Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 ...cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer’s patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.
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•Multimodal atlas of human intestinal development maps 101 cell types onto tissue•Charts developmental origins of diverse cellular compartments and their progenitors•Functional diversity of fibroblasts in stem cell, vasculature, and GALT formation•Resource applied to interrogate pathology of in utero intestinal diseases
Fawkner-Corbett et al. chart human intestinal morphogenesis across time, location, and cellular compartments using a combination of single-cell RNA sequencing and spatial transcriptomics. The resulting online searchable atlas describes the principles of crypt-villus axis formation as well as neural, vascular, mesenchymal morphogenesis, and immune populations of the developing gut.
Cancer is a disease in which cells accumulate genetic aberrations that are believed to confer a clonal advantage over cells in the surrounding tissue. However, the quantitative benefit of frequently ...occurring mutations during tumor development remains unknown. We quantified the competitive advantage of Ape loss, Kras activation, and P53 mutations in the mouse intestine. Our findings indicate that the fate conferred by these mutations is not deterministic, and many mutated stem cells are replaced by wild-type stem cells after biased, but still stochastic events. Furthermore, P53 mutations display a condition-dependent advantage, and especially in colitis-affected intestines, clones harboring mutations in this gene are favored. Our work confirms the previously theoretical notion that the tissue architecture of the intestine suppresses the accumulation of mutated lineages.
The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. ...Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states.Βeta-cells have recently been shown to be heterogeneous with regard to morphology and function. Here, the authors show that β-cells in zebrafish switch from proliferative to functional states with increasing time since β-cell birth, leading to functional and proliferative heterogeneity.
The intestinal epithelium is largely maintained by self-renewing stem cells but with apparently committed progenitors also contributing, particularly following tissue damage. However, the mechanism ...of, and requirement for, progenitor plasticity in mediating pathological response remain unknown. Here we show that phosphorylation of the transcription factor Atoh1 is required for both the contribution of secretory progenitors to the stem cell pool and for a robust regenerative response. As confirmed by lineage tracing, Atoh1+ cells (Atoh1(WT)CreERT2 mice) give rise to multilineage intestinal clones both in the steady state and after tissue damage. In a phosphomutant Atoh1(9S/T-A)CreERT2 line, preventing phosphorylation of ATOH1 protein acts to promote secretory differentiation and inhibit the contribution of progenitors to self-renewal. Following chemical colitis, Atoh1+ cells of Atoh1(9S/T-A)CreERT2 mice have reduced clonogenicity that affects overall regeneration. Progenitor plasticity maintains robust self-renewal in the intestinal epithelium, and the balance between stem and progenitor fate is directly coordinated by ATOH1 multisite phosphorylation.
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•Atoh1+ progenitors contribute to the stem cell pool in homeostasis and regeneration•Multisite phosphorylation of ATOH1 regulates the plasticity of secretory progenitors•Loss of phosphorylation of ATOH1 reduces clonogenic capacity of Atoh1+ cells•Phosphomutant ATOH1 mice are more susceptible to chemical colitis
Tomic et al. report that multisite phosphorylation of ATOH1 regulates the contribution of secretory progenitors to stem cell self-renewal in the small intestine and colon. With damage, the enhanced role of Atoh1+ progenitors in mediating tissue repair is ablated in mice expressing phosphomutant ATOH1 and overall tissue regeneration is impaired.
We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. ...First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.
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•Colonic stem cell dynamics predict lifetime mutant allele frequencies•Mutant clone fixation in colonic crypts takes years due to slow stem cell turnover•Crypt fission enables lateral expansion of mutant clones•Biases in both fixation and expansion increase age-related pro-oncogenic burden
Winton and colleagues describe stem cell dynamics in normal human colon to identify the efficiency of clone fixation within the epithelium and the rate of subsequent lateral expansion. Against these benchmarks biased stem cell behaviors advantaged in both fixation and expansion can be quantified to predict the age-related burden of pro-oncogenic mutation.
How mutations lead to tumor formation is a central question in cancer research. Although cellular changes that follow the occurrence of common mutations are well characterized, much less is known ...about their effects on the population level. Now, two recent studies reveal in what way oncogenic aberrations alter stem cell dynamics to provide cells with an evolutionary advantage over their neighbors (Amoyel et al, ; Baker et al, ).
A new study on clonal tracing in human tissue validates the concept of neutral competition, earlier revealed by genetic manipulation in various model organisms.
Somatic copy number alterations (SCNAs) are an important class of genomic alteration in cancer. They are frequently observed in cancer samples, with studies showing that, on average, SCNAs affect 34% ...of a cancer cell's genome. Furthermore, SCNAs have been shown to be major drivers of tumour development and have been associated with response to therapy and prognosis. Large-scale cancer genome studies suggest that tumours are driven by somatic copy number alterations (SCNAs) or single-nucleotide variants (SNVs). Despite the frequency of SCNAs and their clinical relevance, the use of genomics assays in the clinic is biased towards targeted gene panels, which identify SNVs but provide limited scope to detect SCNAs throughout the genome. There is a need for a comparably low-cost and simple method for high-resolution SCNA profiling. We present conliga, a fully probabilistic method that infers SCNA profiles from a low-cost, simple, and clinically-relevant assay (FAST-SeqS). When applied to 11 high-purity oesophageal adenocarcinoma samples, we obtain good agreement (Spearman's rank correlation coefficient, r.sub.s=0.94) between conliga's inferred SCNA profiles using FAST-SeqS data (approximately pounds sterling14 per sample) and those inferred by ASCAT using high-coverage WGS (gold-standard). We find that conliga outperforms CNVkit (r.sub.s=0.89), also applied to FAST-SeqS data, and is comparable to QDNAseq (r.sub.s=0.96) applied to low-coverage WGS, which is approximately four-fold more expensive, more laborious and less clinically-relevant. By performing an in silico dilution series experiment, we find that conliga is particularly suited to detecting SCNAs in low tumour purity samples. At two million reads per sample, conliga is able to detect SCNAs in all nine samples at 3% tumour purity and as low as 0.5% purity in one sample. Crucially, we show that conliga's hidden state information can be used to decide when a sample is abnormal or normal, whereas CNVkit and QDNAseq cannot provide this critical information. We show that conliga provides high-resolution SCNA profiles using a convenient, low-cost assay. We believe conliga makes FAST-SeqS a more clinically valuable assay as well as a useful research tool, enabling inexpensive and fast copy number profiling of pre-malignant and cancer samples.
Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that ...underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.
Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external ...factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1−/− mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1−/− cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.
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•Hemogenic endothelium (HE) and pro-HSCs emerge in Ncx1−/− embryos lacking circulation•Ncx1−/− pro-HSCs are transcriptionally and functionally impaired•The HE to pro-HSC transition involves a glycolysis to OxPhos metabolic shift•Activation of glycolysis reduces hematopoietic output of E9.5 intraembryonic precursors
Azzoni et al. report that hematopoietic stem cell precursors (pro-HSC) still emerge in the absence of circulation, but are functionally impaired and do not activate the hematopoietic program. Single-cell transcriptomics indicate that the onset of circulation induces a metabolic switch that is required for hematopoietic development.
The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous ...lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.
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•All acinar cells have an equal probability to contribute to tissue renewal•All acinar cells function as bona fide progenitor cells during homeostasis•Acinar fission-like events underlie pancreas regeneration
The dynamics of tissue maintenance in the adult exocrine pancreas are largely unknown. In this study, Lodestijn et al. use a combination of experimental and computational methods to reveal that there is no hierarchy in the adult exocrine pancreas and that all cells can act as bona fide progenitors.
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