Biology subjects

Thyme, S. B.

Publications and source records attributed to Thyme, S. B..

8 recordsLinked to original sources

bioRxiv2026

Zebrafish screen of schizophrenia risk genes reveals convergent dysregulation of cholesterol metabolism

Rare coding variants provide a tractable entry point for understanding the molecular mechanisms underlying schizophrenia risk. Here, we generated and characterized zebrafish lines with mutations in the orthologs of >20 human schizophrenia-associated genes, including eight of the top ten SCHEMA genes, genes disrupted in childhood-onset schizophrenia (COS), and genes located within recurrent copy number variants. Whole-brain phospho-Erk activity mapping and behavioral profiling identified phenotypes in multiple mutant lines. We prioritized a protein-truncating mutation in sp4, which encodes an activity-dependent transcription factor, and a COS-associated missense mutation in atp1a3a, which encodes a Na+/K+ ATPase pump, for additional characterization. Both knockout and point mutations in atp1a3a disrupted brain activity and behavior in larvae and impaired navigation of a Y-maze in juveniles. Bulk RNA sequencing data from adult sp4 and atp1a3a brains highlighted convergent upregulation of sterol biosynthesis pathways, including increased expression of srebf2 and msmo1. Analysis of previously published telencephalon single-cell data demonstrated that cholesterol synthesis genes are enriched in astrocyte-like cells and increase in expression during post-larval development. Consistent with transcriptomic findings, filipin staining indicated increased free cholesterol in juvenile sp4 and atp1a3a mutant brains. Our findings identify dysregulation of glial and sterol-associated programs as a shared molecular consequence of two distinct schizophrenia risk mutations. Although whether sterol pathway dysregulation represents a primary pathogenic mechanism or a secondary response to changes in neuronal activity requires further investigation, the convergence observed between genetic models and developmental stages suggests that disruptions to lipid homeostasis could represent a shared feature of schizophrenia disease biology.

genetics↗

bioRxiv2026

StrIPETrack: a real-time, ROI-flexible tracking platform for high-throughput zebrafish behavior

Quantitative phenotyping is essential to studies of animal behavior, enabling systematic analysis of variation arising from natural diversity or experimental manipulation. High-throughput behavioral assays that can simultaneously test multiple animals support sufficiently powered studies of behavioral variation, but accurate tracking of each animal is critical. Furthermore, behavioral tasks and experimental arenas span a wide range of complexity, from the reaction of a single larval zebrafish to an acoustic stimulus to associative conditioning in cue-rich environments. Here, we developed and validated StrIPETrack (Structural similarity-based Image Processing for Estimation and Tracking), a Python-based, modular animal tracking software designed for flexible region-of-interest (ROI) definitions and extensibility across assays. We show that StrIPETrack measures activity comparably to our previous LabVIEW-based zebrafish tracking software and detects similar behavioral differences between wild-type clutches. In addition, StrIPETrack accurately captures behavior in a complex arena: the Y-maze. Our approach for analyzing Y-maze navigation yields an expanded set of metrics beyond turn count and direction, revealing more subtle behavioral variation. Overall, this versatile software can be applied to monitor the activity of multiple animals in parallel in both simple high-throughput and more complex assays, and can be readily adapted to new paradigms. SummaryOur open-source tracking software provides rich behavioral phenotyping of animals in many behavioral tasks. The flexible ROI design and live tracking makes the software adaptable to diverse paradigms.

animal behavior and cognition↗

bioRxiv2025

Gli3R-mediated inhibition of hedgehog signaling alters the embryonic transcriptome in zebrafish

Hedgehog signaling is a conserved developmental pathway that patterns diverse tissues during vertebrate embryogenesis. In zebrafish, disruptions to the hedgehog pathway cause well-characterized defects in specific cell types including neurons and glia derived from the ventral neural tube. We inhibited hedgehog signaling by overexpressing the Gli3 repressor ubiquitously and performed bulk RNA-seq of 30 hours post-fertilization zebrafish embryos. Consistent with known roles of hedgehog signaling, we observed reduced expression of genes marking lateral floor plate, motor neurons, Kolmer-Agduhr cells, dopaminergic neurons, slow muscle cells, and anterior pituitary. Gene set enrichment analysis using marker genes derived from the Daniocell atlas also revealed downregulation of genes marking H+-ATPase-rich and Na+-K+-ATPase-rich ionocytes, which are located in the embryonic skin and are responsible for osmotic homeostasis. Reduced expression of ionocyte-specific transporter genes and the transcription factor foxi3a suggests that Gli activity may play a previously unrecognized role in the development of this cell type.

developmental biology↗

bioRxiv2025

Single Cell Multiomics Across Nine Mammals Reveals Cell Type Specific Regulatory Conservation in the Brain

