Biology subjects

Appel, B.

Publications and source records attributed to Appel, B..

16 recordsLinked to original sources

bioRxiv2026

Autonomous Dampening of Wnt Signaling Selects Zebrafish Spinal Cord Progenitors for an Oligodendrocyte Fate

Nervous system development relies on coordinated formation of neurons and glia from neural progenitor cells (NPCs). During spinal cord development, NPCs in the pMN domain of the ventral spinal cord produce motor neurons followed by oligodendrocyte precursor cells (OPCs), a subset of which differentiate as oligodendrocytes, the myelinating cell of the vertebrate central nervous system. Whereas prior research has outlined an extensive gene regulatory network driving oligodendrocyte lineage cell differentiation, less is known about the mechanisms that specify NPCs for oligodendrocyte fate. To elucidate these mechanisms, our lab recently generated a single-cell RNA sequencing and single-cell ATAC sequencing multi-omics dataset from the zebrafish spinal cord. The dataset identified a molecularly distinct subset of NPCs that appear fated to the oligodendrocyte lineage, which we refer to as pre-OPCs. Our data revealed that pre-OPCs are enriched for RNAs encoding factors that antagonize Wnt signaling, such as barhl2 and cachd1. The chromatin regions associated with these genes are highly accessible in pre-OPCs, suggesting that their expression is developmentally regulated. Using confocal light microscopy to visualize expression of a Wnt-responsive transgenic reporter, we found that Wnt signaling is downregulated in the spinal cord immediately prior to OPC specification. To explore how modulation of Wnt signaling influences OPC specification, we pharmacologically inhibited or activated Wnt signaling immediately prior to OPC specification. These experiments revealed that embryos treated with a Wnt inhibitor produced excess OPCs whereas those treated with a Wnt agonist had a deficit of OPCs. Additionally, CRISPR/Cas9 targeted mutations of barhl2 and cachd1 impaired OPC production. Finally, we identified wnt4b as a ventral source of Wnt and determined that loss of wnt4b enhances OPC specification. Together, our studies reveal pre-OPC modulation of ventral Wnt signaling as a mechanism of OPC specification in the spinal cord.

developmental biology↗

bioRxiv2025

Gsx2 Regulates Oligodendrocyte Precursor Formation in the Zebrafish Spinal Cord

Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system, later in development. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.

developmental biology↗

bioRxiv2025

Bdnf-Ntrk2 Signaling Promotes but is not Essential for Spinal Cord Myelination in Larval Zebrafish

Myelin, a specialized membrane produced by oligodendroglial cells in the central nervous system, wraps axons to enhance conduction velocity and maintain axon health. Not all axons are myelinated, and not all myelinated axons are uniformly wrapped along their lengths. Several lines of evidence indicate that neuronal activity can influence myelination, however, the cellular and molecular mechanisms that mediate communication between axons and oligodendrocytes remain poorly understood. Prior research showed that the neurotrophic growth factor Bdnf and its receptor Ntrk2 promote myelination in rodents, raising the possibility that Bdnf and Ntrk2 convey myelin-promoting signals from neurons to oligodendrocytes. We explored this possibility using a combination of gene expression analyses, gene function tests, and myelin sheath formation assays in zebrafish larvae. Altogether, our data indicate that, although not essential for myelination, Bdnf-Ntrk2 signaling contributes to the timely formation of myelin in the developing zebrafish spinal cord.

developmental biology↗

bioRxiv2024

Oligodendrocytes use post-synaptic proteins to coordinate myelin formation on axons of distinct neurotransmitter classes

Axon myelination can tune neuronal circuits through placement and modulation of different patterns of myelin sheaths on distinct types of axons. How myelin formation is coordinated on distinct axon classes remains largely unknown. Recent work indicates neuronal activity and vesicle release promote myelin formation, and myelin-producing oligodendrocytes express canonical postsynaptic factors that potentially facilitate oligodendrocyte-axon interaction for myelin ensheathment. Here, we examined whether the inhibitory postsynaptic scaffold protein Gephyrin (Gphn) mediates selective myelination of specific axon classes in the larval zebrafish. Consistent with this possibility, Gphn was enriched in myelin on GABAergic and glycinergic axons. Strikingly, in gphnb deficient larvae, myelin sheaths were longer specifically on GABAergic axons, and the frequency of myelin placement shifted toward glutamatergic axons at the expense of GABAergic axons. Collectively, our results indicate that oligodendrocytes use postsynaptic machinery to coordinate myelin formation in an axon identity-dependent manner.

