The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

SOX9 modulates cancer biomarker and cilia genes in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with high mortality. The cellular origins of PDAC are largely unknown; however, ductal cells, especially centroacinar cells (CACs), have several characteristics in common with PDAC, such as expression of SOX9 and components of the Notch-signaling pathway. Mutations in KRAS and alterations to Notch signaling are common in PDAC, and both these pathways regulate the transcription factor SOX9. To identify genes regulated by SOX9, we performed siRNA knockdown of SOX9 followed by RNA-seq in PANC-1s, a human PDAC cell line. We report 93 differentially expressed (DE) genes, with convergence on alterations to Notch-signaling pathways and ciliogenesis. These results point to SOX9 and Notch activity being in a positive feedback loop and SOX9 regulating cilia production in PDAC. We additionally performed ChIP-seq in PANC-1s to identify direct targets of SOX9 binding and integrated these results with our DE gene list. Nine of the top 10 downregulated genes have evidence of direct SOX9 binding at their promoter regions. One of these targets was the cancer stem cell marker EpCAM. Using whole-mount in situ hybridization to detect epcam transcript in zebrafish larvae, we demonstrated that epcam is a CAC marker and that Sox9 regulation of epcam expression is conserved in zebrafish. Additionally, we generated an epcam null mutant and observed pronounced defects in ciliogenesis during development. Our results provide a link between SOX9, EpCAM and ciliary repression that can be exploited in improving our understanding of the cellular origins and mechanisms of PDAC.

Dissociable effects of visual crowding on the perception of color and motion.

Our ability to recognize objects in peripheral vision is fundamentally limited by crowding, the deleterious effect of clutter that disrupts the recognition of features ranging from orientation and color to motion and depth. Previous research is equivocal on whether this reflects a singular process that disrupts all features simultaneously or multiple processes that affect each independently. We examined crowding for motion and color, two features that allow a strong test of feature independence. "Cowhide" stimuli were presented 15° in peripheral vision, either in isolation or surrounded by flankers to give crowding. Observers reported either the target direction (clockwise/counterclockwise from upward) or its hue (blue/purple). We first established that both features show systematic crowded errors (biased predominantly toward the flanker identities) and selectivity for target-flanker similarity (with reduced crowding for dissimilar target/flanker elements). The multiplicity of crowding was then tested with observers identifying both features. Here, a singular object-selective mechanism predicts that when crowding is weak for one feature and strong for the other that crowding should be all-or-none for both. In contrast, when crowding was weak for color and strong for motion, errors were reduced for color but remained for motion, and vice versa with weak motion and strong color crowding. This double dissociation reveals that crowding disrupts certain combinations of visual features in a feature-specific manner, ruling out a singular object-selective mechanism. Thus, the ability to recognize one aspect of a cluttered scene, like color, offers no guarantees for the correct recognition of other aspects, like motion.

Objective assessment of stored blood quality by deep learning.

Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans' assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

Attenuating the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase-Sex-Determining Region Y-Box 9 Axis Promotes Liver Progenitor Cell-Mediated Liver Regeneration in Zebrafish.

BACKGROUND AND AIMS: The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC-to-hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. APPROACH AND RESULTS: Here, using zebrafish models of LPC-mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC-to-hepatocyte differentiation through the mitogen-activated ERK kinase (MEK)-extracellular signal-regulated kinase (ERK)-sex-determining region Y-box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC-to-hepatocyte differentiation as well as genetic suppression of the EGFR-ERK-SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short-term treatment with EGFR inhibitors resulted in better liver recovery over the long term. CONCLUSIONS: The EGFR-ERK-SOX9 axis suppresses LPC-to-hepatocyte differentiation during LPC-mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

Label-Free Analysis of Red Blood Cell Storage Lesions Using Imaging Flow Cytometry.

