Biology subjects

Margolin, G.

Publications and source records attributed to Margolin, G..

12 recordsLinked to original sources

bioRxiv2025

Specialized gas-exchange endothelium of the zebrafish gill

The pulmonary vasculature plays a critical role in gas exchange and in lung pathologies, but it is challenging to observe and experimentally manipulate deep within the lungs of living mammals. Unlike mammalian lungs, externally located zebrafish gills are readily accessible for high-resolution optical imaging and experimental manipulation, suggesting zebrafish might provide an excellent comparative vertebrate model for studying the development and function of gas-exchange organs and the gas-exchange blood vasculature. To characterize their resident cell populations, we performed single-cell RNA sequencing (scRNAseq) on adult zebrafish gills, revealing numerous cell types with transcriptional similarities to those found in mammalian lungs. We uncovered and characterized several different endothelial cell populations, including distinct clusters of arterial endothelial cells and lymphatic endothelial cells. The largest endothelial cell cluster closely resembles Cap2 or "Aerocyte" endothelial cells, a recently discovered unusual mammalian endothelial cell type found exclusively in lung alveoli. Zebrafish aerocytes localize to the analogous gas-exchange structures in fish, the highly vascularized gill lamellae. We use confocal and super-resolution imaging of transgenic and hybridization chain reaction-probed zebrafish, array tomography, and focused ion beam scanning electron microscopy to carry out a detailed and comprehensive characterization of gill aerocytes including 3-D ultrastructural reconstruction of one of these cells, showing that as in mammals these cells are closely associated with gas-exchange epithelia and that they possess unique properties that may help facilitate their gas-exchange function. Together, our findings help establish a new, experimentally accessible comparative vertebrate model for studying the gas-exchange blood vasculature.

developmental biology↗

bioRxiv2025

Differential expression analysis in single cell and spatial RNASeq without model assumptions

Gene up(down)regulation findings in single cell and spatial RNASeq can be inconsistent despite remarkable progress in technology. False findings in high-quality samples raise concerns about assumptions behind widely accepted data analysis approaches. We therefore propose a weighted averaging approach for data analysis without assuming anything besides randomness of technical noise. This approach is closely related to prior work on statistics of cluster-randomized experiments. We show that weighing transcript counts based on measured noise variances and utilizing weighted rather than standard unweighted tests reduces both false positive and false negative findings. Our approach eliminates the need for parametrizing data distributions and/or rescaling transcript counts, which may cause artifacts by distorting and biasing the data. The resulting analysis is less complex and produces more consistent differential gene expression estimates.

genomics↗

bioRxiv2025

Discovery and performance of DNA methylation panels for cancer detection and classification in blood

Examining DNA in a liquid biopsy for non-invasive cancer detection relies on identifying dilute signal in a high background. This study aims to identify DNA methylation biomarkers for multi-cancer detection. Utilizing large tissue datasets, we apply novel search algorithms to discover confined biomarker panels capable of distinguishing tumor from normal and determining the tissue of origin. We explore the applicability to blood-based testing using targeted methylation sequencing followed by machine learning classification. We present an 8-marker panel, which successfully predicts tumors across 14 types with a 91% average sensitivity, maintaining a low false positive rate (< 0.04%). Additionally, a panel of 39 CpG sites exhibits accuracies ranging from 69% to 98% for identifying tissue of origin. When tested on 114 patient plasma samples (colon, liver, pancreatic, prostate, and stomach cancer), the 8-marker panel obtains an AUC of 0.78 with a 78% sensitivity among 32 early-stage patients (stage I-II), and 60% overall. Using the 39-marker panel in a multi-class classification model selecting only the best match, 54% of tumor samples were on average correctly assigned to the tissue of origin, and up to 80% when allowing more inclusive criteria. Using a limited set of biomarkers, our work contributes to advancing non-invasive cancer diagnostics.