neuroscience↗

bioRxiv2024

Cspg4 sculpts oligodendrocyte precursor cell morphology

The extracellular matrix (ECM) provides critical biochemical and structural cues that regulate neural development. Chondroitin sulfate proteoglycans (CSPGs), a major ECM component, have been implicated in modulating oligodendrocyte precursor cell (OPC) proliferation, migration, and maturation, but their specific roles in oligodendrocyte lineage cell (OLC) development and myelination in vivo remain poorly understood. Here, we use zebrafish as a model system to investigate the spatiotemporal dynamics of ECM deposition and CSPG localization during central nervous system (CNS) development, with a focus on their relationship to OLCs. We demonstrate that ECM components, including CSPGs, are dynamically expressed in distinct spatiotemporal patterns coinciding with OLC development and myelination. We found that zebrafish lacking cspg4 function produced normal numbers of OLCs, which appeared to undergo proper differentiation. However, OPC morphology in mutant larvae was aberrant. Nevertheless, the number and length of myelin sheaths produced by mature oligodendrocytes were unaffected. These data indicate that Cspg4 regulates OPC morphogenesis in vivo, supporting the role of the ECM in neural development.

developmental biology↗

bioRxiv2024

Creation of a novel zebrafish model with low DHA status to study the role of maternal nutrition during neurodevelopment

Docosahexaenoic acid (DHA), a dietary omega-3 fatty acid, is a major building block of brain cell membranes. Offspring rely on maternal DHA transfer to meet their neurodevelopmental needs, but DHA sources are lacking in the American diet. Low DHA status is linked to altered immune responses, white matter defects, impaired vision, and an increased risk of psychiatric disorders during development. However, the underlying cellular mechanisms involved are largely unknown, and advancements in the field have been limited by the existing tools and animal models. Zebrafish are an excellent model for studying neurodevelopmental mechanisms. Embryos undergo rapid external development and are optically transparent, enabling direct observation of individual cells and dynamic cell-cell interactions in a way that is not possible in rodents. Here, we create a novel DHA-deficient zebrafish model by 1) disrupting elovl2, a key gene in the DHA biosynthesis pathway, via CRISPR-Cas9 genome editing, and 2) feeding mothers a DHA-deficient diet. We show that low DHA status during development is associated with a small eye morphological phenotype and demonstrate that even the morphologically normal siblings exhibit dysregulated gene pathways related to vision and stress response. Future work using our zebrafish model could reveal the cellular and molecular mechanisms by which low DHA status leads to neurodevelopmental abnormalities and provide insight into maternal nutritional strategies that optimize infant brain health.

developmental biology↗

bioRxiv2024

Amyloid beta oligomers dysregulate oligodendrocyte differentiation and myelination via PKC in the zebrafish spinal cord

Amyloid {beta} oligomers (A{beta}o) have been proposed as candidates to induce oligodendrocyte (OL) and myelin dysfunctions in early stages of Alzheimers disease (AD) pathology. Nevertheless, little is known about how A{beta}o affect OL differentiation and myelination in vivo, and the underlying molecular mechanisms. In this study, we explored the effects of a brain intraventricular injection of A{beta}o on OLs and myelin in the developing spinal cord of zebrafish larvae. Using quantitative fluorescent in situ RNA hybridization assays, we demonstrated that A{beta}o altered myrf and mbp mRNA levels and the regional distribution of mbp during larval development, suggesting an early differentiation of OLs. Through live imaging of Tg(myrf:mScarlet) and Tg(mbp:tagRFP) zebrafish lines, both crossed with Tg(olig2:EGFP), we found that A{beta}o increased the number of myrf+ and mbp+ OLs in the dorsal spinal cord at 72 hpf and 5 dpf, respectively, without affecting total cell numbers. Furthermore, A{beta}o also increased the number of myelin sheaths per OL and the number of myelinated axons in the dorsal spinal cord compared to vehicle-injected control animals. Interestingly, the treatment of A{beta}o-injected zebrafish with the pan-PKC inhibitor Go6983 restored the aforementioned alterations in OLs and myelin to control levels. Altogether, not only do we demonstrate that A{beta}o induce a precocious oligodendroglial differentiation leading to dysregulated myelination, but we also identified PKC as a key player in A{beta}o-induced pathology.

neuroscience↗

bioRxiv2021

Temporal single-cell transcriptomes of zebrafish spinal cord pMN progenitors reveal distinct neuronal and glial progenitor populations

