Biology subjects

Ruzycki, P. A.

Publications and source records attributed to Ruzycki, P. A..

7 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↗

bioRxiv2025

The long noncoding RNA Peanut (Gm11454) promotes neurogenesis and rod photoreceptor differentiation during postnatal retinal development.

Long noncoding RNAs (lncRNAs) display pervasive expression and function in the developing nervous system. Temporal profiling of gene expression in the retina has demonstrated differential expression of lncRNAs throughout development; however, determinations of lncRNA function during retinal development remain limited. In this study, we identify numerous lncRNAs with dynamic temporal expression and characterize the function of the lncRNA Gm11454, which we have named Peanut. Using overexpression of Peanut in mice retinas, we determine that Peanut promotes rod photoreceptor fate and neurogenesis of retinal progenitor cells (RPCs) via inhibition of Notch signaling and by regulating expression of neighboring gene Tox2. A novel Peanut knockout mouse model demonstrates that Peanut is required for proper visual function and photoreceptor gene expression. Finally, we determined that Peanut is necessary for proper cell cycle progression and neurogenesis. Our results characterize the function of a novel lncRNA as a regulator of RPC neurogenesis and differentiation and support the importance of lncRNAs in the developing retina.

developmental biology↗

bioRxiv2025

A genome-wide in vivo CRISPR screen identifies neuroprotective strategies in the mouse and human retina

Retinitis pigmentosa (RP) is a genetically diverse blinding disorder lacking broadly effective therapies. We performed a genome-wide in vivo CRISPR knockout screen in mice carrying the P23H rhodopsin mutation (the most common cause of autosomal dominant RP in the United States) to systematically identify neuroprotective genes. We discovered multiple knockouts that accelerated rod photoreceptor loss, validated top candidates, and showed that overexpressing two genes--UFD1 and UXT--preserved rods and cones, maintained retinal function, and improved visual behaviors. To accelerate translation, we developed a human P23H RP model in adult retinal explants, recreating key disease features. UFD1 and UXT augmentation prevented photoreceptor loss in human P23H retinas. Our findings establish a pipeline for systematic identification and translational testing of neuroprotective genes in mouse and human RP models, provide a novel set of validated candidate genes, and underscore the therapeutic promise of UFD1 and UXT as mutation-agnostic strategies to preserve vision.

neuroscience↗

bioRxiv2025

Active DNA demethylation is upstream of rod-photoreceptor fate determination and required for retinal development

Retinal cell fate specification from multipotent retinal progenitors is governed by dynamic changes in chromatin structure and gene expression. Methylation at cytosines in DNA (5mC) is actively regulated for proper control of gene expression and chromatin architecture. Numerous genes display active DNA demethylation across retinal development; a process that requires oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and is controlled by the ten-eleven translocation methylcytosine dioxygenase (TET) enzymes. Using an allelic series of conditional TET enzyme mutants, we determine that DNA demethylation is required upstream of NRL and NR2E3 expression for the establishment of rod-photoreceptor fate. Using histological, behavioral, transcriptomic, and base-pair resolution DNA methylation analyses, we establish that inhibition of active DNA demethylation results in global changes in gene expression and methylation patterns that prevent photoreceptor precursors from adopting a rod-photoreceptor fate, instead producing a retina in which all photoreceptors specify as cones. Our results establish the TET enzymes and DNA demethylation as critical regulators of retinal development and cell fate specification, elucidating a novel mechanism required for the specification of rod-photoreceptors.

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↗

bioRxiv2022

Rho enhancers play unexpectedly minor roles in Rhodopsin transcription and rod cell integrity

Enhancers function with a basal promoter to control the transcription of target genes. Enhancer regulatory activity is often studied using reporter-based transgene assays. However, unmatched results have been reported when selected enhancers are silenced in situ. In this study, using genomic deletion analysis in mice, we investigated the roles of two previously identified enhancers and the promoter of the Rho gene that codes for the visual pigment rhodopsin. The Rho gene is robustly expressed by rod photoreceptors of the retina, and essential for the subcellular structure and visual function of rod photoreceptors. Mutations in RHO cause severe vision loss in humans. We found that each Rho regulatory region can independently mediate local epigenomic changes, but only the promoter is absolutely required for establishing active Rho chromatin configuration and transcription and maintaining the cell integrity and function of rod photoreceptors. To our surprise, two Rho enhancers that enable strong promoter activation in reporter assays are largely dispensable for Rho expression in vivo. Only small and age-dependent impact is detectable when both enhancers are deleted. Our results demonstrate context-dependent roles of enhancers and highlight the importance of studying functions of cis-regulatory regions in the native genomic context.

genomics↗

bioRxiv2021

Essential functions of MLL1 and MLL2 in retinal development and cone cell maintenance

MLL1 (KMT2A) and MLL2 (KMT2B) are homologous members of the mixed-lineage leukemia (MLL) family of histone methyltransferases involved in epigenomic transcriptional regulation. Their sequence variants have been associated with neurological and psychological disorders, but little is known about their roles and mechanism of action in CNS development. Using mouse retina as a model, we previously reported MLL1s role in retinal neurogenesis and horizontal cell maintenance. Here we determine roles of MLL2 and MLL1/MLL2 together in retinal development using conditional knockout (CKO) mice. Deleting Mll2 from Chx10+ retinal progenitors resulted in a similar phenotype as Mll1 CKO, but removal of both alleles produced much more severe deficits than each single CKO: 1-month double CKO mutants displayed null light responses in electroretinogram; thin retinal layers, including shorter photoreceptor outer segments with impaired phototransduction gene expression; and reduced numbers of M-cones, horizontal and amacrine neurons, followed by fast retinal degeneration. Despite moderately reduced progenitor cell proliferation at P0, the neurogenic capacity was largely maintained in double CKO mutants. However, upregulated apoptosis and reactive gliosis were detected during postnatal retinal development. Finally, the removal of both MLLs in fated rods produced a normal phenotype, but the CKO in M-cones impaired M-cone function and survival, indicating both cell non-autonomous and autonomous mechanisms. Altogether, our results suggest that MLL1/MLL2 play redundant roles in maintaining specific retinal neurons after cell fate specification and are essential for establishing functional neural networks.

neuroscience↗