Biology subjects

Leible, S.

Publications and source records attributed to Leible, S..

7 recordsLinked to original sources

bioRxiv2025

Direct detection of CRISPR mutations and transcriptional responses at single cell resolution in vivo

CRISPR screens coupled with single-cell RNA sequencing are transforming high-throughput functional genomics. However, applications in vivo remain limited and are confounded by difficulties in identifying and genetically characterizing edited cells. Here we present scPT-seq, a single-cell RNA assay that resolves CRISPR-induced mutations at base-pair resolution and captures transcriptional responses in the same single cells in vivo. scPT-seq comes with a computational analysis suite enabling haplotype-resolved mutation detection and characterization of complex editing outcomes, including splice-junction variation. Applied to the Drosophila intestine, a highly regenerative tissue, scPT-seq distinguishes cell-autonomous from environmental effects by identifying mutant and wild-type cells within tissues, and reveals spatially organized compensatory mechanisms in response to mutations. By using editing outcomes as heritable clonal markers, we identified distinct intestinal stem cell populations with specialized differentiation trajectories. In summary, scPT-seq provides a versatile technology for dissecting gene function and lineage dynamics in complex tissues.

molecular biology↗

bioRxiv2024

Proliferation and differentiation of intestinal stem cells depends on the zinc finger transcription factor BCL11/Chronophage

The molecular programs that drive proliferation and differentiation of intestinal stem cells (ISCs) are essential for organismal fitness. Notch signalling regulates the binary fate decision of ISCs, favouring enterocyte commitment when Notch activity is high and enteroendocrine cell (EE) fate when activity is low. However, the gene regulatory mechanisms that underlie this process on an organ scale remain poorly understood. Here, we find that the expression of the C2H2-type zinc-finger transcription factor Chronophage (Cph), homologous to mammalian BCL11, increases specifically along the ISC-to-EE lineage when Notch is inactivated. We show that the expression of Cph is regulated by the Achaete-Scute Complex (AS-C) gene, scute, which directly binds to multiple sites within the Cph locus to promote its expression. Our genetic and single-cell RNA sequencing experiments demonstrate that Cph maintains the ISC and EE populations and is necessary to remodel the transcriptome of progenitor cells with low Notch activity. By identifying and functionally validating Cph target genes, we uncover a novel role for sugar free frosting (sff) in directing proliferative and lineage commitment steps of ISCs. Our results shed light on the mechanisms by which Cph sustains intestinal epithelial homeostasis and could represent a conserved strategy for balancing proliferation and differentiation in different tissues and species.

developmental biology↗

bioRxiv2024

Restraining Wnt activation and intestinal tumorigenesis by a Rab35 dependent GTPase relay

Maintenance of homeostatic processes ensure curtailment of intestinal tumorigenesis. Inactivating mutations to Adenomatous Polyposis Coli (Apc) result in aberrantly activated Wnt signalling and initiates colorectal cancer (CRC) in approx. 80% of cases, yet our understanding of the subcellular mechanisms that modulate dysregulated pathway activity is limited. Here, using a conditional in vivo genetic screen, we identify Rab35 GTPase as a novel tumour suppressor that modulates regional Wnt activity after loss of Apc in progenitor cells. Single cell analysis revealed that progenitor cells respond to Apc depletion by increasing the expression of a GTPase activating protein, which we named blackbelt, and triggering Rab35 disassociation from the plasma membrane. Mechanistically, we demonstrate that Rab35 controls the localisation and activation of the Rho GTPase, Cdc42, which functions as a relay to regulate JNK signalling. This in turn tunes the Wnt pathway upstream of {beta}-catenin to direct proliferation and differentiation of progenitor cells. Importantly, we show that maintaining active JNK signalling is important for the propagation of Apc mutant mouse colon organoids. Our findings highlight a novel GTPase cascade that sustains aberrant Wnt activity in specific segments of the intestine and provides impetus to therapeutically exploit this pathway to target CRC.

cancer biology↗

bioRxiv2021

Glyoxal as alternative fixative for single cell RNA sequencing

Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.

genomics↗

bioRxiv2020

Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues

Recent advances allowing the genomic analysis of individual cells from a bulk population have provided intriguing new insights into areas such as developmental processes and tumor heterogeneity. Most approaches to date, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Pediatric central nervous system tumors - the leading cause of childhood cancer deaths - represent one such example, where often only frozen rather than native material is available. Due to an increasing need for advanced techniques to understand the heterogeneity of these tumors, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, we applied the established nucleus isolation method to analyze frozen primary tissue from two pediatric central nervous system tumors - one pilocytic astrocytoma and one glioblastoma - allowing the identification of distinct tumor cell populations and infiltrating microglia. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet-based 3 transcriptome amplification (10X Genomics) and plate-based whole transcriptome amplification (Fluidigm C1) single-cell sequencing platforms, thereby dramatically increasing the potential for application of such methods to rare entities.

cancer biology↗

bioRxiv2018

Gene expression atlas of a developing tissue by single cell expression correlation analysis

The Drosophila wing disc has been a fundamental model system for the discovery of key signaling pathways and for our understanding of developmental processes. However, a complete map of gene expression in this tissue is lacking. To obtain a complete gene expression atlas in the wing disc, we employed single-cell sequencing (scRNA-seq) and developed a new method for analyzing scRNA-seq data based on gene expression correlations rather than cell mappings. This enables us to discover 824 genes with spatially restricted expression patterns, and to compute expression maps for all genes in the wing disc. This approach identifies both known and new clusters of genes with similar expression patterns and functional relevance. As proof of concept, we characterize the previously unstudied gene CG5151 and show it regulates Wnt signaling. This novel method will enable the leveraging of scRNA-seq data for generating expression atlases of undifferentiated tissues during development.

developmental biology↗

bioRxiv2017

Sorting & Sequencing Flies By Size: Identification Of Novel TOR Regulators And Parameters For Successful Sorting

As DNA sequencing throughput increases, novel strategies for discovering genes that affect traits of interest become available. One strategy starts with a population of animals and selects individuals over multiple generations for a particular trait. Subsequent whole genome sequencing should identify loci affecting this trait. We apply this strategy by sorting flies for wing length over 18 generations, obtaining two populations that differ in wing length by 20%. Flies with longer wings had increased overall body sizes and elevated TOR activity, suggesting that genetic variation targets TOR signaling to influence body size. High-throughput sequencing of big and small flies identified thousands of single nucleotide polymorphisms that differed between the two populations, leading us to identify five novel regulators of TOR signaling. Surprisingly, stochastic simulations of the process show that large fractions of the genetic differences between the big and small flies are probably biological false positives, selected by chance by random drift. We employ these computer simulations to identify experimental setup parameters to improve the signal-to-noise ratio for successfully running sort-and-sequence experiments - a resource which will hopefully be useful for the community.

genetics↗