Ventral spinal cord progenitor cells, which express the basic helix loop helix transcription factor Olig2, sequentially produce motor neurons and oligodendrocyte precursor cells (OPCs). Following specification some OPCs differentiate as myelinating oligodendrocytes while others persist as OPCs. Though a considerable amount of work has described the molecular profiles that define motor neurons, OPCs, and oligodendrocytes, less is known about the progenitors that produce them. To identify the developmental origins and transcriptional profiles of motor neurons and OPCs, we performed single-cell RNA sequencing on isolated pMN cells from embryonic zebrafish trunk tissue at stages that encompassed motor neurogenesis, OPC specification, and initiation of oligodendrocyte differentiation. Downstream analyses revealed two distinct pMN progenitor populations: one that appears to produce neurons and one that appears to produce OPCs. This latter population, called Pre-OPCs, is marked by expression of GS Homeobox 2 (gsx2), a gene that encodes a homeobox transcription factor. Using fluorescent in situ hybridizations, we identified gsx2-expressing Pre-OPCs in the spinal cord prior to expression of canonical OPC marker genes. Our data therefore reveal heterogeneous gene expression profiles among pMN progenitors, supporting prior fate mapping evidence. HighlightsO_LISingle-cell RNA sequencing reveals the developmental trajectories of neurons and glia that arise from spinal cord pMN progenitor cells in zebrafish embryos C_LIO_LITranscriptionally distinct subpopulations of pMN progenitors are the apparent sources of neurons or oligodendrocytes, consistent with fate mapping data C_LIO_LIgsx2 expression marks pMN progenitors that produce oligodendrocyte lineage cells C_LI

developmental biology↗

bioRxiv2021

The Akt-mTOR pathway drives myelin sheath growth by regulating cap-dependent translation

In the vertebrate central nervous system, oligodendrocytes produce myelin, a specialized proteolipid rich membrane, to insulate and support axons. Individual oligodendrocytes wrap multiple axons with myelin sheaths of variable lengths and thicknesses. Myelin grows at the distal ends of oligodendrocyte processes and multiple lines of work have provided evidence that mRNAs and RNA binding proteins localize to myelin, together supporting a model where local translation controls myelin sheath growth. What signal transduction mechanisms could control this? One strong candidate is the Akt-mTOR pathway, a major cellular signaling hub that coordinates transcription, translation, metabolism, and cytoskeletal organization. Here, using zebrafish as a model system, we found that Akt-mTOR signaling promotes myelin sheath growth and stability during development. Through cell-specific manipulations to oligodendrocytes, we show that the Akt-mTOR pathway drives cap-dependent translation to promote myelination and that restoration of cap-dependent translation is sufficient to rescue myelin deficits in mTOR loss-of-function animals. Moreover, an mTOR-dependent translational regulator co-localized with mRNA encoding a canonically myelin-translated protein in vivo and bioinformatic investigation revealed numerous putative translational targets in the myelin transcriptome. Together, these data raise the possibility that Akt-mTOR signaling in nascent myelin sheaths promotes sheath growth via translation of myelin-resident mRNAs during development. SIGNIFICANCE STATEMENTIn the brain and spinal cord oligodendrocytes extend processes that tightly wrap axons with myelin, a protein and lipid rich membrane that increases electrical impulses and provides trophic support. Myelin membrane grows dramatically following initial axon wrapping in a process that demands protein and lipid synthesis. How protein and lipid synthesis is coordinated with the need for myelin to be generated in certain locations remains unknown. Our study reveals that the Akt-mTOR signaling pathway promotes myelin sheath growth by regulating protein translation. Because we found translational regulators of the Akt-mTOR pathway in myelin, our data raise the possibility Akt-mTOR activity regulates translation in myelin sheaths to deliver myelin on demand to the places it is needed.

