Lineage-tracing approaches, widely used to characterize stem cell populations, rely on the specificity and stability of individual markers for accurate results. We present a method in which genetic ...labeling in the intestinal epithelium is acquired as a mutation-induced clonal mark during DNA replication. By determining the rate of mutation in vivo and combining this data with the known neutral-drift dynamics that describe intestinal stem cell replacement, we quantify the number of functional stem cells in crypts and adenomas. Contrary to previous reports, we find that significantly lower numbers of “working” stem cells are present in the intestinal epithelium (five to seven per crypt) and in adenomas (nine per gland), and that those stem cells are also replaced at a significantly lower rate. These findings suggest that the bulk of tumor stem cell divisions serve only to replace stem cell loss, with rare clonal victors driving gland repopulation and tumor growth.
•Replication-dependent clonal label applied to infer stem cell dynamics•Stem cell numbers and replacement rates are lower than previous estimates•Stem cell dynamics are unchanging with age•Low stem cell numbers but high replacement rates follow oncogenic transformation
A replication-dependent clonal-label approach reveals significantly lower than expected numbers of “working” stem cells in the intestinal epithelium and in adenomas and slower rates of stem cell replacement.
The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly ...cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.
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.
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.
Medulloblastoma is heterogeneous, being characterized by molecular subgroups that demonstrate distinct gene expression profiles. Activation of the WNT or SHH signaling pathway characterizes two of ...these molecular subgroups, the former associated with low-risk disease and the latter potentially targeted by novel SHH pathway inhibitors. This manuscript reports the validation of a novel diagnostic immunohistochemical method to distinguish SHH, WNT, and non-SHH/WNT tumors and details their associations with clinical, pathological and cytogenetic variables. A cohort (
n
= 235) of medulloblastomas from patients aged 0.4–52 years was studied for expression of four immunohistochemical markers: GAB1, β-catenin, filamin A, and YAP1. Immunoreactivity (IR) for GAB1 characterizes only SHH tumors and nuclear IR for β-catenin only WNT tumors. IRs for filamin A and YAP1 identify SHH and WNT tumors. SHH, WNT, and non-SHH/WNT tumors contributed 31, 14, and 55% to the series. All desmoplastic/nodular (D/N) medulloblastomas were SHH tumors, while most WNT tumors (94%) had a classic phenotype. Monosomy 6 was strongly associated with WNT tumors, while
PTCH1
loss occurred almost exclusively among SHH tumors.
MYC
or
MYCN
amplification and chromosome 17 imbalance occurred predominantly among non-SHH/WNT tumors. Among patients aged 3–16 years and entered onto the SIOP PNET3 trial, outcome was significantly better for children with WNT tumors, when compared to SHH or non-SHH/WNT tumors, which showed similar survival curves. However, high-risk factors (M+ disease, LC/A pathology,
MYC
amplification) significantly influenced survival in both SHH and non-SHH/WNT groups. We describe a robust method for detecting SHH, WNT, and non-SHH/WNT molecular subgroups in formalin-fixed medulloblastoma samples. In corroborating other studies that indicate the value of combining clinical, pathological, and molecular variables in therapeutic stratification schemes for medulloblastoma, we also provide the first outcome data based on a clinical trial cohort and novel data on how molecular subgroups are distributed across the range of disease.
Coastal ecosystems host high levels of primary productivity leading to exceptionally dynamic elemental cycling in both water and sediments. In such environments, carbon is rapidly cycled leading to ...high rates of burial as organic matter and/or high rates of loss to the atmosphere and laterally to the coastal ocean in simpler forms, such as carbon dioxide (CO2) and methane (CH4). To better understand carbon cycling across these heterogeneous environments, new technologies beyond discrete sample collection and analysis are needed to characterize spatial and temporal variability. Here, we describe the ChemYak, an autonomous surface vehicle outfitted with a suite of in situ sensors, developed to achieve large spatial scale chemical mapping of these environments. Dissolved methane and carbon dioxide are measured by a laser spectrometer coupled to a gas extraction unit for continuous quantification during operation. The gas-powered vehicle is capable of rapidly surveying the coastal system with an endurance of up to 10 h at operating speeds in excess of 10 km h–1. Here, we demonstrate its ability to spatially characterize distributions of CO2, CH4, oxygen, and nitrate throughout a New England saltmarsh estuary.
Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of ...differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target.
We present a year‐round time series of dissolved methane (CH4), along with targeted observations during ice melt of CH4 and carbon dioxide (CO2) in a river and estuary adjacent to Cambridge Bay, ...Nunavut, Canada. During the freshet, CH4 concentrations in the river and ice‐covered estuary were up to 240,000% saturation and 19,000% saturation, respectively, but quickly dropped by >100‐fold following ice melt. Observations with a robotic kayak revealed that river‐derived CH4 and CO2 were transported to the estuary and rapidly ventilated to the atmosphere once ice cover retreated. We estimate that river discharge accounts for >95% of annual CH4 sea‐to‐air emissions from the estuary. These results demonstrate the importance of resolving seasonal dynamics in order to estimate greenhouse gas emissions from polar systems.
Plain Language Summary
The primary cause of recent global climate change is increasing concentrations of heat‐trapping greenhouse gases in the atmosphere. Ongoing rapid Arctic climate change is affecting the annual cycle of sea ice formation and retreat; however, most published studies of greenhouse gases in Arctic waters have been conducted during ice‐free, summertime conditions. In order to characterize seasonal variability in greenhouse gas distributions, we collected year‐round measurements of the greenhouse gas methane (CH4) in a coastal Arctic system near Cambridge Bay, Nunavut, Canada. We found that during the ice melt season, river water contains methane concentrations up to 2,000 times higher than the wintertime methane concentrations in the coastal ocean. We utilized a novel robotic kayak to conduct high‐resolution mapping of greenhouse gas distributions during ice melt. From these data, we demonstrate that the river water containing elevated levels of methane and carbon dioxide (CO2) flowed into the coastal ocean, and when ice cover melted, these greenhouse gases were rapidly emitted into the atmosphere. We estimate that in this system, more than 95% of all annual methane emissions from the estuary are driven by river inflow.
Key Points
Methane concentrations in an Arctic estuary show strong seasonality; river inflow at the start of the freshet drives elevated concentrations
Observations with a novel robotic kayak demonstrate that methane and carbon dioxide in the estuary are rapidly ventilated following ice melt
River discharge is estimated to account for >95% of annual methane emissions from the estuary
Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the
community voiced near ...unanimous support for a concept of
that represented a clade comprising all agriculturally and clinically important
species, including the
species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus
into seven genera, including the FSSC described as members of the genus
, with subsequent justification in 2018 based on claims that the 2013 concept of
is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of
including the FSSC. We reassert the practical and scientific argument in support of a genus
that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus
were recombined in genus
, and nine others were renamed
Here the global
community voices strong support for the inclusion of the FSSC in
, as it remains the best scientific, nomenclatural, and practical taxonomic option available.
Little is known about the stem cell organisation of the normal oesophagus or Barrett's metaplastic oesophagus. Using non-pathogenic mitochondrial DNA mutations as clonal markers, the authors reveal ...the stem cell organisation of the human squamous oesophagus and of Barrett's metaplasia and determine the mechanism of clonal expansion of mutations.
Mutated cells were identified using enzyme histochemistry to detect activity of cytochrome c oxidase (CCO). CCO-deficient cells were laser-captured and mutations confirmed by PCR sequencing. Cell lineages were identified using immunohistochemistry.
The normal squamous oesophagus contained CCO-deficient patches varying in size from around 30 μm up to about 1 mm. These patches were clonal as each area within a CCO-deficient patch contained an identical mitochondrial DNA mutation. In Barrett's metaplasia partially CCO-deficient glands indicate that glands are maintained by multiple stem cells. Wholly mutated Barrett's metaplasia glands containing all the expected differentiated cell lineages were seen, demonstrating multilineage differentiation from a clonal population of Barrett's metaplasia stem cells. Patches of clonally mutated Barrett's metaplasia glands were observed, indicating glands can divide to form patches. In one patient, both the regenerating squamous epithelium and the underlying glandular tissue shared a clonal mutation, indicating that they are derived from a common progenitor cell.
In normal oesophageal squamous epithelium, a single stem cell clone can populate large areas of epithelium. Barrett's metaplasia glands are clonal units, contain multiple multipotential stem cells and most likely divide by fission. Furthermore, a single cell of origin can give rise to both squamous and glandular epithelium suggesting oesophageal plasticity.
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