Deleterious changes, collectively termed as storage lesions, alter the characteristics of red blood cell (RBC) morphology during in vitro storage. Due to gradual loss of cellular membrane, RBCs lose their original biconcave disk shape and begin a process of spherical deformation that is characterized by well-defined morphological criteria. At the spheroechinocyte stage, the structure of RBC is irreversibly damaged and lacks the elasticity necessary to efficiently deliver oxygen. Quantifying the prevalence of spheroechinocytes could provide an important morphological measure of the quality of stored blood products. Unlike the conventional RBC morphology characterization assay involving light microscopy, we introduce a label-free assay using imaging flow cytometry (IFC). The technique captures 100,000 images of a sample and calculates a relative measure of spheroechinocyte population in a fraction of the time required by the conventional method. A comparative method study, measuring a morphological index for 11 RCC units through storage, found that the two techniques measured similar trends with IFC reporting the metric at an average of 3.9% higher. We monitored 18 RCC units between Weeks 1 and 6 of storage and found that the spheroechinocyte population increased by an average of 26.2%. The large (3.5-64.1%) variation between the units' spheroechinocyte population percentage at Week 1 suggests a possible dependence of blood product quality on donor characteristics. Given our method's ability to rapidly monitor large samples and refine morphological characterization beyond conventional methods, we believe our technique offers good potential for studying the underlying relationships between RBC morphology and blood storage lesions. © 2019 International Society for Advancement of Cytometry.

Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking.

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.

Centroacinar cells: At the center of pancreas regeneration.

The process of regeneration serves to heal injury by replacing missing cells. Understanding regeneration can help us replace cell populations lost during disease, such as the insulin-producing ß cells lost in diabetic patients. Centroacinar cells (CACs) are a specialized ductal pancreatic cell type that act as progenitors to replace ß cells in the zebrafish. However, whether CACs contribute to ß-cell regeneration in adult mammals remains controversial. Here we review the current understanding of the role of CACs as endocrine progenitors during regeneration in zebrafish and mammals.

Sox9b is a mediator of retinoic acid signaling restricting endocrine progenitor differentiation.

Centroacinar cells (CACs) are ductal Notch-responsive progenitors that in the larval zebrafish pancreas differentiate to form new islets and ultimately contribute to the majority of the adult endocrine mass. Uncovering the mechanisms regulating CAC differentiation will facilitate understanding how insulin-producing ß cells are formed. Previously we reported retinoic acid (RA) signaling and Notch signaling both regulate larval CAC differentiation, suggesting a shared downstream intermediate. Sox9b is a transcription factor important for islet formation whose expression is upregulated by Notch signaling in larval CACs. Here we report that sox9b expression in larval CACs is also regulated by RA signaling. Therefore, we hypothesized that Sox9b is an intermediate between both RA- and Notch-signaling pathways. In order to study the role of Sox9b in larval CACs, we generated two cre/lox based transgenic tools, which allowed us to express full-length or truncated Sox9b in larval CACs. In this way we were able to perform spatiotemporal-controlled Sox9b gain- and loss-of-function studies and observe the subsequent effect on progenitor differentiation. Our results are consistent with Sox9b regulating CAC differentiation by being a downstream intermediate of both RA- and Notch-signaling pathways. We also demonstrate that adult zebrafish with only one functional allele of sox9b undergo accelerated ß-cell regeneration, an observation consistent with sox9b regulating CAC differentiation in adults.

Distinct Notch signaling outputs pattern the developing arterial system.

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct developmental windows in which Notch signaling acts to promote artery commitment and maintenance. Together, these findings demonstrate that Notch acts in distinct contexts to initiate and maintain artery identity during embryogenesis.

Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord.

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.

Fate mapping of ptf1a-expressing cells during pancreatic organogenesis and regeneration in zebrafish.

