Biology subjects

Lyons, D.

Publications and source records attributed to Lyons, D..

13 recordsLinked to original sources

bioRxiv2026

3D MERFISH reveals cell-type diversity generated by mRNA partitioning in spiralian embryogenesis

Cell type diversification during development is often attributed to differential gene transcription. Here, we show that, during Crepidula atrasolea embryogenesis, transcriptomically distinct cell types arise through differential distribution of pre-existing mRNAs. Combining immunofluorescence, live imaging, and whole-mount 3D spatial transcriptomics (MERFISH) of over 40 embryos, we show that many mRNAs form subcellular aggregates during mitosis, which segregate asymmetrically between daughter cells through microtubule associations. Cell divisions thus simultaneously increase cell number and diversity, producing six transcriptomically distinct cell quartets in the 24-cell stage embryo. We modeled aggregate compositions across cell divisions using transcript abundance and quantified transcript-specific affinity. These results establish mRNA sorting and inheritance as a scalable cell type patterning mechanism and provide a framework for investigating how mRNA segregation shapes animal development.

molecular biology↗

bioRxiv2024

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development. Summary statementThe study uses sea star embryogenesis as a model of a proliferating epithelium to highlight how cell division induces 3D cell rearrangements during development.

developmental biology↗

bioRxiv2023

C9ORF72 deficiency results in degeneration of the zebrafish retina in vivo

G4C2 Hexanucleotide repeat expansions within the gene C9ORF72 are the most common cause of the neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). This disease-causing expansion leads to a reduction in C9ORF72 expression levels in patients, suggesting haploinsufficiency could contribute to disease. To further understand the consequences of C9ORF72 deficiency in vivo, we generated a c9orf72 mutant zebrafish line. Analysis of the spinal cord revealed no appreciable neurodegenerative pathology such as loss of motor neurons, or increased levels of neuroinflammation. However, detailed examination of c9orf72-/- retinas showed prominent neurodegenerative features, including a decrease in retinal thickness, gliosis, and an overall reduction in neurons of all subtypes. Structurally, analysis of rod and cone cells within the photoreceptor layer showed a disturbance in the outer cells of the retina and rhodopsin mis-localisation from rod outer segments to their cell bodies and synaptic endings. Thus, C9ORF72 may play a previously unappreciated role in retinal homeostasis and suggests C9ORF72 deficiency can induce tissue specific neuronal loss.

neuroscience↗

bioRxiv2023

Sleep pressure modulates single-neuron synapse dynamics in zebrafish

Sleep is a nearly universal behaviour with unclear functions1. The Synaptic Homeostasis Hypothesis (SHY) proposes that sleep is required to renormalize the increases in synaptic number and strength that occur during wakefulness2. Some studies examining either large neuronal populations3 or small patches of dendrites4 have found evidence consistent with SHY, but whether sleep merely serves as a permissive state or actively promotes synaptic downregulation at the scale of whole neurons is unknown. Here, by repeatedly imaging all excitatory synapses on single neurons across sleep/wake states of zebrafish larvae, we show that synapses are gained during periods of wake (either spontaneous or forced) and lost during sleep in a neuron-subtype dependent manner. However, synapse loss is greatest during sleep associated with high sleep pressure following prolonged wakefulness and low in the latter half of the night. Conversely, sleep induced pharmacologically during periods of low sleep pressure is insufficient to trigger synapse loss unless adenosine levels are boosted while noradrenergic tone is inhibited. We conclude that sleep-dependent synapse loss is regulated by sleep pressure at the level of the single neuron and that not all sleep periods are equally capable of fulfilling the functions of synaptic homeostasis.

neuroscience↗

bioRxiv2023

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes

How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum has long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdes, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. The final assembled and filtered Berghia genome is comparable to other high quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes), and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

genomics↗

bioRxiv2023

Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

Central nervous system neurons have axons that vary over 100-fold in diameter. This diversity contributes to the regulation of myelination and conduction velocity along axons, which is critical for proper nervous system function. Despite this importance, technical challenges have limited our understanding of mechanisms controlling axon diameter growth, preventing systematic dissection of how manipulating diameter affects conduction along individual axons. Here, we establish zebrafish as system to investigate the regulation of axon diameter and axonal conduction in vivo. We identify a role for importin 13b specifically in axon diameter growth, that is independent of axonal length and overall cell size. Impairment of diameter growth along individual myelinated axons reduced conduction velocity in proportion to the diameter, but had no effect on the precision of action potential propagation or ability to fire at high frequencies. This work highlights an axon diameter-specific mechanism of growth that regulates conduction speeds along myelinated axons.