neuroscience↗

bioRxiv2021

Fmrp regulates oligodendrocyte lineage cell specification and differentiation

Neurodevelopment requires the precise integration of a wide variety of neuronal and glial cell types. During early embryonic development, motor neurons and then oligodendrocyte precursor cells (OPCs) are specified from neural progenitors residing in the periventricular pMN progenitor domain of the spinal cord. Following gliogenesis, OPCs can differentiate as oligodendrocytes (OLs) - the myelinating glial cells of the central nervous system - or remain as OPCs. To generate unique cell types capable of highly divergent functions, these specification and differentiation events require specialized gene expression programs. RNA binding proteins (RBPs) regulate mRNA localization and translation in the developing nervous system and are linked to many neurodevelopmental disorders. One example is Fragile X syndrome (FXS), caused by the loss of the RBP fragile X mental retardation protein (FMRP). Importantly, infants with FXS have reduced white matter and we previously showed that zebrafish Fmrp is autonomously required in OLs to promote myelin sheath growth. We now find that Fmrp regulates cell specification in pMN progenitor cells and subsequently promotes differentiation of OPCs, such that fmr1 mutant zebrafish embryos generate excess OPCs and fewer differentiating OLs in the developing spinal cord. Although the early patterning of spinal progenitor domains appears largely normal in fmr1 mutants during early embryogenesis, Shh signaling is greatly diminished. Taken together, these results suggest cell stage-specific requirements for Fmrp in the specification and differentiation of oligodendrocyte lineage cells.

developmental biology↗

bioRxiv2021

Zebrafish spinal cord oligodendrocyte formation requires boc function

The axis of the vertebrate neural tube is patterned, in part, by a ventral to dorsal gradient of Shh signaling. In the ventral spinal cord, Shh induces concentration-dependent expression of transcription factors, subdividing neural progenitors into distinct domains that subsequently produce distinct neuronal and glial subtypes. In particular, progenitors of the pMN domain express the bHLH transcription factor Olig2 and produce motor neurons followed by oligodendrocytes, the myelinating glial cell type of the central nervous system. In addition to its role in patterning ventral progenitors, Shh signaling must be maintained through development to specify pMN progenitors for oligodendrocyte fate. Using a forward genetic screen in zebrafish for mutations that disrupt development of oligodendrocytes, we identified a new mutant allele of boc, which encodes a type I transmembrane protein that functions as a coreceptor for Shh. Embryos homozygous for the bocco25 allele, which creates a missense mutation in a Fibronectin type III domain that binds Shh, have normally patterned spinal cords but fail to maintain pMN progenitors, resulting in a deficit of oligodendrocytes. Using a sensitive fluorescent detection method for in situ RNA hybridization, we found that spinal cord cells express boc in a graded fashion that is inverse to the gradient of Shh signaling activity and that boc function is necessary to maintain pMN progenitors by shaping the Shh signaling gradient.

developmental biology↗

bioRxiv2020

Prdm8 regulates pMN progenitor specification for motor neuron and oligodendrocyte fates by modulating Shh signaling response

Spinal cord pMN progenitors sequentially produce motor neurons and oligodendrocyte precursor cells (OPCs). Some OPCs differentiate rapidly as myelinating oligodendrocytes whereas others remain into adulthood. How pMN progenitors switch from producing motor neurons to OPCs with distinct fates is poorly understood. pMN progenitors express prdm8, which encodes a transcriptional repressor, during motor neuron and OPC formation. To determine if prdm8 controls pMN cell fate specification, we used zebrafish as a model system to investigate prdm8 function. Our analysis revealed that prdm8 mutant embryos have a deficit of motor neurons resulting from a premature switch from motor neuron to OPC production. Additionally, prdm8 mutant larvae have excess oligodendrocytes and a concomitant deficit of OPCs. Notably, pMN cells of mutant embryos have elevated Shh signaling coincident with the motor neuron to OPC switch. Inhibition of Shh signaling restored the number of motor neurons to normal but did not rescue the proportion of oligodendrocytes. These data suggest that Prdm8 regulates the motor neuron-OPC switch by controlling the level of Shh activity in pMN progenitors and also regulates allocation of oligodendrocyte lineage cell fates. Summary StatementPrdm8 regulates the timing of a motor neuron-oligodendrocyte switch and oligodendrocyte lineage cell identity in the zebrafish spinal cord.

developmental biology↗

bioRxiv2019

Developmental myelination is modified by microglial pruning

During development, oligodendrocytes contact and wrap neuronal axons with myelin. Similar to neurons and synapses, excess myelin sheaths are produced and selectively eliminated. However, unlike these other structures, almost nothing is known about myelin sheath elimination. Microglia, the resident immune cells of the CNS, refine the developing CNS by engulfing surplus neurons and synapses. To determine if microglia also prune myelin sheaths, we used zebrafish to visualize and manipulate interactions between microglia, oligodendrocytes, and neurons during development. We found that microglia closely associate with oligodendrocytes and specifically phagocytose myelin sheaths. Silencing neuronal activity with botulinum toxin (BoNT/B) increased myelin engulfment by microglia. Furthermore, oligodendrocytes maintained excessive myelin sheaths following microglial ablation. Our work reveals a neuronal activity-regulated role for microglia in regulating myelination by oligodendrocytes.

