Biology subjects

Redhai, S.

Publications and source records attributed to Redhai, S..

4 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↗

bioRxiv2019

Mating Induces Switch From Hormone-Dependent to -Independent Steroid Receptor-Mediated Growth in Drosophila Prostate-Like Cells

Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate grows throughout adult life, stimulated by environmentally regulated endocrine and local androgens. We previously showed that in each fly accessory gland, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid, ecdysone, whose concentration reflects socio-sexual experience in adults. BMP signalling regulates ecdysone receptor (EcR) levels post-transcriptionally, partly via EcRs N-terminus. Nuclear growth in virgin males is ecdysone-dependent. However, mating activates genome endoreplication to drive additional BMP-mediated nuclear growth via a cell type-specific form of hormone-independent EcR signalling. In virgin males with low ecdysone levels, this mechanism ensures resources are conserved. However, by switching to hormone-independence after mating, this control is overridden to hyper-activate growth of secretory secondary cells. Our data suggest parallels between this physiological, behaviour-induced switch and altered pathological signalling associated with prostate cancer progression.

cell biology↗