The emergence of circadian timekeeping in the intestine.

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

Circadian regulation of digestive and metabolic tissues.

The circadian clock is a self-sustained molecular timekeeper that drives 24-h (circadian) rhythms in animals. The clock governs important aspects of behavior and physiology including wake/sleep activity cycles that regulate the activity of metabolic and digestive systems. Light/dark cycles (photoperiod) and cycles in the time of feeding synchronize the circadian clock to the surrounding environment, providing an anticipatory benefit that promotes digestive health. The availability of animal models targeting the genetic components of the circadian clock has made it possible to investigate the circadian clock's role in cellular functions. Circadian clock genes have been shown to regulate the physiological function of hepatocytes, gastrointestinal cells, and adipocytes; disruption of the circadian clock leads to the exacerbation of liver diseases and liver cancer, inflammatory bowel disease and colorectal cancer, and obesity. Previous findings provide strong evidence that the circadian clock plays an integral role in digestive/metabolic disease pathogenesis, hence, the circadian clock is a necessary component in metabolic and digestive health and homeostasis. Circadian rhythms and circadian clock function provide an opportunity to improve the prevention and treatment of digestive and metabolic diseases by aligning digestive system tissue with the 24-h day.

Fluorescent Reporters for Studying Circadian Rhythms in Drosophila melanogaster.

Circadian rhythms are daily oscillations in physiology and gene expression that are governed by a molecular feedback loop known as the circadian clock. In Drosophila melanogaster, the core clock consists of transcription factors clock (Clk) and cycle (cyc) which form protein heterodimers that activate transcription of their repressors, period (per) and timeless (tim). Once produced, protein heterodimers of per/tim repress Clk/cyc activity. One cycle of activation and repression takes approximately ("circa") 24-h ("diem") and repeats even in the absence of external stimuli. The circadian clock is active in many cells throughout the body; however, tracking it dynamically represents a challenge. Traditional fluorescent reporters are slowly degraded and consequently cannot be used to assess dynamic temporal changes exhibited by the circadian clock. The use of rapidly degraded fluorescent protein reporters containing destabilized GFP (dGFP) that report transcriptional activity in vivo at a single-cell level with ~1-h temporal resolution can circumvent this problem. Here we describe the use of circadian clock reporter strains of Drosophila melanogaster, ClockPER and ClockTIM, to track clock transcriptional activity using the intestine as a tissue of interest. These methods may be extended to other tissues in the body.

BMAL1 Regulates the Daily Timing of Colitis.

Many physiological functions exhibit circadian rhythms: oscillations in biological processes that occur in a 24-hour period. These daily rhythms are maintained through a highly conserved molecular pacemaker known as the circadian clock. Circadian disruption has been proposed to cause increased risk of Inflammatory Bowel Disease (IBD) but the underlying mechanisms remain unclear. Patients with IBD experience chronic inflammation and impaired regeneration of intestinal epithelial cells. Previous animal-based studies have revealed that colitis models of IBD are more severe in mice without a circadian clock but the timing of colitis, and whether its inflammatory and regenerative processes have daily rhythms, remains poorly characterized. We tested circadian disruption using Bmal1-/- mutant mice that have a non-functional circadian clock and thus no circadian rhythms. Dextran Sulfate Sodium (DSS) was used to induce colitis. The disease activity of colitis was found to exhibit time-dependent variation in Bmal1+/+ control mice but is constant and elevated in Bmal1-/- mutants, who exhibit poor recovery. Histological analyses indicate worsened colitis severity in Bmal1-/- mutant colon, and colon infiltration of immune system cells shows a daily rhythm that is lost in the Bmal1-/- mutant. Similarly, epithelial proliferation in the colon has a daily rhythm in Bmal1+/+ controls but not in Bmal1-/- mutants. Our results support a critical role of a functional circadian clock in the colon which drives 24-hour rhythms in inflammation and healing, and whose disruption impairs colitis recovery. This indicates that weakening circadian rhythms not only worsens colitis, but delays healing and should be taken into account in the management of IBD. Recognition of this is important in the management of IBD patients required to do shift work.

The Circadian Clock Gene, Bmal1, Regulates Intestinal Stem Cell Signaling and Represses Tumor Initiation.

