Biology subjects

Hill, C. J.

Publications and source records attributed to Hill, C. J..

7 recordsLinked to original sources

bioRxiv2025

Human NKX2.2 influences islet endocrine cell fate choices through regulation of WNT pathway genes

Transcriptional regulation is a key central mechanism of cell fate determination in developing tissues. The homeobox transcription factor NKX2.2 is an essential regulator of mouse and human pancreatic endocrine development, however its precise molecular role in a human system has not been previously investigated. In this study we generated NKX2.2 null (NKX2.2KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9 technologies and differentiated them towards a pancreatic {beta} cell fate using a stem cell-derived {beta} cell differentiation protocol. Functional and transcriptomic analyses of the hESC-derived pancreatic endocrine cells lacking NKX2.2 revealed similarities and differences compared to the molecular functions of NKX2.2 in mice. In the absence of NKX2.2, the {beta} cell differentiations result in reduced numbers of insulin-producing cells, and the differentiations become skewed towards polyhormonal fates, including cells co-expressing insulin, ghrelin and somatostatin. Deletion of NKX2.2 also eliminates the off-target formation of enterochromaffin cells. Single cell transcriptome analysis of the early endocrine cell population revealed a marked disruption of metabolic pathways that was confirmed by comparative metabolite tracing, providing novel insights into the regulation of early endocrine lineage decisions. Furthermore, NKX2.2 directly regulates several genes in the WNT signaling pathway, suggesting this is a key molecular mechanism through which NKX2.2 regulates these islet cell fate decisions in the human system.

developmental biology↗

bioRxiv2025

The Chromatin Remodeler Protein CHD4 Cooperates With NKX2.2 to Regulate Pancreatic Beta Cell Integrity

NKX2.2 is a transcription factor that regulates pancreatic islet beta ({beta}) cell identity and function; however, cofactor proteins that modulate the functional activity of NKX2.2 in {beta} cells are relatively unexplored. An unbiased proteomics screen identified chromodomain helicase DNA-binding protein 4 (CHD4) as an NKX2.2 interacting partner. CHD4 is a nucleosome remodeler that directs the appropriate differentiation, maturation and function of many cell types. To characterize the roles of CHD4 in {beta} cells, we generated Chd4 {beta}KO mice. Deletion of Chd4 substantially impaired the function of {beta} cells. The Chd4 {beta}KO mice became diabetic due to the disruption of islet integrity, calcium signaling and downregulation of essential {beta} cell regulatory genes. We also discovered CHD4 is required to bind at and repress non-beta cell genes, including Kcnj5, the gene that encodes the GIRK4 potassium channel in {beta} cells. Aberrant upregulation of GIRK4 causes impaired glucose-stimulated insulin secretion. These studies demonstrate that CHD4 is an essential transcriptional cofactor of NKX2.2 that is required for the proper maturation and function of pancreatic {beta} cells. Article HighlightsO_LINKX2.2 interacts with the Nucleosome Remodeling and Deacetylase (NuRD) complex through its interaction with CHD4. C_LIO_LIDeletion of CHD4 from developing pancreatic {beta} cells in mice causes diabetes due to a loss of islet integrity, disrupted calcium signaling and impaired insulin secretion. C_LIO_LIBeta cells lacking CHD4 inappropriately upregulate the GIRK4 potassium channel; inhibition of GIRK4 rescues the insulin secretion defect. C_LI

developmental biology↗

bioRxiv2025

Optimizing Single-Cell Long-Read Sequencing for Enhanced Isoform Detection in Pancreatic Islets

Alternative splicing is an essential mechanism for generating protein diversity by producing distinct isoforms from a single gene. Dysregulation of splicing that affects pancreatic function and immune tolerance has been linked to both type 1 and type 2 diabetes. Next-generation sequencing technologies, with their short read lengths, are limited in their ability to accurately detect splice variants. Long-read sequencing technologies offer the potential to overcome these limitations by providing full-length transcript information; however, their application in single-cell RNA sequencing has been hindered by technical challenges, including insufficient read lengths and higher error rates. Furthermore, cell types that produce high levels of a single transcript, such as islet endocrine cells, can obscure identification of lower abundance transcripts. In this study, we optimized a protocol for single-cell long-read sequencing in pancreatic islets to improve read length and transcript detection. Our findings demonstrate that 5 library preparation protocols outperform 3 protocols, resulting in better transcript identification. Furthermore, we show that targeted depletion of insulin transcripts enhances the detection of informative reads, highlighting the utility of transcript depletion strategies. This optimized protocol enables isoform-specific gene expression analysis and reveals differential transcript usage across the various cell types in pancreatic islets. By leveraging this approach, we gain deeper insights into the transcriptomic complexity and cellular heterogeneity within pancreatic islets. Article HighlightsO_LIThis study addresses the limitations of current single-cell long-read RNA-sequencing (sclrRNA-seq) technologies in detecting full-length transcripts and isoform diversity, particularly in pancreatic islets. C_LIO_LIWe demonstrate that optimizing single-cell library preparation protocols reproducibly enhances read length and transcript identification in pancreatic islets. C_LIO_LI5 capture methods, combined with targeted insulin depletion and extended reverse transcription, significantly improved read length and isoform detection compared to standard protocols, while maximizing the number of informative reads. C_LIO_LIThese improvements yield longer reads in single-cell experiments, substantially enhancing transcript identification and enabling more accurate analysis of isoform diversity. C_LI

