Biology subjects

Wythe, J. D.

Publications and source records attributed to Wythe, J. D..

7 recordsLinked to original sources

bioRxiv2026

Cross-Species Efficacy of Combinatorial Gene Therapy for Osteoarthritis and Correction of Neuro-Inflammatory Pain Mechanisms

Osteoarthritis is a leading cause of chronic pain and disability, which lacks disease-modifying treatment. Given the complex multi-tissue and multifactorial drivers behind disease progression, effective treatments will require simultaneously targeting several mechanisms underlying joint degeneration and pain. Here, we developed and evaluated a combinatorial gene therapy, consisting of a high-capacity adenoviral vector carrying two therapeutic genes to target distinct pathological mechanisms: inflammation (IL-1Ra) and chondrocyte health (PRG4). Intra-articular delivery of this treatment improved functional, structural, and pain outcomes in murine and equine osteoarthritis models. In addition, treatment normalized inflammatory environments in joint tissues, as well as in the dorsal root ganglia (DRG) known to harbor joint-innervating sensory neurons. Moreover, gene therapy reversed OA-induced molecular signatures of neural hyperexcitability, suggesting amelioration of peripheral sensitization. Collectively, these findings support combinatorial gene therapy as a promising treatment for osteoarthritis, while identifying neuroinflammatory signatures for correction of disease progression and pain. One Sentence SummaryA single intra-articular injection of a combinatorial gene therapy slows OA progression and reduces pain in small and large animal models.

systems biology↗

bioRxiv2025

KneEZ Clear, an Effective Tissue Clearing Protocol to Study Musculoskeletal Tissues in the Mouse

Wholemount, 3-dimensional (3D) tissue imaging holds significant promise for analyzing heterogeneous musculoskeletal tissues, such as knee joints, that demand time- and labor-intensive processing using traditional histological methods. Current musculoskeletal clearing protocols rely on either solvent-based tissue clearing, which substantially alters the size and architecture of cleared tissues, possibly compromising downstream quantification and perhaps more importantly reducing signal from endogenous fluorescent reporters, or on expensive and time-consuming hydrogel-based approaches that requires specialized equipment. While aqueous-based clearing overcomes these challenges, there is a clear need for a method that is optimized for clearing musculoskeletal tissues and that can easily be implemented in a standard lab environment. Here, we present KneEZ Clear, a simple, rapid, and flexible aqueous-based method that renders mineralized and non-mineralized tissues of murine knee joints optically transparent. We show that KneEZ Clear, which is based on the EZ Clear method, is highly flexible, demonstrating efficacy in a wide range of murine musculoskeletal tissues including the vertebral column, hindlimb, skull, and teeth. Critically, KneEZ Clear does not require specialized equipment and retains endogenous signal from fluorophores and fluorescent proteins. Additionally, following clearing and wholemount imaging, precious samples can still be processed for subsequent 2D histological analyses for validation or further study. Finally, we show that KneEZ Clear can be applied to samples of disease models to reveal alterations in tissue architecture and homeostasis. The simplicity, versatility, and efficiency of KneEZ Clear for optical clearing of musculoskeletal tissues will accelerate our understanding of cellular interactions and dynamics in homeostasis and disease.

physiology↗

bioRxiv2024

MEK signaling represents a viable therapeutic vulnerability of KRAS-driven somatic brain arteriovenous malformations

Brain arteriovenous malformations (bAVMs) are direct connections between arteries and veins that remodel into a complex nidus susceptible to rupture and hemorrhage. Most sporadic bAVMs feature somatic activating mutations within KRAS, and endothelial-specific expression of the constitutively active variant KRASG12D models sporadic bAVM in mice. By leveraging 3D-based micro-CT imaging, we demonstrate that KRASG12D-driven bAVMs arise in stereotypical anatomical locations within the murine brain, which coincide with high endogenous Kras expression. We extend these analyses to show that a distinct variant, KRASG12C, also generates bAVMs in predictable locations. Analysis of 15,000 human patients revealed that, similar to murine models, bAVMs preferentially occur in distinct regions of the adult brain. Furthermore, bAVM location correlates with hemorrhagic frequency. Quantification of 3D imaging revealed that G12D and G12C alter vessel density, tortuosity, and diameter within the mouse brain. Notably, aged G12D mice feature increased lethality, as well as impaired cognition and motor function. Critically, we show that pharmacological blockade of the downstream kinase, MEK, after lesion formation ameliorates KRASG12D-driven changes in the murine cerebrovasculature and may also impede bAVM progression in human pediatric patients. Collectively, these data show that distinct KRAS variants drive bAVMs in similar patterns and suggest MEK inhibition represents a non-surgical alternative therapy for sporadic bAVM.

molecular biology↗

bioRxiv2021

FOXO1 represses Sprouty2 and Sprouty4 expressionin endothelial cells to promote arterial specification and vascular remodeling in the mouse yolk sac