cancer biology↗

bioRxiv2025

A novel transgenic reporter to study vertebrate epigenetics

Epigenetic reprogramming contributes to the generation of cellular diversity during vertebrate development but the mechanisms directing this are still not well understood. Large-scale genetic screens have been highly successful in identifying epigenetic regulatory genes in invertebrates such as worms and flies, but similar large-scale genetic screens to identify epigenetic regulators have not been carried out in vertebrates. Here we report a newly generated "EpiTag" zebrafish transgenic reporter line that permits easy cellular-level visualization of epigenetic silencing or activation in living animals during development, gametogenesis, and regeneration. We use the EpiTag reporter to carry out an F3 ENU mutagenesis screen for epigenetic silencing or activating mutants, identifying relevant vertebrate tissue-specific epigenetic regulatory genes including a new epigenetic model for metabolic dysfunction-associated fatty liver disease (MAFLD). The EpiTag reporter line represents a powerful new tool for genetic and experimental analysis of tissue-specific epigenetic gene regulation in vertebrates. One Sentence SummaryEpiTag transgenic zebrafish provide a powerful new tool for visualizing and studying epigenetic regulation in living vertebrate animals.

genomics↗

bioRxiv2025

Early epigenetic priming of regeneration studied with a novel transgenic epigenetic reporter

Tissue regeneration requires previously differentiated cells to regain developmental plasticity. However, the upstream mechanisms initiating this process remain poorly understood. Here, we leverage a novel "EpiTag" transgenic zebrafish reporter line that enables real-time visualization of epigenetic silencing and activation to identify and carry out a comprehensive multi-omics analysis of cells undergoing epigenetic reprogramming during caudal fin regeneration. EpiTag GFP expression is transiently activated in cells contributing to regeneration between 12 to 16 hours post amputation (hpa), preceding the expression of canonical blastema markers. Single-cell RNA-seq reveals that GFP+ cells are restricted to regeneration-competent lineages such as pre-osteoblasts, proliferating cells, and wound epithelium. Integrated bulk RNA-seq, time-course RNA-seq, ATAC-seq, and bisulfite-seq on FACS-isolated GFP+ cells uncovers an early gene expression module enriched for chromatin regulators and a late gene expression module enriched for morphogenesis genes. Chromatin accessibility and DNA methylation changes are strongly associated with these late-expressed genes, suggesting epigenetic priming. We identify a number of epigenetic factors upregulated in the early gene expression module and show that ruvbl1 and ruvbl2, components of ATP-dependent chromatin remodeling complexes, are required for proper regeneration in both adult fins and larval tails. Our results establish EpiTag transgenics as a powerful in vivo tool for studying epigenetic reprogramming and highlight early chromatin remodeling events that enable activation of regenerative gene expression programs.

genomics↗

bioRxiv2025

Anatomical and Molecular Characterization of the Zebrafish Meninges

The meninges are a set of connective tissue layers that surround the central nervous system, protecting the brain from mechanical shock, supporting its buoyancy, guarding it from infection and injury, and maintaining brain homeostasis. Despite their critical role, the molecular identity, developmental origins, and functional properties of the cell types populating the meninges remain poorly characterized. This is in large part due to lack of cell type specific markers and difficulty in visualizing and studying these structures through the thick mammalian skull. Here, we show that the zebrafish, a genetically and experimentally accessible vertebrate, possesses an easily imaged mammalian-like meninges. Anatomical and cellular characterization of its composition via histology, electron microscopy, and confocal imaging shows that the adult zebrafish possesses complex multilayered meninges with double-layered dura mater and intricate leptomeningeal layers. Using single cell transcriptomics, we define the molecular identities of meningeal cell populations, including a unique ependymin (epd)-expressing cell population that constitutes the major cellular component of the leptomeningeal barrier and is essential for brain development and survival. These findings support the use of zebrafish as a useful comparative model for studying the meninges, provide a foundational description for future zebrafish meningeal research, and identify a new Leptomeningeal Barrier Cell that serves as the primary epithelial cell component of the leptomeninges.

developmental biology↗

bioRxiv2025

Elevated body temperature exacerbates arrhythmia and seizure-like activity in a zebrafish model of Timothy syndrome

