Biology subjects

Sauka-Spengler, T.

Publications and source records attributed to Sauka-Spengler, T..

At least 19 recordsLinked to original sources

bioRxiv2025

Precise engineering of chimeric antigen receptor expression levels defines T cell identity and function

Chimeric Antigen Receptor (CAR) T therapy is a potent treatment for haematological malignancies, but T cell exhaustion reduces its efficacy in many patients. Although high CAR transgene levels appear to drive T cell exhaustion, the relationship between CAR expression levels, T cell function, and transcriptional identity is yet to be mapped at high resolution. Here, we harness a high-resolution microRNA-based control system to precisely modulate CAR transgene expression levels and assess the impact on T cell activation, gene expression and function. By post-transcriptionally modulating CAR abundance, we show that differential CAR levels significantly impact T cell proliferation, cytokine production and tonic signalling. T cells with high CAR expression become strongly activated even at low target antigen densities, while those with low CAR expression are triggered only by high concentrations of their target. Single-cell RNA sequencing of primary T cells expressing a broad range of CAR transcript levels revealed global transcriptional programmes that become dysfunctional with increased CAR abundance, expanding our understanding of T cell exhaustion. Notably, we identified a narrow CAR expression range where the exhaustion transcriptional state is not triggered, demonstrating that T cell exhaustion can be controlled by fine-tuning CAR levels. This work demonstrates that CAR expression levels are key determinants of T cell transcriptional identity and function and introduces a tractable method to precisely tune CAR expression and T cell activity.

synthetic biology↗

bioRxiv2024

RegVelo: gene-regulatory-informed dynamics of single cells

RNA velocity has emerged as a popular approach for modeling cellular change along the phenotypic landscape but routinely omits regulatory interactions between genes. Conversely, methods that infer gene regulatory networks (GRNs) do not consider the dynamically changing nature of biological systems. To integrate these two currently disconnected fields, we present RegVelo, an end-to-end dynamic, interpretable, and actionable deep learning model that learns a joint model of splicing kinetics and gene regulatory relationships and allows us to perform in silico perturbation predictions. When applied to datasets of the cell cycle, human hematopoiesis, and murine pancreatic endocrinogenesis, RegVelo demonstrates superior predictive power for interactions and perturbation simulations, for example, compared to methods that focus solely on dynamics or GRN inference. To leverage RegVelos full potential, we studied the dynamics of zebrafish neural crest development and underlying regulatory mechanisms using our Smart-seq3 dataset and shared gene expression and chromatin accessibility measurements. Using RegVelos in silico perturbation predictions, validated by CRISPR/Cas9-mediated knockout and single-cell Perturb-seq, we establish transcription factor tfec as an early driver and elf1 as a novel regulator of pigment cell fate and propose a gene-regulatory circuit involving tfec and elf1 interactions via the toggle-switch model.

developmental biology↗

bioRxiv2024

Robust CRISPR/dCas13 RNA blockers specifically perturb miRNA-target interactions and rescue type 1 myotonic dystrophy pathology

While RNA-targeting strategies are powerful tools for disease therapy, challenges, including low target engagement and off-target collateral effects, currently limit their efficacy. Here, we report the engineering and optimisation of a CRISPR/dCas13 RNA steric blocker (CRISPR-Lock) that prevents mRNA translation, shields mRNAs from miRNA-mediated silencing, and blocks RNA-protein interactions. By tuning the spatial resolution and mismatch tolerance of CRISPR-Lock, we develop a high-resolution perturbation approach that employs genetically encoded CRISPR-Lock as a miRNA target protector. This system enables precise spatiotemporal control of miRNA:mRNA interactions, offering broader applicability compared to phosphorodiamidate mor-pholino (PMO) target protectors. Moreover, we demonstrate the potential therapeutic application of CRISPR-Lock for blocking pathological RNA-protein interactions in type 1 myotonic dystro-phy (DM1). Optimising CRISPR-Lock to target expanded repeat RNAs corrects approximately 85% of clinically relevant splicing biomarkers in patient-derived myotubes and significantly out-performs third-generation PMO antisense oligonucleotides. Finally, by delivering a miniaturised AAV-encoded CRISPR-Lock system into an established DM1 mouse model, we demonstrate the dose-dependent correction of intranuclear foci and splicing dysregulation, underscoring the potential therapeutic application of this technology.

bioengineering↗

bioRxiv2024

Single-cell multi-omics, spatial transcriptomics and systematic perturbation decode circuitry of neural crest fate decisions

