Biology subjects

MacDonald, R. B.

Publications and source records attributed to MacDonald, R. B..

13 recordsLinked to original sources

bioRxiv2026

Long read sequencing of retinal RNA improves killifish transcriptome annotation

Purpose The African Turquoise Killifish has recently emerged as a powerful model for aging and age-related disease research studies. However, molecular based investigations have been limited by preliminary genome and transcriptome builds with incomplete reference genome sequence, fragmented chromosome assembly, and missing gene annotations. These issues make primary (alignment and quantification) and secondary (Gene Ontology, Gene Set Enrichment Analysis, cross-species comparisons) analyses difficult to reliably implement and interpret. This study seeks to generate a complete retinal reference transcriptome to facilitate future killifish transcriptomic, epigenetic, and proteomic studies of the visual system. Methods We generated an enhanced retina transcriptome using long-read PacBio RNAseq data that was processed using a robust computational pipeline to merge reads, classify genes, and annotate with nearest orthologous gene names from other species. This new annotation was compared to available references and validated using bulk and single cell RNAseq datasets. Results Comparison of the widely used Nfu_20140520 and the newly released NfurGRZ-RIMD1 genome builds identified NfurGRZ-RIMD1 to be more contiguous and complete. However, we identified limitations with both transcriptomes, including the lack of annotation of certain retina specific genes and many uninformative gene names. Using long-read PacBio sequencing of RNA collected from young and old Killifish retinas, we annotated a deep retinal transcriptome onto the NfurGRZ-RIMD1 reference genome. This analysis identified thousands of previously unannotated transcripts from retinas of young and old killifish. By matching each translated protein sequence to its nearest ortholog, we increased the number and proportion of genes with meaningful gene names. Mapping of bulk and single-cell RNAseq data showed substantial increase in mapping rate and identified hundreds of genes and transcripts with age-dependent expression dynamics. Conclusions Assembly of an enhanced retinal transcriptome for the killifish improved both primary and secondary analyses of bulk and single cell RNAseq data. Improvements will benefit future studies investigating the mechanisms of aging in the killifish and to best utilize this powerful model to understand human disease.

genomics↗

bioRxiv2026

An early CYP26A1/CRYAA progenitor-glial domain marks the presumptive macular region during human retinal development

The human macula, a high-acuity retinal region essential for central vision, emerges early in development, but the cellular basis of its regional specialisation remains incompletely resolved. Building on recent studies that identified CYP26A1-mediated retinoic acid (RA) suppression in the presumptive macula, we define a CRYAA-positive progenitor-glial compartment in the temporal human retina from post-conception week (pcw) 7 onward. Single-cell RNA sequencing, immunohistochemistry and spatial morphometry indicate that this compartment is linked to regional gliogenic maturation and later corresponds to a specialised macular Muller glial population, rather than simply reflecting uniform pan-retinal developmental timing. Spatial mapping shows that the CYP26A1-positive domain remains delimited as the surrounding retina expands. Adult tissue analysis shows that CYP26A1 expression is retained in primate macular Muller glia and in corresponding acute-zone regions of visual-streak species. These findings extend current models of CYP26A1-mediated RA modulation in human acute zone/macular development by linking the early CYP26A1 domain to a regionally specialised CRYAA-positive progenitor-glial compartment. We propose that early macular patterning includes a spatially restricted glial programme that may contribute to later regional architecture and disease vulnerability.

developmental biology↗

bioRxiv2026

Glia regulate local retinoic acid levels to specify neuronal specialisation for high-acuity vision

Neurons of the same type can be differentially tuned to perform distinct functional roles, yet how such specialisation arises during development remains poorly understood. Here, we show that in the zebrafish retina, heterogeneity within a single glial population generates local gradients of retinoic acid (RA) signalling that fine-tune neural function within a single neuronal type. The acute zone (AZ), a ventro-temporal region of the retina, is specialised for prey capture in the upper frontal visual field. In this region, UV cones exhibit enhanced light sensitivity through elongation of their outer segments (OS), the cellular compartment responsible for phototransduction. We found that cyp26 genes, which encode RA-degrading enzymes, are expressed in Muller glia in a region-specific manner, and that this expression pattern closely matches the spatial variation in cone OS length. Inhibition of Cyp26 activity prevented OS elongation in the AZ. This effect is mediated by RA signalling, as direct activation of RA receptors using RAR agonists similarly reduced OS length in this region. Notably, inhibition of Cyp26 predominantly activated RA signalling in Muller glia rather than in cones, indicating that RA signalling regulates cone OS length in a cell-non-autonomous manner via Muller glia. Consistent with this model, blocking Muller glia genesis abolished cone specialisation across all regions of the developing retina. Together, these findings identify Muller glia as key regulators of cone functional specialisation through the spatial control of RA signalling.

