Cell-type-specific signaling networks in heterocellular organoids

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids

The Transcription co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation into Enterocyte vs Secretory Lineages

BACKGROUND & AIMS: Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established. METHODS: We sorted cells from Lgr5-Gfp knock-in intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription PCR and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing. We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16. RESULTS: The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt-villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode delta-like canonical Notch ligand and other secretory-specific transcription factors. CONCLUSIONS: In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1

SH3BP4 Regulates Intestinal Stem Cells and Tumorigenesis by Modulating β-Catenin Nuclear Localization

SUMMARY Wnt signals at the base of mammalian crypts play a pivotal role in intestinal stem cell (ISC) homeostasis, whereas aberrant Wnt activation causes colon cancer. Precise control of Wnt signal strength is governed by a number of negative inhibitory mechanisms acting at distinct levels of the cascade. Here, we identify the Wnt negative regulatory role of Sh3bp4 in the intestinal crypt. We show that the loss of Sh3bp4 increases ISC and Paneth cell numbers in murine intestine and accelerates adenoma development in Apcmin mice. Mechanistically, human SH3BP4 inhibits Wnt signaling downstream of b-catenin phosphorylation and ubiquitination. This Wnt inhibitory role is dependent on the ZU5 domain of SH3BP4. We further demonstrate that SH3BP4 is expressed at the perinuclear region to restrict nuclear localization of b-catenin. Our data uncover the tumor-suppressive role of SH3BP4 that functions as a negative feedback regulator of Wnt signaling through modulating b-catenin’s subcellular localization

Mathematical and Statistical Techniques for Systems Medicine: The Wnt Signaling Pathway as a Case Study

The last decade has seen an explosion in models that describe phenomena in systems medicine. Such models are especially useful for studying signaling pathways, such as the Wnt pathway. In this chapter we use the Wnt pathway to showcase current mathematical and statistical techniques that enable modelers to gain insight into (models of) gene regulation, and generate testable predictions. We introduce a range of modeling frameworks, but focus on ordinary differential equation (ODE) models since they remain the most widely used approach in systems biology and medicine and continue to offer great potential. We present methods for the analysis of a single model, comprising applications of standard dynamical systems approaches such as nondimensionalization, steady state, asymptotic and sensitivity analysis, and more recent statistical and algebraic approaches to compare models with data. We present parameter estimation and model comparison techniques, focusing on Bayesian analysis and coplanarity via algebraic geometry. Our intention is that this (non exhaustive) review may serve as a useful starting point for the analysis of models in systems medicine.Comment: Submitted to 'Systems Medicine' as a book chapte

The analysis of PIK3CA mutations in gastric carcinoma and metanalysis of literature suggest that exon-selectivity is a signature of cancer type

BACKGROUND: PIK3CA is one of the genes most frequently mutated in human cancers and it is a potential target for personalized therapy. The aim of this study was to assess the frequency and type of PIK3CA mutations in gastric carcinoma and compare them with their clinical pathological correlates. METHODS: We analysed 264 gastric cancers, including 39 with microsatellite instability (MSI), for mutations in the two PIK3CA hotspots in exons 9 and 20 by direct sequencing of DNA obtained from microdissected cancer cells. RESULTS: The cases harbouring mutations were 42 (16%). All were heterozygous missense single base substitutions; the most common was H1047R (26/42; 62%) in exon 20 and the second was Q546K (4/42; 9.5%) in exon 9. All the mutated MSI cases (8/39) carried the H1047R mutation. No other association between PI3KCA mutations and clinical pathological covariates was found. A metanalysis of the mutations occurring in the same regions in 27 publications showed that ratio between exon 20 and exon 9 prevalences was 0.6 (95% CI: 0.5 -0.8) for colon, 1.6 (95% CI: 1.1 -2.3) for breast, 2.7 (95% CI: 1.6 -4.9) for gastric and 4.1 (95% CI: 1.9 -10.3) for endometrial cancer. CONCLUSIONS: The overall prevalence of PIK3CA mutations implies an important role for PIK3CA in gastric cancer. The lack of association with any clinical-pathological condition suggests that mutations in PIK3CA occur early in the development of cancer. The metanalysis showed that exon-selectivity is an important signature of cancer type reflecting different contexts in which tumours arise

Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease

Intestinal Stem Cell Niche: The Extracellular Matrix and Cellular Components

The intestinal epithelium comprises a monolayer of polarised columnar cells organised along the crypt-villus axis. Intestinal stem cells reside at the base of crypts and are constantly nourished by their surrounding niche for maintenance, self-renewal, and differentiation. The cellular microenvironment including the adjacent Paneth cells, stromal cells, smooth muscle cells, and neural cells as well as the extracellular matrix together constitute the intestinal stem cell niche. A dynamic regulatory network exists among the epithelium, stromal cells, and the matrix via complex signal transduction to maintain tissue homeostasis. Dysregulation of these biological or mechanical signals could potentially lead to intestinal injury and disease. In this review, we discuss the role of different intestinal stem cell niche components and dissect the interaction between dynamic matrix factors and regulatory signalling during intestinal stem cell homeostasis

Investigation of signaling pathways that regulate human colon crypt maturation and their dysregulation in tumorigenesis

Intestinal epithelial cell polarity and lineages along the crypt-villus axis provide an excellent model in studying genetic programs controlling cellular proliferation and differentiation. Multipotent stem cells residing at the bottom of the intestinal crypts undergo cell division, where daughter cells will migrate upwards and undergo cell cycle arrest and differentiation. Although the biological and genetic regulation of these processes still remains unclear, there has been report on several key pathways regarding this regulation, including Wnt, Notch and TGF signaling pathways. This study is based on the hypothesis that perturbation of the signaling pathways that regulate cellular polarity and maturation along the colon crypt axis is closely related to tumor development and progression in gastrointestinal tract cancers. To study the differential gene expression between upper and bottom part of colon crypt, a total of 18 cDNA microarray experiments were performed using RNA extracted from 9 paired upper and bottom parts of the colon crypts. Gene expression data were extracted, and Significance Analysis of Microarrays (SAM) was performed to identify genes with significant correlating expression at bottom compartment of the crypts. 969 cDNA clones with significant differential expression were identified, in which 367 and 602 cDNA clones were preferentially upregulated and downregulated respectively in bottom proliferative compartment of the crypts. A significant correlation of these 969 SAM list genes with WNT target genes (obtained from inducible dominant negative TCF4 transfection in CRC cell lines by van de Watering et al) was observed, demonstrating that colon development is tightly regulated by WNT-signaling pathway through TCF4 transcription regulation. Biological pathway analysis using GenMapp illustrated that the colon crypt development involved multiple molecular pathways, including cell cycle, apoptosis, MAP kinase, WNT and BMP pathways etc. Strikingly, we have identified a set of genes that code for secretory proteins of several key pathways where most of them have been previously demonstrated to be expressed in connective tissues, postulating that intestinal myofibroblasts were playing important roles in maintaining the microenvironment for epithelial cells development. With the help of the colon top/bottom array data set obtained here, we hope to provide a comprehensive picture for understanding the global regulation in colonic epithelial cells maturation by in depth analysis of the gene expression profile

An evaluation of a pilot counselling model in Hong Kong for individuals with mild-to-moderate psychological issues

Title on author's file: An evaluation of the effectiveness of a pilot 3-session counselling model with individuals with mild and moderate psychological issues in Hong Kong202305 bcwwAccepted ManuscriptSelf-fundedPublishe

Study of the mechanisms underlying heritable germline epimutation of MSH2 in a Hereditary Nonpolyposis Colorectal Cancer family

Colorectal cancer (CRC) is one of the most common cancers world-wide. Among various forms of familial cancers, Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is the most frequent type of hereditary cancer syndromes. It is well known that inactivation of DNA mismatch repair (MMR) genes are responsible for HNPCC. Germline mutations in either the MSH2 or the MLH1 genes can be detected in the majority of HNPCC patients. Alternatively, methylation of the MLH1 promoter in the germline have been observed in a few individuals with HNPCC. More recently, we have first identified a family with inheritance in three successive generations, of germline allele-specific and mosaic hypermethylation of the MSH2 gene promoter, without evidence of DNA mismatch repair gene mutation. Three siblings carrying the germline methylation developed early-onset colorectal (CRC) or endometrial cancers, all with microsatellite instability and MSH2 protein loss [Chan et al Nat Genet 2006]. To further elucidate the underlying mechanism of this germline epimutation, we have performed linkage analysis using microsatellite markers flanking the MSH2 gene. Based on the presence of recombination events, we have successfully narrowed down the chromosomal region to less than 20Mb that is linked with the occurrence of germline MSH2 methylation. Methylation status of the genes with CpG island within this region was examined. The promoter of all genes located upstream of MSH2 were unmethylated. The MSH6 gene which is located downstream of MSH2 also showed no sign of methylation. Our result suggests a possible existence of a cis-acting element that may contribute to the MSH2 methylation. Further study of this chromosomal region may reveal novel mechanisms involved in the regulation of methylation in humans