neuroscience↗

bioRxiv2022

Obesity medication lorcaserin requires brainstem GLP-1 neurons to reduce food intake in mice

Overweight and obesity are rapidly becoming the "new normal" in developed countries, which promotes a widespread negative impact on human health. Amongst recently developed obesity medications are the serotonin 2C receptor (5-HT2CR) agonist lorcaserin and glucagon-like peptide-1 receptor (GLP-1R) agonists, but the brain circuits employed by these medications to produce their therapeutic effects remain to be fully defined. 5-HT2CRs and GLP-1Rs are widely distributed in the brain, including in the key homeostatic region the nucleus of the solitary tract (NTS) where GLP-1 is produced by preproglucagon (PPGNTS) neurons. PPGNTS cells were profiled using histochemistry and single nucleus RNA sequencing (Nuc-Seq) of mouse brainstem. Transcriptomic analyses revealed 5-HT2CR expression was widespread in PPGNTS clusters. Demonstrating the functional significance of this co-expression, lorcaserin required PPGNTS to reduce food intake. Analysis of second order neurons revealed that local GLP1-R neurons within the NTS are necessary for 5-HT2CRNTS food intake suppression. In contrast, GLP-1RNTS were not required for GLP-1R agonist liraglutide and exendin-4s short term feeding reduction, suggesting scope for lorcaserin and GLP1-R agonist combination therapy. In support of this, lorcaserin+liraglutide and lorcaserin+exendin-4 produced greater reductions in food intake when administered in combination as compared to monotherapies. These data provide insight into the therapeutic mechanisms of lorcaserin and identify a combination strategy to improve the therapeutic profile of lorcaserin and GLP1-R agonists.

neuroscience↗

bioRxiv2022

Design and validation of the first family of photo-activatable ligands for melatonin receptors

Melatonin is a neurohormone released in a circadian manner with peak levels during the night. In mammals, melatonin mediates its effects mainly through G protein-coupled MT1 and MT2 receptors. Drugs acting on melatonin receptors are indicated for circadian rhythm- and sleep-related disorders and major depression. Pharmacological tools to study the activation of these receptors with high temporal resolution are lacking. Here, we synthesized a family of light-activatable caged melatonin compounds by covalently attaching o-nitrobenzyl (o-NB) or coumarin photocleavable groups to the N-position of melatonin. All caged compounds showed the expected decrease in binding affinity for MT1 and MT2. Among them, the o-NB derivative MCS-0382 showed the best uncaging and biological properties upon light activation with a significant increase (2.5 log left-shift) in affinity and potency in melatonin receptor binding and signaling, respectively. Generation of biologically active melatonin from MCS-0382 was further demonstrated by its ability to modulate the excitation of SCN neurons in rat brain slices. MCS-0382 is now available to study melatonin effects in a temporally controlled manner in cellular and physiological settings.

pharmacology and toxicology↗

bioRxiv2022

Cell size asymmetries in the sea star embryo

Cell size asymmetries are often linked to cell fate decisions, due to cell volumes and cell fate determinants being unequally partitioned during asymmetric cell divisions. A clear example is found in the sea urchin embryo, where a characteristic and obvious unequal 4th cleavage generates micromeres, which are necessary for mesendoderm cell fate specification. Unlike sea urchin development, sea star development is generally thought to have only equal cleavage. However, subtle cell size asymmetries can be observed in sea star embryos; whether those cell size asymmetries are consistently produced during sea star development and if they are involved in cell fate decisions remains unknown. Using confocal live imaging of early embryos we quantified cell size asymmetries in 16-cell stage embryos of two sea star species, Patiria miniata and Patiriella regularis. Using photoconversion to perform lineage tracing, we find that the position of the smallest cells of P. miniata embryos is biased toward anterior ventral tissues. However, both blastomere dissociation and mechanical removal of one small cell do not prevent dorsoventral (DV) axis formation, suggesting that embryos compensate for the loss of those cells and asymmetric partitioning of maternal determinants is not strictly necessary for DV patterning. Finally, we show that manipulating cell size to introduce artificial cell size asymmetries is not sufficient to direct the positioning of the future DV axis in P. miniata embryos. Our results show that although cell size asymmetries are consistently produced during sea star early cleavage and may be predictive of the DV axis, they are not necessary to instruct DV axis formation.