neuroscience↗

bioRxiv2025

Gelatinase activity is required for Muller glia morphogenesis and neuronal maintenance in the retina

Glial cells are indispensable support elements of the nervous system, yet the mechanisms by which they integrate into densely packed, extracellular matrix (ECM)-rich cellular environments remain poorly understood. In the retina, Muller glia (MG) serve as the principal support cells, performing roles analogous to astrocytes in the brain. As late-born retinal cells, MG undergo morphogenesis within a complex extracellular milieu composed of neighbouring neurons, synapses, and ECM components. Using the zebrafish retina, we show that developing MG dynamically interact with the ECM across all retinal layers while expressing the ECM-remodelling enzymes matrix metalloproteinases (Mmp) 2 and 9. Although Mmps are known to contribute to tissue remodelling and disease processes such as glioma invasion, their developmental roles in glia remain less defined. Here, we demonstrate that Mmp2 and Mmp9 are required for MG morphogenesis and glia-neuron integration. Pharmacological inhibition or genetic loss of mmp2 and mmp9 impaired MG outgrowth and reduced process complexity within synaptic layers, leading to decreased MG-synapse associations. Double mutants (mmp2-/-;mmp9-/-) displayed normal neuronal development and visual function at larval stages but exhibited gliosis, premature neurodegeneration, and visual deficits in adulthood. Together, these findings reveal that Mmp2 and Mmp9 are critical for establishing MG-synapse interactions during development and for maintaining neuronal integrity and visual function in the mature retina.

neuroscience↗

bioRxiv2025

Turquoise killifish naturally develop hallmarks of age-related macular degeneration with advancing age

Ageing is a major risk factor for developing vision loss diseases such as age-related macular degeneration (AMD). Unfortunately, we do not have the ability to effectively prevent, slow, or stop onset and progression of AMD long term. These challenges with therapeutic development result from poor understanding of disease mechanism and pathogenesis due to a lack of animal models that manifest the hallmark features of disease. Here, we investigated the rapidly ageing turquoise killifish (Nothobranchius furzeri) retina for features of human ageing and AMD. We report that the ageing killifish retina expresses genes associated with human retinal disease in the photoreceptors and retinal pigment epithelium (RPE). Our characterisation of the retina identified that killifish spontaneously develop many hallmark features of AMD and human ageing, including photoreceptor deterioration, lipid deposits, outer retinal inflammation, and ceramide accumulation in the RPE with advancing age. Further, we identify a sex-specific difference in the severity of phenotypes. We propose that the turquoise killifish is a highly suitable model for investigating ageing and AMD-related disease mechanisms across the lifespan.

neuroscience↗

bioRxiv2024

Iterative Bleaching Extends Multiplexity (IBEX) imaging facilitates simultaneous identification of all cell types in the vertebrate retina

The vertebrate retina is a complex multicellular tissue made up of distinct neuron types and glia, arranged in a stereotypic layered organisation to facilitate vision. Understanding how these cell types come together to form precise circuits during development requires the ability to simultaneously discriminate between multiple cell types and their spatial position in the same tissue. Currently, we have a limited capacity to resolve all constitutive cell types and their relationships to one another due to our limited ability to combine multiple cellular markers. To extend this capacity, we have adapted a highly multiplexed immunohistochemistry technique known as Iterative Bleaching Extends Multiplexity (IBEX) and applied it to the development of the zebrafish (Danio rerio) retina. IBEX allows for multiple rounds of cellular labelling to be performed, before imaging and integration of data, resulting in the ability to visualise multiple markers on the same tissue. We have optimised IBEX in zebrafish using fluorescent micro-conjugation of known antibody markers to label the complete retina with up to 11 cell-specific antibodies. We have further adapted the IBEX technique to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and wholemount immunohistochemistry (WMIHC). We then took advantage of IBEX to explore the multicellular relationships in the developing retina between glial cells and neurons and photoreceptor subtypes. Finally, we tested IBEX on retinas from the emerging ageing model, the killifish (Nothobranchius furzeri), and developmental model, the African clawed frog (Xenopus laevis), demonstrating the usefulness of the technique across multiple species. The techniques described here can be applied to any tissue in any organism where antibodies are readily available to efficiently explore cellular relationships in the context of development, ageing or disease.

