (OLs) make numerous myelin sheaths on multiple axons, whereas in the peripheral nervous system (PNS) myelinating Schwann cells (SCs) make just one myelin sheath on a single axon. Why the myelinating potentials of OLs and SCs are so fundamentally different is unclear. Here, we find that loss of Fbxw7, an E3 ubiquitin ligase component, enhances the myelinating potential of SCs. Fbxw7 mutant SCs are seen myelinating multiple axons in a fashion reminiscent of OLs as well as aberrantly myelinating large axons while simultaneously ensheathing small unmyelinated axons - typically distinct roles of myelinating SCs and non-myelinating Remak SCs, respectively. We found that several of the Fbxw7 mutant phenotypes, including the ability to generate thicker myelin sheaths, were due to dysregulation of mTOR. However, the remarkable ability of mutant SCs to either myelinate multiple axons or myelinate some axons while simultaneously encompassing other unmyelinated axons is independent of mTOR signaling. This indicates distinct roles for Fbxw7 in regulating multiple aspects of SC behavior and that novel Fbxw7-regulated mechanisms control modes of myelination previously thought to fundamentally distinguish myelinating SCs from non-myelinating SCs and OLs. Our data reveal unexpected plasticity in the myelinating potential of SCs, which may have important implications for our understanding of both PNS and CNS myelination and myelin repair.

neuroscience↗