BACKGROUND: Pancreas development in zebrafish shares many features with mammals, including the participation of epithelial progenitor cells expressing pancreas transcription factor 1a (ptf1a). However, to date it has remained unclear whether, as in mammals, ptf1a-expressing zebrafish pancreatic progenitors are able to contribute to multiple exocrine and endocrine lineages. To delineate the lineage potential of ptf1a-expressing cells, we generated ptf1a:creER(T2) transgenic fish and performed genetic-inducible lineage tracing in developmental, regenerating, and ptf1a-deficient zebrafish pancreas. RESULTS: In addition to their contribution to the acinar cell lineage, ptf1a-expressing cells give rise to both pancreatic Notch-responsive-cells (PNCs) as well as small numbers of endocrine cells during pancreatic development. In fish with ptf1a haploinsufficiency, a higher proportion of ptf1a lineage-labeled cells are traced into the PNC and endocrine compartments. Further reduction of ptf1a gene dosage converts pancreatic progenitor cells to gall bladder and other non-pancreatic cell fates. CONCLUSIONS: Our results confirm the presence of multipotent ptf1a-expressing progenitor cells in developing zebrafish pancreas, with reduced ptf1a dosage promoting greater contributions towards non-acinar lineages. As in mammals, loss of ptf1a results in conversion of nascent pancreatic progenitor cells to non-pancreatic cell fates, underscoring the central role of ptf1a in foregut tissue specification.

Retinoic acid plays an evolutionarily conserved and biphasic role in pancreas development.

As the developing zebrafish pancreas matures, hormone-producing endocrine cells differentiate from pancreatic Notch-responsive cells (PNCs) that reside within the ducts. These new endocrine cells form small clusters known as secondary (2°) islets. We use the formation of 2° islets in the pancreatic tail of the larval zebrafish as a model of ß-cell neogenesis. Pharmacological inhibition of Notch signaling leads to precocious endocrine differentiation and the early appearance of 2° islets in the tail of the pancreas. Following a chemical screen, we discovered that blocking the retinoic acid (RA)-signaling pathway also leads to the induction of 2° islets. Conversely, the addition of exogenous RA blocks the differentiation caused by Notch inhibition. In this report we characterize the interaction of these two pathways. We first verified that signaling via both RA and Notch ligands act together to regulate pancreatic progenitor differentiation. We produced a transgenic RA reporter, which demonstrated that PNCs directly respond to RA signaling through the canonical transcriptional pathway. Next, using a genetic lineage tracing approach, we demonstrated these progenitors produce endocrine cells following inhibition of RA signaling. Lastly, inhibition of RA signaling using a cell-type specific inducible cre/lox system revealed that RA signaling acts cell-autonomously in PNCs to regulate their differentiation. Importantly, the action of RA inhibition on endocrine formation is evolutionarily conserved, as shown by the differentiation of human embryonic stem cells in a model of human pancreas development. Together, these results revealed a biphasic function for RA in pancreatogenesis. As previously shown by others, RA initially plays an essential role during embryogenesis as it patterns the endoderm and specifies the pancreatic field. We reveal here that later in development RA is involved in negatively regulating the further differentiation of pancreatic progenitors and expands upon the developmental mechanisms by which this occurs.

Ribosomal biogenesis genes play an essential and p53-independent role in zebrafish pancreas development.

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

A twin and molecular genetics study of sleep paralysis and associated factors.

Sleep paralysis is a relatively common but under-researched phenomenon. In this paper we examine prevalence in a UK sample and associations with candidate risk factors. This is the first study to investigate the heritability of sleep paralysis in a twin sample and to explore genetic associations between sleep paralysis and a number of circadian expressed single nucleotide polymorphisms. Analyses are based on data from the Genesis1219 twin/sibling study, a community sample of twins/siblings from England and Wales. In total, data from 862 participants aged 22-32 years (34% male) were used in the study. This sample consisted of monozygotic and dizygotic twins and siblings. It was found that self-reports of general sleep quality, anxiety symptoms and exposure to threatening events were all associated independently with sleep paralysis. There was moderate genetic influence on sleep paralysis (53%). Polymorphisms in the PER2 gene were associated with sleep paralysis in additive and dominant models of inheritance-although significance was not reached once a Bonferroni correction was applied. It is concluded that factors associated with disrupted sleep cycles appear to be associated with sleep paralysis. In this sample of young adults, sleep paralysis was moderately heritable. Future work should examine specific polymorphisms associated with differences in circadian rhythms and sleep homeostasis further in association with sleep paralysis.

Adoption of the Q transcriptional regulatory system for zebrafish transgenesis.

The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio rerio. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos; however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish.

