Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

An inducible model of chronic hyperglycemia.

Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute ß-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain ß-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.

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.

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.

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.

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.

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.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish.

Liver duct paucity is characteristic of children born with Alagille Syndrome (ALGS), a disease associated with JAGGED1 mutations. Here, we report that zebrafish embryos with compound homozygous mutations in two Notch ligand genes, jagged1b (jag1b) and jagged2b (jag2b) exhibit a complete loss of canonical Notch activity and duct cells within the liver and exocrine pancreas, whereas hepatocyte and acinar pancreas development is not affected. Further, animal chimera studies demonstrate that wild-type endoderm cells within the liver and pancreas can rescue Notch activity and duct lineage specification in adjacent cells lacking jag1b and jag2b expression. We conclude that these two Notch ligands are directly and solely responsible for all duct lineage specification in these organs in zebrafish. Our study uncovers genes required for lineage specification of the intrahepatopancreatic duct cells, challenges the role of duct cells as progenitors, and suggests a genetic mechanism for ALGS ductal paucity.The hepatopancreatic duct cells connect liver hepatocytes and pancreatic acinar cells to the intestine, but the mechanism for their lineage specification is unclear. Here, the authors reveal that Notch ligands Jagged1b and Jagged2b induce duct cell lineage in the liver and pancreas of the zebrafish.

Inducible Knockout of Mouse Zfhx3 Emphasizes Its Key Role in Setting the Pace and Amplitude of the Adult Circadian Clock.

The transcription factor zinc finger homeobox 3 (ZFHX3) plays a key role in coupling intracellular transcriptional-translational oscillations with intercellular synchrony in mouse suprachiasmatic nucleus (SCN). However, like many key players in central nervous system function, ZFHX3 serves an important role in neurulation and neuronal terminal differentiation while retaining discrete additional functions in the adult SCN. Recently, using a dominant missense mutation in mouse Zfhx3, we established that this gene can modify circadian period and sleep in adult animals. Nevertheless, we were still concerned that the neurodevelopmental consequences of ZFHX3 dysfunction in this mutant may interfere with, or confound, its critical adult-specific roles in SCN circadian function. To circumvent the developmental consequences of Zfhx3 deletion, we crossed a conditional null Zfhx3 mutant to an inducible, ubiquitously expressed Cre line (B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J). This enabled us to assess circadian behavior in the same adult animals both before and after Cre-mediated excision of the critical Zfhx3 exons using tamoxifen treatment. Remarkably, we found a strong and significant alteration in circadian behavior in tamoxifen-treated homozygous animals with no phenotypic changes in heterozygous or control animals. Cre-mediated excision of Zfhx3 critical exons in adult animals resulted in shortening of the period of wheel-running in constant darkness by more than 1 h in the majority of homozygotes while, in 30% of animals, excision resulted in complete behavioral arrhythmicity. In addition, we found that homozygous animals reentrain almost immediately to 6-h phase advances in the light-dark cycle. No additional overt phenotypic changes were evident in treated homozygous animals. These findings confirm a sustained and significant role for ZFHX3 in maintaining rhythmicity in the adult mammalian circadian system.

Genetic polymorphisms and their association with brain and behavioural measures in heterogeneous stock mice.

Although the search for quantitative trait loci for behaviour remains a considerable challenge, the complicated genetic architecture of quantitative traits is beginning to be understood. The current project utilised heterogeneous stock (HS) male mice (n = 580) to investigate the genetic basis for brain weights, activity, anxiety and cognitive phenotypes. We identified 126 single nucleotide polymorphisms (SNPs) in genes involved in regulation of neurotransmitter systems, nerve growth/death and gene expression, and subsequently investigated their associations with changes in behaviour and/or brain weights in our sample. We found significant associations between four SNP-phenotype pairs, after controlling for multiple testing. Specificity protein 2 (Sp2, rs3708840), tryptophan hydroxylase 1 (Tph1, rs262731280) and serotonin receptor 3A (Htr3a, rs50670893) were associated with activity/anxiety behaviours, and microtubule-associated protein 2 (Map2, rs13475902) was associated with cognitive performance. All these genes except for Tph1 were expressed in the brain above the array median, and remained significantly associated with relevant behaviours after controlling for the family structure. Additionally, we found evidence for a correlation between Htr3a expression and activity. We discuss our findings in the light of the advantages and limitations of currently available mouse genetic tools, suggesting further directions for association studies in rodents.

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.

The Zfhx3-Mediated Axis Regulates Sleep and Interval Timing in Mice.

An AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3 significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3(Sci/+), we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3(Sci/+) mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3(Sci/+)-dependent network.

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.

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.

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.

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.

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.

Centroacinar Cells Are Progenitors That Contribute to Endocrine Pancreas Regeneration.

Diabetes is associated with a paucity of insulin-producing ß-cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in ß-cell neogenesis and regeneration. To facilitate discovery of such mechanisms, we use a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notch-responsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells (CACs). Adult CACs are also Notch responsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors, we took two complementary approaches: 1) We established the transcriptome for adult CACs. Using gene ontology, transgenic lines, and in situ hybridization, we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing, we demonstrated that CACs do form new endocrine cells after ß-cell ablation or partial pancreatectomy. We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model to study both ß-cell neogenesis and ß-cell regeneration. Furthermore, we show that in cftr loss-of-function mutants, there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis.

First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic ß-cell mass.

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing ß cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of ß-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating ß-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling ß-cell mass, potential therapeutic targets for treating diabetes.

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.