Timothy syndrome (TS) is a multisystem disorder with autistic-like features, seizures and arrhythmia as the main symptoms. Most TS cases are caused by a de novo single amino acid substitution G406R in the CACNA1C gene that encodes the pore-forming subunit of the voltage-gated L-type calcium channel CaV1.2. We generated a zebrafish model of TS with a homologous amino acid substitution in the cacna1c-encoded protein. Unlike patients, heterozygous mutants showed only mild impairments with no changes in mortality. However, homozygous mutants showed increased mortality, arrhythmias, neural activity and sensitivity to pentylenetetrazole-induced seizure-like behavior. Mutants also exhibited microcephaly, cerebellar hypotrophy and abnormal development of GABAergic neuron populations, and transcriptomic analysis revealed dysregulated expression of neuropeptide genes (including bdnf and vgf). Consistent with the idea that CaV1.2 channels activate during fever, we found that heterozygous larvae manifested arrhythmia and seizure-like behavior when exposed to elevated environmental temperature, and homozygous larvae switched from bradycardia to tachycardia. These data provide a basis for using zebrafish to study the etiology of TS abnormalities and suggest t hat fever may be a particular risk for TS patients.

genetics↗

bioRxiv2024

Mutations in the microexon splicing regulator srrm4 have minor phenotypic effects on zebrafish neural development

Achieving a diversity of neuronal cell types and circuits during brain development requires alternative splicing of developmentally regulated mRNA transcripts. Microexons are a type of alternatively spliced exon that are 3-27 nucleotides in length and are predominantly expressed in neuronal tissues. A key regulator of microexon splicing is the RNA-binding protein Serine/arginine repetitive matrix 4 (Srrm4). Srrm4 is a highly conserved, vertebrate splicing factor that is part of an ancient family of splicing proteins. To better understand the function of Srrm4 during brain development, we examined neural expression of zebrafish srrm4 from days 1-5 of development using fluorescence in situ hybridization. We found that srrm4 has a dynamically changing expression pattern, with expression in diverse cell types and stages during development. We then used CRISPR-based mutagenesis to generate zebrafish srrm4 mutants. Unlike previously described morphant phenotypes, srrm4 mutants did not show overt morphological defects. Moreover, sequencing of wild-type and mutant transcriptomes revealed only minor changes in splicing. srrm4 thus appears to have a limited role in zebrafish neural development.

developmental biology↗

bioRxiv2024

Parp1 deletion rescues cerebellar hypotrophy in xrcc1 mutant zebrafish

Defects in DNA single-strand break repair are associated with neurodevelopmental and neurodegenerative disorders. One such disorder is that resulting from mutations in XRCC1, a scaffold protein that plays a central role in DNA single-strand base repair. XRCC1 is recruited at sites of single-strand breaks by PARP1, a protein that detects and is activated by such breaks and is negatively regulated by XRCC1 to prevent excessive PARP binding and activity. Loss of XRCC1 leads to the toxic accumulation and activity of PARP1 at single-strand breaks leading to base excision repair defects, a mechanism that may underlie pathological changes in patients carrying deleterious XRCC1 mutations. Here, we demonstrate that xrcc1 knockdown impairs development of the cerebellar plate in zebrafish. In contrast, parp1 knockdown alone does not significantly affect neural development, and instead rescues the cerebellar defects observed in xrcc1 mutant larvae. These findings support the notion that PARP1 inhibition may be a viable therapeutic candidate in neurological disorders.

neuroscience↗

bioRxiv2023

Single-cell analysis of shared signatures and transcriptional diversity during zebrafish development

During development, animals generate distinct cell populations with specific identities, functions, and morphologies. We mapped transcriptionally distinct populations across 489,686 cells from 62 stages during wild-type zebrafish embryogenesis and early larval development (3-120 hours post-fertilization). Using these data, we identified the limited catalog of gene expression programs reused across multiple tissues and their cell-type-specific adaptations. We also determined the duration each transcriptional state is present during development and suggest new long-term cycling populations. Focused analyses of non-skeletal muscle and the endoderm identified transcriptional profiles of understudied cell types and subpopulations, including the pneumatic duct, individual intestinal smooth muscle layers, spatially distinct pericyte subpopulations, and homologs of recently discovered human best4+ enterocytes. The transcriptional regulators of these populations remain unknown, so we reconstructed gene expression trajectories to suggest candidates. To enable additional discoveries, we make this comprehensive transcriptional atlas of early zebrafish development available through our website, Daniocell.

developmental biology↗

bioRxiv2019

A Methylation Density Binary Classifier for Predicting and Optimizing the Performance of Methylation Biomarkers in Clinical Samples