The cranial neural crest (NC) is a migratory embryonic population ideal for studying cell plasticity, motility, and fate establishment. Although NC migration has been linked to changes in cell adhesion, polarity, and signaling, the gene regulatory circuitry governing these processes remained obscure. Using time-resolved single-cell multi-omics, spatial transcriptomics, and gene regulatory network reconstruction, we identified ten programs underlying 23 NC cell states and three spatial trajectories. Using in silico perturbation and systematic CRISPR/Cas9-mediated Perturb-seq, we uncovered novel lineage drivers and an endothelial-like program controlling NC migration, distinct from the epithelial-to-mesenchymal transition (EMT) program. We show that endothelial-like regulons (fli1a, elk3) drive migration through direct or tiered activation via the "FOX:ETS-Ebf3a-targets" axis, while ETS suppressors (erf, erfl3) maintain cell plasticity. Using the newly developed SyncReg tool, we identify functional redundancy among ETS regulons, which has thus far obscured their critical roles in NC migration, and we quantify their synergy with retinoic acid receptors, also essential for this process. Our GRN model, combined with novel velocity-embedded simulations, accurately predicted the functions of all major regulons, which were confirmed by in vivo functional perturbations. This study provides a comprehensive, validated cranial NC regulatory landscape, resolving heterogeneous regulatory circuits underlying NC cell motility and plasticity.

developmental biology↗

bioRxiv2023

Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. The engineered sgRNAs become activated upon recognising complementary RNAs, thus enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimisations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAs in vivo. Our sensors open up novel opportunities for developing new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers.

synthetic biology↗

bioRxiv2022

Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to mutations in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, it remains elusive how this seemingly ubiquitous factor is specifically upregulated in neural crest cells. Here, we use epigenomic profiling of chick neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7. To our knowledge, this is the first report of tissue-specific regulation of a chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Collectively our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

developmental biology↗

bioRxiv2021

Integrative annotation of genomic features reveal functional elements and epigenetic landscapes in the developing zebrafish

Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals.

developmental biology↗

bioRxiv2021

Segregation of neural crest specific lineage trajectories from a heterogeneous neural plate border territory only emerges at neurulation

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harbouring neural crest and cranial placode progenitors. Here we profile avian epiblast cells as a function of time using single-cell RNA-seq to define transcriptional changes in the emerging neural plate border. The results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridisation. Developmental trajectory analysis shows that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined lineages.

developmental biology↗

bioRxiv2021

Distinct epicardial gene regulatory programmes drive development and regeneration of the zebrafish heart

Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart can fully regenerate following injury. Reactivation of cardiac developmental programmes is considered key to successfully regenerating the heart, yet the regulatory elements underlying the response triggered upon injury and during development remain elusive. Organ-wide activation of the epicardium is essential for zebrafish heart regeneration and is considered a potential regenerative source to target in the mammalian heart. Here we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardium by integrating gene expression profiles with open chromatin ATAC-seq data. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programmes driving each of these processes, which were largely distinct. We identified wt1a, wt1b, and the AP-1 subunits junbb, fosab and fosb as central regulators of the developing network, whereas hif1ab, zbtb7a, tbx2b and nrf1 featured as putative central regulators of the regenerating epicardial network. By interrogating developmental gene regulatory networks that drive cell-specific transcriptional heterogeneity, we tested novel subpopulation-related epicardial enhancers in vivo. Taken together, our work revealed striking differences between the regulatory blueprint deployed during epicardial development and regeneration. These findings challenge the dogma that heart regeneration is essentially a reactivation of developmental programmes, and provide important insights into epicardial regulation that can assist in developing therapeutic approaches to enable tissue regeneration in the adult mammalian heart.

