Biology subjects

Tangeman, J. A.

Publications and source records attributed to Tangeman, J. A..

5 recordsLinked to original sources

bioRxiv2026

Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for Pleurodeles waltl

Advances in transcriptomic technologies have transformed the study of complex biological processes, including tissue regeneration, by enabling high-resolution characterization of gene expression programs. In regenerative vertebrate models such as the Iberian ribbed newt (Pleurodeles waltl), these approaches can provide critical insight into the molecular mechanisms underlying retina and lens regeneration. However, single-cell and single-nucleus RNA sequencing studies lack spatial resolution, therefore the ability to validate gene expression patterns within ocular tissues is essential and requires optimization. In this study, we optimized hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) for use in P. waltl eyes. HCR-FISH enables sensitive and specific detection of mRNA transcripts through split-initiator probes and hairpin-based signal amplification with automatic background suppression. In addition, because incomplete genome annotation in emerging model organisms complicates transcript selection and probe design, we optimized an optional in silico workflow to support transcript screening, orthology confirmation, and split-initiator probe generation. We systematically optimized fixation duration, proteinase K concentration, and tissue processing parameters to preserve tissue integrity while enhancing signal quality. To overcome imaging constraints imposed by highly pigmented ocular tissues, we implemented a whole-mount protocol with optional bleaching followed by cryosectioning, enabling improved visualization without compromising spatial localization. Using this workflow, we successfully detected key retinal markers including SLC1A3 (Muller glia cells) and RPE65 (retinal pigment epithelium) within the newt eye. Notably, the RPE65 probe was designed in house and showed comparable detection to a standard Molecular Instruments probe across two sample-preparation protocols. This study presents a reproducible framework for spatial transcript detection in an emerging eye regenerative model and facilitates integration of transcriptomic and anatomical data. Together, the integrated design-to-detection pipeline will strengthen spatial validation of RNA sequencing profiles in P. waltl.

developmental biology↗

bioRxiv2024

miR-26 deficiency causes alterations in lens transcriptome and results in adult-onset cataract

PurposeDespite strong evidence demonstrating that normal lens development requires regulation governed by miRNAs, the functional role of specific miRNAs in mammalian lens development remains largely unexplored. MethodsA comprehensive analysis of miRNA transcripts in the newborn mouse lens, exploring both differential expression between lens epithelial cells and lens fiber cells and overall miRNA abundance was conducted by miRNA-seq. Mouse lenses lacking each of three abundantly expressed lens miRNAs: miR-184, miR-26 and miR-1 were analyzed to explore the role of these miRNAs in lens development. ResultsMice lacking all three copies of miR-26 (miR-26TKO) developed postnatal cataracts as early as 4-6 weeks of age. RNA-seq analysis of neonatal lenses from miR-26TKO mice exhibited abnormal reduced expression of a cohort of genes found to be lens-enriched and linked to cataract (e.g. Foxe3, Hsf4, Mip, Tdrd7, and numerous crystallin genes), and abnormal elevated expression of genes related to neural development (Lhx3, Neurod4, Shisa7, Elavl3), inflammation (Ccr1, Tnfrsf12a, Csf2ra), the complement pathway, and epithelial to mesenchymal transition (Tnfrsf1a, Ccl7, Stat3, Cntfr). ConclusionmiR-1, miR-184 and miR-26 are each dispensable for normal embryonic lens development. However, loss of miR-26 causes lens transcriptome changes and drives cataract formation.

developmental biology↗

bioRxiv2023

DISTINCT METABOLIC STATES DIRECT RETINAL PIGMENT EPITHELIUM CELL FATE DECISIONS

During tissue regeneration, proliferation, dedifferentiation, and reprogramming are necessary to restore lost structures. However, it is not fully understood how metabolism intersects with these processes. Chicken embryos can regenerate their retina through retinal pigment epithelium (RPE) reprogramming when treated with fibroblast factor 2 (FGF2). Using transcriptome profiling, we uncovered extensive regulation of gene sets pertaining to proliferation, neurogenesis, and glycolysis throughout RPE-to-neural retina reprogramming. By manipulating cell media composition, we determined that glucose, glutamine, or pyruvate are sufficient to support RPE reprogramming identifying glycolysis as a requisite. Conversely, the induction of oxidative metabolism by activation of pyruvate dehydrogenase induces Epithelial-to-mesenchymal transition (EMT), while simultaneously blocking the activation of neural retina fate. We also identify that EMT is partially driven by an oxidative environment. Our findings provide evidence that metabolism controls RPE cell fate decisions and provide insights into the metabolic state of RPE cells, which are prone to fate changes in regeneration and pathologies, such as proliferative vitreoretinopathy.

developmental biology↗

bioRxiv2023

Integrated single-cell multiomics uncovers foundational regulatory mechanisms of lens development and pathology

Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataract. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq, and CUT&RUN-seq to discover novel mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Further, we divulge a conserved epigenetic paradigm of cellular differentiation, defined by progressive loss of H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation.

developmental biology↗

bioRxiv2021

Transcriptome profiling of embryonic retinal pigment epithelium reprogramming

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in an FGF2-dependent manner. To characterize the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from 1) intact RPE, 2) transiently reprogrammed RPE (t-rRPE) 6 hours post-retinectomy, and 3) reprogrammed RPE (rRPE) 6 hours post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for MAPK-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis were used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) regulators, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

developmental biology↗