The establishment of a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss of function mouse models have shown that FOXO1, a Forkhead family transcription factor, is required for yolk sac vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that loss of FoxO1 in E8.25 endothelial cells results in increased Sprouty2 and Sprouty4 transcripts, reduced expression of arterial genes, and decreased Flk1/Vegfr2 mRNA levels without affecting overall endothelial cell identity, survival, or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, Delta like 4 (Dll4), is significantly reduced in the yolk sac of FoxO1 mutants without being substantially affected in the embryo proper. We show that in the yolk sac, FOXO1 not only binds directly to a subset of previously identified Sprouty2 gene regulatory elements (GREs), as well as newly identified, evolutionarily conserved Sprouty4 GREs, but can also repress their expression. Additionally, over expression of Sprouty4 in transient transgenic embryos largely recapitulates reduced expression of arterial genes seen in endothelial FoxO1 mutant mouse embryos. Together, these data reveal a novel role for FOXO1 as a key early transcriptional repressor controlling both pre-flow arterial specification and subsequent vessel remodeling within the murine yolk sac.

developmental biology↗

bioRxiv2019

Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that Vascular endothelial growth factor A (Vegfa) from the epithelium specifies a distinct endothelial cell (EC) population in the postnatal mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single cell RNA-seq identified 15-20% lung ECs as transcriptionally distinct and marked by Carbonic anhydrase 4 (Car4), which are specifically lost upon epithelial Vegfa deletion. Car4 ECs, unlike bulk ECs, have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without intervening pericytes. Without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct transcriptional profiles. These findings support a signaling role of AT1 cells and shed light on alveologenesis.

developmental biology↗

bioRxiv2018

FishNET: An automated relational database for zebrafish colony management.

The zebrafish Danio rerio is a powerful model system to study the genetics of development and disease. However, maintenance of zebrafish husbandry records is both time intensive and laborious, and a standardized way to manage and track the large amount of unique lines in a given laboratory or centralized facility has not been embraced by the field. Here we present FishNet, an intuitive, open source, relational database for managing data and information related to zebrafish husbandry and maintenance. By creating a \"virtual facility\", FishNET enables users to remotely inspect the rooms, racks, tanks and lines within a given facility. Importantly, FishNET scales from one laboratory, to an entire facility with several laboratories, to multiple facilities, generating a cohesive laboratory and community-based platform. Automated data entry eliminates confusion regarding line nomenclature and streamlines maintenance of individual lines, while flexible query forms allow researchers to retrieve database records based on user-defined criteria. FishNet also links associated embryonic and adult biological samples with data, such as genotyping results or confocal images, to enable robust and efficient colony management and storage of laboratory information. A shared calendar function with email notifications and automated reminders for line turnover, automated tank counts and census reports promote communication with both end-users and administrators. The expected benefits of FishNET are improved vivaria efficiency, increased quality control for experimental numbers, and flexible data reporting and retrieval. FishNets easy, intuitive record management and open source, end user-modifiable architecture provides an efficient solution to real-time zebrafish colony management for users throughout a facility and institution, and in some cases across entire research hubs.

genetics↗

bioRxiv2017

A Novel Reporter Allele For Monitoring Dll4 Expression Within The Embryonic And Adult Mouse

Canonical Notch signaling requires the presence of a membrane bound ligand and a corresponding transmembrane Notch receptor. Receptor engagement induces multiple proteolytic cleavage events culminating in the nuclear accumulation of the Notch intracellular domain and its binding to a transcriptional co-factor to mediate gene expression. Notch signaling networks are essential regulators of vascular patterning and angiogenesis, as well as myriad other biological processes. Delta-like 4 (Dll4) encodes the earliest Notch ligand detected in arterial cells, and is enriched in sprouting endothelial tip cells. Dll4 expression has often been inferred by proxy using a lacZ knockin reporter allele. This is problematic, as a single copy of Dll4 is haploinsufficient. Additionally, Notch activity regulates Dll4 transcription, making it unclear whether these reporter lines accurately reflect Dll4 expression. Accordingly, accurately defining Dll4 expression is essential for determining its role in development and disease. To address these limitations, we generated a novel BAC transgenic allele with a nuclear-localized {beta}-galactosidase reporter (Dll4-BAC-nlacZ). Through a comparative analysis, we show the BAC line overcomes previous issues of haploinsufficiency, it recapitulates Dll4 expression in vivo, and allows superior visualization and imaging. As such, this novel Dll4 reporter is an important addition to the growing Notch toolkit.\n\nSummary Statement:\n\nWe have developed a novel reporter line, free from complicating factors associated with previous alleles, for monitoring Dll4 expression, at a cellular resolution, in the developing and adult mouse.

developmental biology↗