BACKGROUND & AIMS: Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known. We tested the nonredundant clock gene Bmal1 in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer. METHODS: Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod (day/night cycle) disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal nontransformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal. RESULTS: Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors show high Yes-associated protein 1 (Hippo signaling) activity but show low Wnt (Wingless and Int-1) activity. Intestinal organoid assays show that loss of Bmal1 increases self-renewal in a Yes-associated protein 1-dependent manner. CONCLUSIONS: Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation. Transcript profiling: GEO accession number: GSE157357.

Impaired ECM Remodeling and Macrophage Activity Define Necrosis and Regeneration Following Damage in Aged Skeletal Muscle.

Regenerative capacity of skeletal muscle declines with age, the cause of which remains largely unknown. We investigated extracellular matrix (ECM) proteins and their regulators during early regeneration timepoints to define a link between aberrant ECM remodeling, and impaired aged muscle regeneration. The regeneration process was compared in young (three month old) and aged (18 month old) C56BL/6J mice at 3, 5, and 7 days following cardiotoxin-induced damage to the tibialis anterior muscle. Immunohistochemical analyses were performed to assess regenerative capacity, ECM remodeling, and the macrophage response in relation to plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), and ECM protein expression. The regeneration process was impaired in aged muscle. Greater intracellular and extramyocellular PAI-1 expression was found in aged muscle. Collagen I was found to accumulate in necrotic regions, while macrophage infiltration was delayed in regenerating regions of aged muscle. Young muscle expressed higher levels of MMP-9 early in the regeneration process that primarily colocalized with macrophages, but this expression was reduced in aged muscle. Our results indicate that ECM remodeling is impaired at early time points following muscle damage, likely a result of elevated expression of the major inhibitor of ECM breakdown, PAI-1, and consequent suppression of the macrophage, MMP-9, and myogenic responses.

Time after time: circadian clock regulation of intestinal stem cells.

Daily fluctuations in animal physiology, known as circadian rhythms, are orchestrated by a conserved molecular timekeeper, known as the circadian clock. The circadian clock forms a transcription-translation feedback loop that has emerged as a central biological regulator of many 24-h processes. Early studies of the intestine discovered that many digestive functions have a daily rhythm and that intestinal cell production was similarly time-dependent. As genetic methods in model organisms have become available, it has become apparent that the circadian clock regulates many basic cellular functions, including growth, proliferation, and differentiation, as well as cell signalling and stem cell self-renewal. Recent connections between circadian rhythms and immune system function, and between circadian rhythms and microbiome dynamics, have also been revealed in the intestine. These processes are highly relevant in understanding intestinal stem cell biology. Here we describe the circadian clock regulation of intestinal stem cells primarily in two model organisms: Drosophila melanogaster and mice. Like all cells in the body, intestinal stem cells are subject to circadian timing, and both cell-intrinsic and cell-extrinsic circadian processes contribute to their function.

Testing the expression of circadian clock genes in the tissues of Chinook salmon, Oncorhynchus tshawytscha.

Animals have an endogenous circadian clock that temporally regulates 24 hour (h) oscillations in behavior and physiology. This highly conserved mechanism consists of two positive regulators, Bmal and Clock, and two negative regulators, Cry and Per, that run with a 24-h cycle that synchronizes itself with environmental changes in light, food, and temperature. We examined the circadian clock in Chinook salmon (Oncorhynchus tshawytscha), a non-model organism in which the function of the clock has not been studied. Recent studies indicate that clock genes in Chinook salmon play a role in its evolution of local adaptation, possibly by influencing migration timing. We designed real-time quantitative PCR (RT-qPCR) assays to quantify the transcription of components of the clock system, and validated these for PCR efficiency and specificity in detecting Chinook target genes. Chinook salmon tissue samples were collected in 3-h intervals, over the course of 24 h, from five different organs. Our data indicate that the circadian clock functions differently in each of these tissues. In the liver, positive and negative regulators exhibit anti-phasic peaking in the evening and morning, respectively. However, in the heart, these same regulators peak and trough with a different timing, indicating that the liver and heart are not synchronous. The digestive tract displays yet another difference: simultaneous phases in the expression of positive and negative clock regulators, and we do not observe significant rhythms in clock gene expression in the retina. Our data show that there is a functional clock in Chinook salmon tissues, but that this clock behaves in a tissue-specific manner, regardless of the whole animal being exposed to the same environmental cues. These results highlight the adaptive role of the clock in Chinook salmon and that it may have different positive and negative effects depending on tissue function.

Intestinal Stem Cells Exhibit Conditional Circadian Clock Function.