BackgroundVariation in intercellular methylation patterns can complicate the use of methylation biomarkers for clinical diagnostic applications such as blood-based cancer testing. Here, we describe development and validation of a methylation density binary classification method called EpiClass (available for download at https://github.com/Elnitskilab/EpiClass), that can be used to predict and optimize the performance of methylation biomarkers, particularly in challenging, heterogeneous samples such as liquid biopsies. This approach is based upon leveraging statistical differences in single-molecule sample methylation density distributions to identify ideal thresholds for sample classification. ResultsWe developed and tested the classifier using reduced representation bisulfite sequencing (RRBS) data derived from ovarian carcinoma tissue DNA and controls. We used these data to perform in silico simulations using methylation density profiles from individual epiallelic copies of ZNF154, a genomic locus known to be recurrently methylated in numerous cancer types. From these profiles, we predicted the performance of the classifier in liquid biopsies for the detection of epithelial ovarian carcinomas (EOC). In silico analysis indicated that EpiClass could be leveraged to better identify cancer-positive liquid biopsy samples by implementing precise thresholds with respect to methylation density profiles derived from circulating cell-free DNA (cfDNA) analysis. These predictions were confirmed experimentally using DREAMing to perform digital methylation density analysis on a cohort of low volume (1-mL) plasma samples obtained from 26 EOC-positive and 41 cancer-free women. EpiClass performance was then validated in an independent cohort of 24 plasma specimens, derived from a longitudinal study of 8 EOC-positive women, and 12 plasma specimens derived from 12 healthy women, respectively, attaining a sensitivity/specificity of 91.7%/100.0%. Direct comparison of CA-125 measurements with EpiClass demonstrated that EpiClass was able to better identify EOC-positive women than standard CA-125 assessment. Finally, we used independent whole genome bisulfite sequencing (WGBS) datasets to demonstrate that EpiClass can also identify other cancer types as well or better than alternative methylation-based classifiers. ConclusionsOur results indicate that assessment of intramolecular methylation density distributions calculated from cfDNA facilitate the use of methylation biomarkers for diagnostic applications. Furthermore, we demonstrated that EpiClass analysis of ZNF154 methylation was able to outperform CA-125 in the detection of etiologically-diverse ovarian carcinomas, indicating the broad utility of ZNF154 for use as a biomarker of ovarian cancer.

bioinformatics↗

bioRxiv2017

Significant Associations Between Driver Gene Mutations And DNA Methylation Alterations Across Many Cancer Types

Recent evidence shows that mutations in several driver genes can cause aberrant methylation patterns, a hallmark of cancer. In light of these findings, we hypothesized that the landscapes of tumor genomes and epigenomes are tightly interconnected. We measured this relationship using principal component analyses and methylation-mutation associations applied at the nucleotide level and with respect to genome-wide trends. We found a few mutated driver genes were associated with genome-wide patterns of aberrant hypomethylation or CpG island hypermethylation in specific cancer types. We identified associations between 737 mutated driver genes and site-specific methylation changes. Moreover, using these mutation-methylation associations, we were able to distinguish between two uterine and two thyroid cancer subtypes. The driver gene mutation-associated methylation differences between the thyroid cancer subtypes were linked to differential gene expression in JAK-STAT signaling, NADPH oxidation, and other cancer-related pathways. These results establish that driver-gene mutations are associated with methylation alterations capable of shaping regulatory network functions. In addition, the methodology presented here can be used to subdivide tumors into more homogeneous subsets corresponding to their underlying molecular characteristics, which could improve treatment efficacy.\n\nAuthor summaryMutations that alter the function of driver genes by changing DNA nucleotides have been recognized as a key player in cancer progression. Recent evidence showed that DNA methylation, a molecular signature that is used for controlling gene expression and that consists of cytosine residues with attached methyl groups in the context of CG dinucleotides, is also highly dysregulated in cancer and contributes to carcinogenesis. However, whether those methylation alterations correspond to mutated driver genes in cancer remains unclear. In this study, we analyzed 4,302 tumors from 18 cancer types and demonstrated that driver gene mutations are inherently connected with the aberrant DNA methylation landscape in cancer. We showed that those driver gene-associated methylation patterns can classify heterogeneous tumors in a cancer type into homogeneous subtypes and have the potential to influence the genes that contribute to tumor growth. This finding could help us to better understand the fundamental connection between driver gene mutations and DNA methylation alterations in cancer and to further improve the cancer treatment.

cancer biology↗