developmental biology↗

bioRxiv2021

Neuromesodermal progenitor origin of trunk neural crest in vivo

Neural crest (NC) is a vertebrate-specific population of multipotent embryonic cells predisposed to diverse derivatives along the anteroposterior (A-P) axis. Only cranial NC progenitors give rise to ectomesenchymal cell types, whereas trunk NC is biased for neuronal cell fates. By integrating multimodal single-cell analysis, we provide evidence for divergent embryonic origins of cranial vs. trunk NC that explain this dichotomy. We show that the NC regulator foxd3 is heterogeneously expressed across the A-P axis and identify its specific cranial and trunk autoregulatory enhancers. Whereas cranial-specific enhancer is active in the bona fide NC, the trunk foxd3 autoregulatory element surprisingly marked bipotent tailbud neuromesodermal progenitors (NMps). We integrated NMp single cell epigemomics and trasncriptomics data and for the first time reconstructed anamniote NMp gene regulatory network. Moreover, using pseudotime and developmental trajectory analyses of NMps and NC during normal development and in foxd3 mutants, we demonstrate an active role for foxd3 in balancing non-cranial NC and NMp fates during early embryonic development. Strikingly, we show that a portion of posterior NC in the developing zebrafish embryo is derived from the pro-neural NMps. This suggests a common embryonic origin of trunk NC and NM progenitors that is distinct from cranial NC anlage, and elucidates pro-neural bias of trunk NC.

developmental biology↗

bioRxiv2019

The repertoire of serous ovarian cancer non-genetic heterogeneity revealed by single-cell sequencing of normal fallopian tube epithelial cells

The inter-differentiation between cell states promotes cancer cell survival under stress and fosters non-genetic heterogeneity (NGH). NGH is, therefore, a surrogate of tumor resilience but its quantification is confounded by genetic heterogeneity. Here we show that NGH can be accurately measured when informed by the molecular signatures of the normal cells of origin. We surveyed the transcriptomes of [~] 4000 normal fallopian tube epithelial (FTE) cells, the cells of origin of serous ovarian cancer (SOC), and identified six FTE subtypes. We used subtype signatures to deconvolute SOC expression data and found substantial intra-tumor NGH that was previously unrecognized. Importantly, NGH-based stratification of [~]1700 tumors robustly predicted survival. Our findings lay the foundation for accurate prognostic and therapeutic stratification of SOC.\n\nHighlightsO_LIThe projection of FTE subtypes refines the molecular classification of serous OC\nC_LIO_LIComprehensive single-cell profiling of FTE cells identifies 6 molecular subtypes\nC_LIO_LISubstantial non-genetic heterogeneity of HGSOC identified in 1700 tumors\nC_LIO_LIA mesenchymal-high HGSOC subtype is robustly correlated with poor prognosis\nC_LI

cancer biology↗

bioRxiv2019

Nanoscale dynamics of cholesterol in the cell membrane

Cholesterol constitutes approximately 30-40% of the mammalian plasma membrane -- a larger fraction than any other single component. It is a major player in numerous signalling processes as well as molecular membrane architecture. However, our knowledge on dynamics of cholesterol in the plasma membrane is limited which restricts our understanding of the mechanisms regulating its involvement in cell signalling. Here, advanced fluorescence imaging and spectroscopy approaches were applied on in vitro (model membranes) and in vivo (live cells and embryos) membranes to systematically study the nanoscale dynamics of cholesterol in biological membranes. The results show that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. The data indicate that diffusion of cholesterol and phospholipids is not correlated with membrane domain partitioning. Instead, our data show that the fast diffusion of cholesterol is due to its nanoscale interactions and localization in the membrane.

biophysics↗

bioRxiv2018

Reconstruction of the global neural crest gene regulatory network in vivo

Precise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and transient dynamic molecular interactions. Here we present a genome-wide in vivo reconstruction of the GRN underlying development of neural crest (NC), an emblematic embryonic multipotent cell population. By coupling NC-specific epigenomic and single-cell transcriptome profiling with genome/epigenome engineering in vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, novel trans-factors and cis-signatures. Assembling the NC regulome has allowed the comprehensive reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates. Our integrated approach, allowing dissection of cell-type-specific regulatory circuits in vivo, has broad implications for GRN discovery and investigation.

developmental biology↗

bioRxiv2018

Functional heterogeneity within the developing zebrafish epicardium

The epicardium is essential during cardiac development, homeostasis and repair and yet fundamental insights into its underlying cell biology, notably epicardium formation, lineage heterogeneity and functional cross-talk with other cell types in the heart, are currently lacking. In this study, we investigated epicardial heterogeneity and the functional diversity of discrete epicardial subpopulations in the developing zebrafish heart. Single-cell RNA-sequencing uncovered three epicardial subpopulations with specific genetic programmes and distinctive spatial distribution within the developing heart. Perturbation of unique gene signatures uncovered distinct functions associated with each subpopulation and established novel epicardial roles in cell adhesion, migration, and chemotaxis as a mechanism for recruitment of leukocytes into the heart. This work elucidates the mutual spatiotemporal relationships between different epicardial subpopulations and assigns unique function to each during cardiac development. Understanding which mechanisms cells employ to establish a functional epicardium and to communicate with other cardiovascular cell types during development will bring us closer to repairing cellular relationships that are disrupted during cardiovascular disease.