The circadian clock is a molecular pacemaker that produces 24-hr physiological cycles known as circadian rhythms. How the clock regulates stem cells is an emerging area of research with many outstanding questions. We tested clock function in vivo at the single cell resolution in the Drosophila intestine, a tissue that is exquisitely sensitive to environmental cues and has circadian rhythms in regeneration. Our results indicate that circadian clocks function in intestinal stem cells and enterocytes but are downregulated during enteroendocrine cell differentiation. Drosophila intestinal cells are principally synchronized by the photoperiod, but intestinal stem cell clocks are highly responsive to signaling pathways that comprise their niche, and we find that the Wnt and Hippo signaling pathways positively regulate stem cell circadian clock function. These data reveal that intestinal stem cell circadian rhythms are regulated by cellular signaling and provide insight as to how clocks may be altered during physiological changes such as regeneration and aging.

The Circadian Clock Gene BMAL1 Coordinates Intestinal Regeneration.

BACKGROUND & AIMS: The gastrointestinal syndrome is an illness of the intestine caused by high levels of radiation. It is characterized by extensive loss of epithelial tissue integrity, which initiates a regenerative response by intestinal stem and precursor cells. The intestine has 24-hour rhythms in many physiological functions that are believed to be outputs of the circadian clock: a molecular system that produces 24-hour rhythms in transcription/translation. Certain gastrointestinal illnesses are worsened when the circadian rhythms are disrupted, but the role of the circadian clock in gastrointestinal regeneration has not been studied. METHODS: We tested the timing of regeneration in the mouse intestine during the gastrointestinal syndrome. The role of the circadian clock was tested genetically using the BMAL1 loss of function mouse mutant in vivo, and in vitro using intestinal organoid culture. RESULTS: The proliferation of the intestinal epithelium follows a 24-hour rhythm during the gastrointestinal syndrome. The circadian clock runs in the intestinal epithelium during this pathologic state, and the loss of the core clock gene, BMAL1, disrupts both the circadian clock and rhythmic proliferation. Circadian activity in the intestine involves a rhythmic production of inflammatory cytokines and subsequent rhythmic activation of the JNK stress response pathway. CONCLUSIONS: Our results show that a circadian rhythm in inflammation and regeneration occurs during the gastrointestinal syndrome. The study and treatment of radiation-induced illnesses, and other gastrointestinal illnesses, should consider 24-hour timing in physiology and pathology.

Evasion of anti-growth signaling: A key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds.

The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting.

Surfaceome profiling reveals regulators of neural stem cell function.

The composition of cell-surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry-based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor-ß signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N-cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c-kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain-derived populations and identifies a host of other candidates that may regulate NSCs.

The circadian clock gates the intestinal stem cell regenerative state.

The intestine has evolved under constant environmental stresses, because an animal may ingest harmful pathogens or chemicals at any time during its lifespan. Following damage, intestinal stem cells (ISCs) regenerate the intestine by proliferating to replace dying cells. ISCs from diverse animals are remarkably similar, and the Wnt, Notch, and Hippo signaling pathways, important regulators of mammalian ISCs, are conserved from flies to humans. Unexpectedly, we identified the transcription factor period, a component of the circadian clock, to be critical for regeneration, which itself follows a circadian rhythm. We discovered hundreds of transcripts that are regulated by the clock during intestinal regeneration, including components of stress response and regeneration pathways. Disruption of clock components leads to arrhythmic ISC divisions, revealing their underappreciated role in the healing process.

A genome-scale shRNA resource for transgenic RNAi in Drosophila.

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.

The search for putative precursor cells within the pancreas has been the focus of extensive research. Previously, we identified rare pancreas-derived multipotent precursor (PMP) cells in the mouse with the intriguing capacity to generate progeny in the pancreatic and neural lineages. Here, we establish the embryonic pancreas as the developmental source of PMPs through lineage-labeling experiments. We also show that PMPs express insulin and can contribute to multiple pancreatic and neural cell types in vivo. In addition, we have isolated PMPs from adult human islet tissue that are also capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types. Finally, both mouse and human PMP-derived cells ameliorated diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin(+) multipotent stem cells and suggest that these cells may provide a promising line of investigation toward potential therapeutic benefit.

All for one, and one for all: the clonality of the intestinal stem cell niche.