genomics↗

bioRxiv2025

Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

Peritoneal endometriosis causes pelvic pain and infertility, but the underlying mechanisms related to these symptoms are not fully understood. Endometriosis diagnosis is typically delayed; thus, patient samples are unsuitable to study early endometriosis formation in situ. This study aimed to generate a 3D co-culture model of early peritoneal endometriosis using patient-derived primary cells, providing unique opportunities to examine endometriotic lesion initiation and progression. Peritoneal wash fluid, fallopian tube mesentery and endometrial biopsies were collected from patients undergoing laparoscopic surgery to isolate primary cells. A composite 3D hydrogel construct was assembled by embedding human peritoneal fibroblasts (HPFs) in a Matrigel-collagen I matrix and subsequent seeding with a layer of human peritoneal mesothelial cells (HPMCs). Immunohistological investigation of the composite hydrogel construct confirmed the successful assembly of a simple peritoneum layer model comprising a mesothelial monolayer, basement membrane and underlying fibroblasts, while secretion of tissue plasminogen activator demonstrated functional mesothelial physiology. Endometrial epithelial organoids (EEOs) were co-cultured with endometrial stromal cells (ESCs) to form endometrial assembloids mimicking shed endometrial tissue fragments at menstruation. When transplanted onto the peritoneal layer model, endometrial assembloids adhered, thus simulating early endometriotic lesion formation. Histological analysis demonstrated direct cell-cell contacts between HPMC, HPF and ESC at the endometrial-peritoneal interface, suggesting the involvement of those cell types in lesion initiation. Our modifiable superficial endometriosis model allows for further refinement by the addition of hormones, cytokines and/or other cell types to determine the underlying molecular mechanism involved in endometriotic lesion formation. HighlightO_LISimple peritoneal layer model resembles parietal peritoneum structure and function. C_LIO_LICombined endometrial and peritoneal model mimics early endometriosis formation. C_LIO_LIThe patient-derived multi-cellular model is suitable to study early endometriosis. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/643554v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@8e0639org.highwire.dtl.DTLVardef@3ab7a7org.highwire.dtl.DTLVardef@ef6e81org.highwire.dtl.DTLVardef@13fe704_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Vault particles are common contaminants of extracellular vesicle preparations

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

cell biology↗

bioRxiv2020

Olfactory rod cells: a rare cell type in the larval zebrafish olfactory epithelium with an actin-rich apical projection

We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending about 10 m from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. The cells also differ from secondary sensory cells such as hair cells of the inner ear or lateral line, or sensory cells in the taste bud, as they are not associated with established synaptic terminals. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium.

developmental biology↗

bioRxiv2019

Macrophages utilize mitochondrial fission to enhance mROS production during responses to Streptococcus pneumoniae

Immunometabolism and regulation of mitochondrial reactive oxygen species (mROS) are critical determinants of the immune effector phenotype of differentiated macrophages. Mitochondrial function requires dynamic fission and fusion, but whether effector function is associated with altered dynamics during bacterial responses is unknown. We show that macrophage mitochondria undergo fission after 12 h of progressive ingestion of live Streptococcus pneumoniae (pneumococci). Fission is associated with progressive reduction in oxidative phosphorylation but increased mROS generation. Fission is enhanced by mROS production, PI3K{gamma} signaling and by cathepsin B, but not by inflammasome activation or IL-1{beta} generation. Reduced fission following PI3K{gamma} or cathepsin B inhibition is associated with reduced mROS generation and bacterial killing. Fission is associated with Parkin recruitment to mitochondria, but not mitophagy. Fission occurs upstream of apoptosis induction and independently of caspase activation. During macrophage innate responses to live bacteria mitochondria shift from oxidative phosphorylation and ATP generation to mROS production and microbicidal responses with a progressive shift towards mitochondrial fission.

immunology↗