neuroscience↗

bioRxiv2019

Neuronal Activity Enhances mRNA Localization to Myelin Sheaths During Development

Myelin is a specialized membrane produced by oligodendrocytes that insulates and supports axons. Oligodendrocytes extend numerous cellular processes, as projections of the plasma membrane, and simultaneously wrap multiple layers of myelin membrane around target axons. Notably, myelin sheaths originating from the same oligodendrocyte are variable in size, suggesting local mechanisms regulate myelin sheath growth. Purified myelin contains ribosomes and hundreds of mRNAs, supporting a model that mRNA localization and local protein synthesis regulate sheath growth and maturation. However, the mechanisms by which mRNAs are selectively enriched in myelin sheaths are unclear. To investigate how mRNAs are targeted to myelin sheaths, we tested the hypothesis that transcripts are selected for myelin enrichment through consensus sequences in the 3 untranslated region (3 UTR). Using methods to visualize mRNA in living zebrafish larvae, we identified candidate 3 UTRs that were sufficient to localize mRNA to sheaths and enriched near growth zones of nascent membrane. We bioinformatically identified motifs common in 3 UTRs from three myelin-enriched transcripts and determined that these motifs are required for mRNA transport to myelin sheaths. Finally, we show that one motif is highly enriched in the myelin transcriptome, suggesting that this sequence is a global regulator of mRNA localization during developmental myelination.

neuroscience↗

bioRxiv2018

Synaptic proteins expressed by oligodendrocytes mediate CNS myelination

Oligodendrocytes ensheath neuronal axons with myelin, a proteolipid-rich membrane that increases conduction velocity and provides trophic support. Our lab and others have provided evidence that vesicular release from neurons promotes myelin sheath growth. Complementarily, transcriptomic and proteomic approaches have revealed that oligodendrocytes express many proteins that allow dendrites to sense and respond to vesicular release at synapses. Do axon-myelin contacts use similar communication mechanisms as nascent synapses to form myelin sheaths on axons? To test this, we used fusion proteins to track synaptic vesicle localization and membrane fusion within spinal cord axons of zebrafish larvae during developmental myelination. Additionally, we used a CRISPR/Cas9-mediated GAL4 enhancer trap and genetically-encoded intrabody to detect expression and localization of PSD-95, a component of dendritic postsynaptic complexes, within oligodendrocytes. We found that synaptic vesicles accumulate at ensheathment sites over time and are exocytosed with variable patterning underneath myelin sheaths. Accordingly, we also found that most, but not all sheaths localized PSD-95 with patterning similar to exocytosis site location within the axon. By querying published transcriptome databases, we found that oligodendrocytes express numerous transsynaptic adhesion molecules that function across synapses to promote synapse formation and maturation. Disruption of candidate PDZ-binding transsynaptic adhesion proteins in oligodendrocytes revealed that these proteins have variable effects on sheath length and number. We focused on one candidate, Cadm1b (SynCAM1), and demonstrated that it localized to myelin sheaths where both its PDZ binding and extracellular adhesion to axons are required for myelin sheath growth. Our work reveals shared mechanisms of synaptic and myelin plasticity and provides new targets for mechanistic unraveling of activity-regulated myelination.

neuroscience↗

bioRxiv2018

Sequential Specification of Oligodendrocyte and NG2 Cell Fates by Distinct Levels of Hedgehog Signaling

During development of the central nervous system oligodendrocyte precursor cells (OPCs) give rise to both myelinating oligodendrocytes and NG2 glia, which are the most proliferative cells in the adult mammalian brain. NG2 glia retain characteristics of OPCs, and some NG2 glia produce oligodendrocytes, but many others persist throughout adulthood. Why some OPCs differentiate as oligodendrocytes during development whereas others persist as OPCs and acquire characteristics of NG2 glia is not known. Using zebrafish spinal cord as a model, we found that OPCs that differentiate rapidly as oligodendrocytes and others that remain as OPCs arise in sequential waves from distinct neural progenitors. Additionally, oligodendrocyte and persistent OPC fates are specified during a defined critical period by small differences in Shh signaling and Notch activity, which modulates Shh signaling response. Thus, our data indicate that OPCs fated to produce oligodendrocytes or remain as OPCs during development are specified as distinct cell types, raising the possibility that the myelinating potential of OPCs is set by graded Shh signaling activity.

developmental biology↗