Intestinal epithelia are maintained by intestinal stem cells (ISCs) that divide to replace dying absorptive and secretory cells that make up this tissue. Lineage labeling studies, both in vertebrates and Drosophila, have revealed the relationships between ISCs and their progeny. In addition, a number of signaling pathways involved in ISC proliferation and differentiation have been identified. Further studies will clarify the signals originating from the ISC niche and determine the processes that control the number and uniform distribution of niches throughout the epithelium.

The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration.

Identification of the signaling pathways that control the proliferation of stem cells (SCs), and whether they act in a cell or non-cell autonomous manner, is key to our understanding of tissue homeostasis and cancer. In the adult Drosophila midgut, the Jun N-Terminal Kinase (JNK) pathway is activated in damaged enterocyte cells (ECs) following injury. This leads to the production of Upd cytokines from ECs, which in turn activate the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway in Intestinal SCs (ISCs), stimulating their proliferation. In addition, the Hippo pathway has been recently implicated in the regulation of Upd production from the ECs. Here, we show that the Hippo pathway target, Yorkie (Yki), also plays a crucial and cell-autonomous role in ISCs. Activation of Yki in ISCs is sufficient to increase ISC proliferation, a process involving Yki target genes that promote division, survival and the Upd cytokines. We further show that prior to injury, Yki activity is constitutively repressed by the upstream Hippo pathway members Fat and Dachsous (Ds). These findings demonstrate a cell-autonomous role for the Hippo pathway in SCs, and have implications for understanding the role of this pathway in tumorigenesis and cancer stem cells.

The germline stem cells of Drosophila melanogaster partition DNA non-randomly.

The Immortal Strand Hypothesis proposes that asymmetrically dividing stem cells cosegregate chromatids to retain ancestral DNA templates. Using both pulse-chase and label retention assays, we show that non-random partitioning of DNA occurs in germline stem cells (GSCs) in the Drosophila ovary as these divide asymmetrically to generate a new GSC and a differentiating cystoblast. This process is disrupted when GSCs are forced to differentiate through the overexpression of Bag of Marbles, a factor that impels the terminal differentiation of cystoblasts. When Decapentaplegic, a ligand which maintains the undifferentiated state of GSCs, is expressed ectopically the non-random partitioning of DNA is similarly disrupted. Our data suggest asymmetric chromatid segregation is coupled to mechanisms specifying cellular differentiation via asymmetric stem cell division.

E-Cadherin regulates neural stem cell self-renewal.

E-Cadherin, a cell adhesion protein, has been shown to take part in the compartmentalization, proliferation, survival, and differentiation of cells. E-Cadherin is expressed in the adult and embryonic forebrain germinal zones in vivo, and in clonal colonies of cells derived from these regions and grown in vitro. Mice carrying E-Cadherin floxed genes crossed to mice expressing Cre under the Nestin promoter demonstrate defects in the self-renewal of neural stem cells both in vivo and in vitro. The functional role of E-Cadherin is further demonstrated using adhesion-blocking antibodies in vitro, which specifically target cadherin extracellular adhesive domains. Adult neural stem cell colonies decrease in the presence of E-Cadherin antibodies in a dosage-dependent manner, in contrast to P-Cadherin antibody. On overexpression of normal E-Cadherin and a mutated E-Cadherin, containing no intracellular binding domain, an increased number of clonal adult neural stem cell colonies are observed. These data suggest it is specifically E-Cadherin adhesion that is responsible for these self-renewal effects. These data show the importance of E-Cadherin in the neural stem cell niche and suggest E-Cadherin regulates the number of these cells.

Don't look: growing clonal versus nonclonal neural stem cell colonies.

Recent reports have challenged the clonality of the neurosphere assay in assessing neural stem cell (NSC) numbers quantitatively. We tested the clonality of the neurosphere assay by culturing mixtures of differently labeled neural cells, watching single neural cells proliferate using video microscopy, and encapsulating single NSCs and their progeny. The neurosphere assay gave rise to clonal colonies when using primary cells plated at 10 cells/microl or less; however, when using passaged NSCs, the spheres were clonal only if plated at 1 cell/microl. Most important, moving the plates during the growth phase (to look at cultures microscopically) greatly increased the incidence of nonclonal colonies. To ensure clonal sphere formation and investigate nonautonomous effects on clonal sphere formation frequencies, single NSCs were encapsulated in agarose and proliferated as clonal free-floating spheres. We demonstrate that clonal neurospheres can be grown by avoiding movement-induced aggregation, by single-cell tracking, and by encapsulation of single cells. Disclosure of potential conflicts of interest is found at the end of this article.