developmental biology↗

bioRxiv2021

Clusters of neuronal Neurofascin prefigure node of Ranvier position along single axons

The spacing of nodes of Ranvier crucially affects conduction properties along myelinated axons. It has been assumed that node position is primarily driven by the growth of myelin sheaths. Here, we reveal an additional mechanism of node positioning that is driven by the axon. We show through longitudinal live imaging of node formation dynamics that stable clusters of the cell adhesion molecule Neurofascin A accumulate at specific sites along axons prior to myelination. While some of these clusters change position upon encounter with growing myelin sheaths, others restrict sheath extension and are therefore predictive of future node position. Animals that lack full-length Neurofascin A showed increased internodal distances and less regular spacing of nodes along single axons. Together, our data reveal the existence of an axonal mechanism to position its nodes of Ranvier that does not depend on regulation of myelin sheath length.

neuroscience↗

bioRxiv2020

Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury

Acute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by prescreening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest. HIGHLIGHTS- Synthetic CRISPR gRNAs are highly active - in vivo pre-screening allows rapid assessment of CRISPR gRNA activity - Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration - tgfb1a promotes spinal regeneration by controlling inflammation

neuroscience↗

bioRxiv2019

TET1-mediated DNA hydroxy-methylation regulates adult remyelination

Adult myelination is essential for brain function and response to injury, but the molecular mechanisms remain elusive. Here we identify DNA hydroxy-methylation, an epigenetic mark catalyzed by Ten-Eleven translocation (TET) enzymes, as necessary for adult myelin repair. While DNA hydroxy-methylation and high levels of TET1 are detected in young adult mice during myelin regeneration after demyelination, this process is defective in old mice. Constitutive or inducible lineage-specific ablation of Tet1 (but not of Tet2) recapitulate the age-related decline of DNA hydroxy-methylation and inefficient remyelination. Genome-wide hydroxy-methylation and transcriptomic analysis identify numerous TET1 targets, including several members of the solute carrier (Slc) gene family. Lower transcripts for Slc genes, including Slc12a2, are observed in Tet1 mutants and old mice and are associated with swelling at the neuroglial interface, a phenotype detected also in zebrafish slc12a2b mutants. We conclude that TET1-mediated DNA hydroxy-methylation is necessary for adult remyelination after injury.

neuroscience↗

bioRxiv2018

Olfactory and Vomeronasal Receptor Feedback Employ Divergent Mechanisms of PERK Activation

Mutually-exclusive chemoreceptor expression in olfactory and vomeronasal sensory neurons (OSNs and VSNs) enables odorant discrimination. This configuration involves chemoreceptor mediated activation of the endoplasmic reticulum (ER)-resident kinase PERK. PERK drives translation of the transcription factor ATF5 to preclude additional chemoreceptor expression. ATF5 translation is transient in OSNs but persistent in VSNs, suggesting chemoreceptor-specific modes of PERK activation. Herein, we showed that the ER-lumenal domain (LD) of PERK recognized vomeronasal receptor (VR)-derived peptides, suggesting direct PERK activation drives persistent ATF5 translation in VSNs. In contrast, PERK LD did not recognize olfactory receptor (OR)-derived peptides in vitro, and facilitating OR maturation in vivo prevented PERK activation, suggesting that ORs activate PERK indirectly through a failure to exit the ER. Importantly, impairing or prolonging ATF5 expression drove specific chemoreceptor repertoire biases. Together, these results demonstrate mechanistic divergence in chemoreceptor feedback and establish that differences in PERK activation promote qualitatively different gene regulatory results.

cell biology↗