developmental biology↗

bioRxiv2024

Age-related dysregulation of the retinal transcriptome in African turquoise killifish

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNA-seq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterised, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNA-seq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasises the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies. HighlightsO_LIThe aged killifish retina displays several ageing hallmarks, such as oxidative stress, gliosis and inflammageing, at the transcriptome level. C_LIO_LIRisk genes for neurodegenerative disorders show dysregulation in the old killifish retina. C_LIO_LIAll vertebrate retinal cell types are present in the killifish retina. C_LIO_LITranscriptional dysregulation in the aged killifish retina is observed across cell types. C_LI

neuroscience↗

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↗

bioRxiv2022

Unique functions of two overlapping PAX6 retinal enhancers

Enhancers play a critical role in development by precisely modulating spatial, temporal, and cell type-specific gene expression. Sequence variants in enhancers have been implicated in disease, however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development, and has a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive, redundant, or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers, HS5 and NRE. Using a unique combination of live imaging and single-cell RNA sequencing in dual enhancer-reporter zebrafish embryos, we find significant differences in the spatiotemporal activity of these enhancers, and show that HS5 and NRE are active in distinct cell types of the developing retina. Our results show that although overlapping, these enhancers have distinct activities in different cell types and therefore likely non-redundant functions. This work demonstrates that unique cell type-specific functions can be uncovered for apparently similar enhancers when investigated at high resolution in vivo.

genetics↗

bioRxiv2022

GliaMorph: A modular image analysis toolkit to quantify Müller glial cell morphology

Cell morphology is critical for all cell functions. This is particularly true for glial cells as they rely on their complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this gap in quantification approaches, we developed an analysis pipeline called "GliaMorph". GliaMorph is a modular image analysis toolkit developed to perform (i) image pre-processing, (ii) semi-automatic region-of-interest (ROI) selection, (iii) apicobasal texture analysis, (iv) glia segmentation, and (v) cell feature quantification. Muller Glia (MG) are the principal retinal glial cell type with a stereotypic shape linked to their maturation and physiological status. We here characterized MG on three levels, including (a) global image-level, (b) apicobasal texture, and (c) apicobasal vertical-to-horizontal alignment. Using GliaMorph, we show structural changes occurring in the developing retina. Additionally, we study the loss of cadherin2 in the zebrafish retina, as well as a glaucoma mouse disease model. The GliaMorph toolkit enables an in-depth understanding of MG morphology in the developing and diseased retina. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/490765v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@198fca6org.highwire.dtl.DTLVardef@f319fcorg.highwire.dtl.DTLVardef@2f0fadorg.highwire.dtl.DTLVardef@1ae4ce7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGlial morphology is complex, making it challenging to accurately quantify 3D cell shape. C_LIO_LIWe developed the GliaMorph toolkit for image pre-processing, glial segmentation, and quantification of Muller glial cells. C_LIO_LIMuller glia elaborate their morphology and rearrange subcellular features during embryonic development. C_LIO_LIGliaMorph accurately identifies subcellular changes in models with disrupted glia cells, including zebrafish cadherin2 loss of function and a mouse glaucoma model. C_LI

developmental biology↗

bioRxiv2020

Age-related degeneration leads to gliosis but not regeneration in the zebrafish retina