Epigenome-Wide DNA Methylation Analysis of Monozygotic Twins Discordant for Diurnal Preference.

Diurnal preference is an individual's preference for daily activities and sleep timing and is strongly correlated with the underlying circadian clock and the sleep-wake cycle validating its use as an indirect circadian measure in humans. Recent research has implicated DNA methylation as a mechanism involved in the regulation of the circadian clock system in humans and other mammals. In order to evaluate the extent of epigenetic differences associated with diurnal preference, we examined genome-wide patterns of DNA methylation in DNA from monozygotic (MZ) twin-pairs discordant for diurnal preference. MZ twins were selected from a longitudinal twin study designed to investigate the interplay of genetic and environmental factors in the development of emotional and behavioral difficulties. Fifteen pairs of MZ twins were identified in which one member scored considerably higher on the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ) than the other. Genome-wide DNA methylation patterns were assessed in twins' buccal cell DNA using the Illumina Infinium HumanMethylation450 BeadChips. Quality control and data pre-processing was undertaken using the wateRmelon package. Differentially methylated probes (DMPs) were identified using an analysis strategy taking into account both the significance and the magnitude of DNA methylation differences. Our data indicate that DNA methylation differences are detectable in MZ twins discordant for diurnal preference. Moreover, downstream gene ontology (GO) enrichment analysis on the top-ranked diurnal preference associated DMPs revealed significant enrichment of pathways that have been previously associated with circadian rhythm regulation, including cell adhesion processes and calcium ion binding.

Amyloid precursor protein (APP) contributes to pathology in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS), the progressive loss of motor neurons is accompanied by extensive muscle denervation, resulting in paralysis and ultimately death. Upregulation of amyloid beta (A4) precursor protein (APP) in muscle fibres coincides with symptom onset in both sporadic ALS patients and the SOD1(G93A) mouse model of familial ALS. We have further characterized this response in SOD1(G93A) mice and also revealed elevated levels of ß-amyloid (Aß) peptides in the SOD1(G93A) spinal cord, which were predominantly localized within motor neurons and their surrounding glial cells. We therefore examined the effect of genetic ablation of APP on disease progression in SOD1(G93A) mice, which significantly improved multiple disease parameters, including innervation, motor function, muscle contractile characteristics, motor unit and motor neuron survival. These results therefore strongly suggest that APP actively contributes to SOD1(G93A)-mediated pathology. Together with observations from ALS cases, this study indicates that APP may contribute to human ALS pathology.

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing ß-cells.

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing ß-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.

Polymorphisms in the circadian expressed genes PER3 and ARNTL2 are associated with diurnal preference and GNß3 with sleep measures.

Sleep and circadian rhythms are intrinsically linked, with several sleep traits, including sleep timing and duration, influenced by both sleep homeostasis and the circadian phase. Genetic variation in several circadian genes has been associated with diurnal preference (preference in timing of sleep), although there has been limited research on whether they are associated with other sleep measurements. We investigated whether these genetic variations were associated with diurnal preference (Morningness-Eveningness Questionnaire) and various sleep measures, including: the global Pittsburgh Sleep Quality index score; sleep duration; and sleep latency and sleep quality. We genotyped 10 polymorphisms in genes with circadian expression in participants from the G1219 sample (n = 966), a British longitudinal population sample of young adults. We conducted linear regressions using dominant, additive and recessive models of inheritance to test for associations between these polymorphisms and the sleep measures. We found a significant association between diurnal preference and a polymorphism in period homologue 3 (PER3) (P < 0.005, recessive model) and a novel nominally significant association between diurnal preference and a polymorphism in aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) (P < 0.05, additive model). We found that a polymorphism in guanine nucleotide binding protein beta 3 (GNß3) was associated significantly with global sleep quality (P < 0.005, recessive model), and that a rare polymorphism in period homologue 2 (PER2) was associated significantly with both sleep duration and quality (P < 0.0005, recessive model). These findings suggest that genes with circadian expression may play a role in regulating both the circadian clock and sleep homeostasis, and highlight the importance of further studies aimed at dissecting the specific roles that circadian genes play in these two interrelated but unique behaviours.