developmental biology↗

bioRxiv2018

Re-purposing Ac/Ds transgenic system for CRISPR/dCas9 modulation of enhancers and non-coding RNAs in zebrafish

Due to its genetic amenability coupled with recent advances in genome editing, the zebrafish serves as an excellent model to examine the function of both coding and non-coding elements. Recently, the non-coding genome has gained prominence due to its critical role in development and disease. Here, we have re-purposed the Ac/Ds maize transposition system to reliably screen and efficiently characterise zebrafish enhancers, with or without germline propagation. We further utilised the system to stably express guide RNAs in microinjected embryos enabling tissue-specific CRISPR/dCas9-interference (CRISPRi) knockdown of lncRNA and enhancer activity without disrupting the underlying genetic sequence. Our study highlights the utility of Ac/Ds transposition for transient epigenome modulation of non-coding elements in zebrafish.\n\nSummary statementWe adapted the Ac/Ds transposition system, which enables continuous expression of guide RNAs for CRISPR/dCas9 perturbation, to examine the function of non-coding RNAs and enhancer elements in zebrafish.

developmental biology↗

bioRxiv2018

A genome-wide assessment of the ancestral neural crest gene regulatory network

The neural crest is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional profiles and chromatin accessibility of neural crest cells in the basal sea lamprey, in order to gain insight into the ancestral state of the neural crest gene regulatory network (GRN) at the dawn of vertebrates. Transcriptome analyses reveal clusters of co-regulated genes during neural crest specification and migration that show high conservation across vertebrates for dynamic programmes like Wnt modulation during the epithelial to mesenchymal transition, but also reveal novel transcription factors and cell-adhesion molecules not previously implicated in neural crest migration. ATAC-seq analysis refines the location of known cis-regulatory elements at the Hox-2 locus and uncovers novel cis-regulatory elements for Tfap2B and SoxE1. Moreover, cross-species deployment of lamprey elements in zebrafish reveals that the lamprey SoxE1 enhancer activity is deeply conserved, mediating homologous expression in jawed vertebrates. Together, our data provide new insight into the core elements of the GRN that are conserved to the base of the vertebrates, as well as expose elements that are unique to lampreys.

developmental biology↗

bioRxiv2017

From pioneer to repressor: Bimodal foxd3 activity dynamically remodels neural crest regulatory landscape in vivo

The neural crest (NC) is a transient embryonic stem cell population characterised by its multipotency and broad developmental potential. Here, we perform NC-specific transcriptional and epigenomic profiling of foxd3-mutant versus wild type cells in vivo to define the gene regulatory circuits controlling NC specification. Together with global binding analysis obtained by foxd3 biotin-ChIP and single cell profiles of foxd3-expressing premigratory NC, our analysis shows that during early steps of NC formation, foxd3 acts globally as a pioneer factor to prime the onset of genes regulating NC specification and migration by re-arranging the chromatin landscape, opening cis-regulatory elements and reshuffing nucleosomes. Strikingly, foxd3 then switches from an activator to its canonical role as a transcriptional repressor. Taken together, these results demonstrate that foxd3 acts bimodally in the neural crest as a switch from permissive to repressive nucleosome/chromatin organisation to maintain stemness and define cell fates.

developmental biology↗

bioRxiv2017

Embryonic circular RNAs at tandem duplicated genes in zebrafish present new paradigm in gene expression

Circular RNAs are a puzzling class of RNAs with covalently closed loop structure, lacking 5 and 3 ends. Present in all cells, no unifying theme has emerged regarding their function. Here, we use transcriptional data obtained by biotagging in zebrafish to uncover a high-resolution cohort of embryonic circRNAs expressed in nuclear and polysomal subcellular compartments in three developmental cell types. The ample presence of embryonic circRNAs on polysomes contradicts previous reports suggesting predominant nuclear localisation. We uncover a novel class of circRNAs, significantly enriched at tandem duplicated genes. Using newly-developed NGS-based approach, we simultaneously resolve the full sequence of putative \"tandem-circRNAs\" and their underlying mRNAs. These form by long-range cis-splicing events often between distant tandem duplicated genes, resulting in chimaeric mRNA transcripts and circRNAs containing their supernumerary excised exons, integrated from multiple tandem loci. Taken together, our results suggest that circularisation events rather than circRNAs themselves are functionally important.

molecular biology↗