Understanding the gene regulatory mechanisms underlying brain function is crucial for advancing knowledge of the genetic basis of neurologic diseases. Cis-regulatory elements (CREs) play a pivotal role in gene regulation, and their evolutionary conservation can offer valuable insights. Importantly, the function and evolution of CREs are affected not only by primary sequence, but also by the cis- and trans-regulatory context. However, comparative functional analyses across species have been limited, leaving how these regulatory landscapes evolve in the brain largely unresolved. Here, we generated single-nucleus multiomic (snRNA- and snATAC-seq) data from cortex tissue across nine mammalian species and identified candidate CREs (cCREs) in a cell type-specific manner. We developed a multidimensional framework of conservation to assess sites of shared function that integrates sequence, chromatin accessibility, and enhancer-gene associations. Using massively parallel reporter assays (MPRA) in human neural progenitor cells and neurons, we measured activity of cCREs including both conserved and human-specific regions. CRISPR interference validated conserved enhancer function, including at neurodevelopmentally important genes like FAM181B. Motif enrichment identified transcription factors distinguishing conserved versus recent cCREs. Linkage disequilibrium score regression indicated that both conserved and human-specific cCREs were enriched for neuropsychiatric GWAS risk, while neurodegenerative risk was confined to conserved elements. Our findings define functional dimensions of enhancer conservation and demonstrate how regulatory evolution shapes human brain biology and disease susceptibility.

genomics↗

bioRxiv2025

Genetic context of transgene insertion can influence neurodevelopment in zebrafish

The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish, the Tg(elavl3:KalTA4) (HuC) Gal4 line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tg(elavl3:KalTA4) zebrafish larvae compared to wildtype siblings, which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tg(elavl3:KalTA4) larvae showed alterations in brain activity, brain morphology, and behavior, including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA-seq analysis revealed dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis, we used economical low-pass whole genome sequencing to map the Tol2-mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga, a known cell cycle regulator, is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan-neuronal lines include the length of the elavl3 promoter (over 8 kb) and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines, we cloned five neuronal promoters (atp6v0cb, smaller elavl3, rtn1a, sncb, and stmn1b) ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase-based pIGLET system. Our study highlights the importance of using appropriate genetic controls and interrogating potential positional effects in new transgenic lines. ARTICLE SUMMARYThe Gal4/UAS system is a genetic tool that allows researchers to overexpress specific genes in specific cell types by pairing a Gal4 "driver" line with a UAS "reporter" line. In zebrafish, more than 500 transgenic Gal4 lines have been generated using transgenesis methods that insert DNA into random sites in the genome. Here, the authors report that a pan-neuronal Gal4 zebrafish line, Tg(elavl3:KalTA4), has neurobehavioral phenotypes, including changes in brain activity, brain structure, and behavior. These findings emphasize the importance of using appropriate controls and highlight the utility of methods for targeted transgenesis.

genetics↗

bioRxiv2024

Diencephalic and Neuropeptidergic Dysfunction in Zebrafish with Autism Risk Mutations

Hundreds of human mutations are linked to autism and related disorders, yet the functions of many of these mutated genes during vertebrate neurodevelopment are unclear. We generated 27 zebrafish mutants with presumptive protein-truncating mutations or specific missense variants corresponding to autism-risk alleles in 17 human genes. We observed baseline and stimulus-driven behavioral changes at larval stages, as well as social behavior differences in lines tested as juveniles. Imaging whole-brain activity revealed a near identical activity map for mutations in the unrelated genes kmt5b and hdlbpa, defined by increased activity mainly in the diencephalon. Mutating 7 of the 17 risk genes resulted in substantial brain size differences. Using RNA sequencing, we further defined molecular drivers of the observed phenotypes, identifying targetable disruptions in neuropeptide signaling, neuronal maturation, and cell proliferation. This multi-modal screen nominated brain regions, cell types, and molecular pathways that may contribute to autism susceptibility. TeaserZebrafish screen uncovers diencephalon, social interaction, and neuropeptidergic signaling phenotypes in ASD risk mutants.

genetics↗

bioRxiv2020

A Customizable Low-Cost System for Massively Parallel Zebrafish Behavior Phenotyping

High-throughput behavioral phenotyping is critical to genetic or chemical screening approaches. Zebrafish larvae are amenable to high-throughput behavioral screening because of their rapid development, small size, and conserved vertebrate brain architecture. Existing commercial behavior phenotyping systems are expensive and not easily modified for new assays. Here, we describe a modular, highly adaptable, and low-cost behavior system. Along with detailed assembly and operation instructions, we provide data acquisition software and a robust, parallel analysis pipeline. We validate our approach by analyzing stimulus response profiles in larval zebrafish, confirming prepulse inhibition phenotypes of two previously isolated mutants, and highlighting best practices for growing larvae prior to behavioral testing. Our new design thus allows rapid construction and streamlined operation of many large-scale behavioral setups with minimal resources and fabrication expertise, with broad applications to other aquatic organisms.

neuroscience↗

bioRxiv2018

Phenotypic landscape of schizophrenia-associated genes defines candidates and their shared functions

Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologues of 132 human schizophrenia-associated genes and created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities. Phenotypes were diverse but specific, including altered forebrain development and decreased prepulse inhibition. Exploration of these datasets identified promising candidates in more than 10 gene-rich regions, including the magnesium transporter cnnm2 and the translational repressor gigyf2, and revealed shared anatomical sites of activity differences, including the pallium, hypothalamus or tectum. Single-cell RNA sequencing uncovered an essential role for the understudied transcription factor znf536 in the development of forebrain neurons implicated in social behavior and stress. This phenotypic landscape of schizophrenia-associated genes prioritizes more than 30 candidates for further study and provides hypotheses to bridge the divide between genetic association and biological mechanism.

genetics↗