Ageing is a significant risk factor for degeneration of the retina. Harnessing the regenerative potential of Muller glia cells (MG) in the retina offers great promise for the treatment of blinding conditions. Yet, the impact of ageing on MG regenerative capacity has not yet been considered. Here we show that the zebrafish retina undergoes telomerase-independent age-related neurodegeneration. Yet, this progressive neuronal loss in the ageing retina is insufficient to stimulate the MG regenerative response. Instead, age-related neurodegeneration leads to MG gliosis and loss of vision, similarly to humans. Nevertheless, gliotic MG cells retain Yap expression and the ability to regenerate neurons after acute light damage. Therefore, we identify key differences in the MG response to acute versus chronic damage in the zebrafish retina and show that aged gliotic MG can be stimulated to repair damaged neurons in old age. SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/174821v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1a184daorg.highwire.dtl.DTLVardef@1193750org.highwire.dtl.DTLVardef@7e65c0org.highwire.dtl.DTLVardef@4a2fe3_HPS_FORMAT_FIGEXP M_FIG C_FIG Our data suggest there are key differences between mechanisms driving regeneration in response to acute damage versus age-related chronic damage. It may be that either the number of cells dying in natural ageing is not enough to stimulate MG to proliferate, or the low number of microglia and respective signals released are not sufficient to trigger MG proliferation. Importantly, we show that gliotic MG cells can be stimulated to repair damaged neurons in old zebrafish retina.

neuroscience↗

bioRxiv2019

Sodium nitroprusside prevents the detrimental effects of glucose on the neurovascular unit and behaviour in zebrafish

Diabetes is associated with dysfunction of the neurovascular unit, although the mechanisms of this are incompletely understood, and currently no treatment exists to prevent these negative effects. We previously found that the NO donor sodium nitroprusside (SNP) prevents the detrimental effect of glucose on neurovascular coupling in zebrafish. We therefore sought to establish the wider effects of glucose exposure on both the neurovascular unit and on behaviour in zebrafish and the ability of SNP to prevent these.\n\nWe incubated 4 days post fertilisation (dpf) zebrafish embryos in 20mM glucose or mannitol for five days until 9dpf, with or without 0.1mM SNP co-treatment for 24h (8-9dpf), and quantified vascular nitric oxide reactivity, vascular mural cell number, expression of a klf2a reporter, glial fibrillary acidic protein (GFAP) and TRPV4, as well as spontaneous neuronal activation at 9dpf, all in the optic tectum. We also assessed the effect on light/dark preference and locomotory characteristics during free-swimming studies.\n\nWe find that glucose exposure significantly reduced nitric oxide reactivity, klf2a reporter expression, vascular mural cell number and TRPV4 expression, while significantly increasing spontaneous neuronal activation and GFAP expression (all in the optic tectum). Furthermore, when we examined larval behaviour we found glucose exposure significantly altered light/dark preference and high and low speed locomotion while in light. Co-treatment with SNP reversed all these molecular and behavioural effects of glucose exposure.\n\nOur findings comprehensively describe the negative effects of glucose exposure on the vascular anatomy, molecular phenotype, and function of the optic tectum and on whole organism behaviour. We also show that SNP or other NO donors may represent a therapeutic strategy to ameliorate the complications of diabetes on the neurovascular unit.

neuroscience↗

bioRxiv2018

Genetic control of cellular morphogenesis in Müller glia

Of all the cells in the body, those with the greatest variety of shapes reside in the central nervous system yet they all start their postmitotic lives as simple elongated cells of the neuroepithelium1.The molecular processes by which these, or indeed any, cells gain their particular cell-specific anatomies remain largely unexplored.We, therefore, developed a strategy to identify the genes involved in cellular morphogenesis using Muller glial (MG) cells in the vertebrate retina as a model system.These radially oriented cells, discovered by Heinrich Muller in 1851 and named in his honour2, are astonishingly complex yet, as the great neurohistologist Ramon y Cajal first noted, they share a conserved set of key anatomical features3.Using genomic and CRISPR based strategies in zebrafish, combined with a temporal dissection of the process, we found more than 40 genes involved in MG cell morphogenesis.Strikingly, the sequential steps of anatomical feature addition are regulated by successive expression of cohorts of interrelated genes, revealing unprecedented insights into the developmental genetics of cellular morphogenesis.

cell biology↗