DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas

Cell lineage analysis is critical in understanding the relationship between progenitors and differentiated cells as well as the mechanism underlying the process of differentiation. In order to study the zebrafish endocrine pancreas cell lineage, transgenic expression of diphtheria toxin gene A chain (DTA) under two cell type-specific promoters derived from the insulin (ins) and somatostatin2 (sst2) genes was used to ablate the two types of endocrine cells: insulin-producing β-cells and somatostatin-producing δ-cells, respectively. We found that ablation of β-cells resulted in a reduction of not only β-cells but also glucagon-producing α-cells; in contrast, δ-cells were largely unaffected. Ablation of δ-cells led to reduction of all three types of endocrine cells: α-, β-, and δ. Interestingly, α-cells were more profoundly affected in both β- and δ-cell ablations and were frequently reduced together with β- and δ-cells. By taking advantage of Tg(ins:gfp) and Tg(sst2:gfp) lines, we also monitored the changes of different types of endocrine cells in vivo after ablation and found that both β- and δ-cell populations significantly recovered by 3 dpf after their ablation and it seemed that δ-cells had a better capability of recovery than β-cells. Thus, our current observations indicated differential interdependence of these three cell lineages. The development of zebrafish α-cells, but not δ-cells, is dependent on β-cells, while the development of both α- and β-cells is dependent on δ-cells. In contrast, the development of δ-cells was independent of β-cells.     

Biphasic effect of insulin on beta cell apoptosis depending on glucose deprivation

Insulin resistant states are associated with an increase in the beta cell mass and also high levels of circulating insulin. Ultimately the beta cells undergo a failure that leads to diabetes. At this stage, a question arises if those persistent high levels of circulating insulin may contribute to beta cell damage. To address this important issue, we submitted beta cells to a prolonged effect of increasing concentrations of insulin. We observed that a prolonged effect of high levels of insulin on the presence of serum (15-24 h) in glucose-deprived beta cells induced apoptosis. This apoptotic effect was both dose- and cycloheximide-dependent.     

Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent

Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo.     

Characterization of the KG1a cell line for use in a cell migration based screening assay

High-throughput screening has become a popular method used to identify new “leads” for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of variousin-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.     

Pancreatic beta cells colocalize insulin and pronesfatin immunoreactivity in rodents

Nesfatin-1 is a recently discovered feeding inhibitory peptide encoded in the precursor protein, nucleobindin 2 (pronesfatin). Previous studies have shown pronesfatin expression in the brain, stomach and pancreas. However, the identity of cells that express nesfatin in the pancreas remain unknown. The objective of this study was to determine which cells in the pancreas of mice and rats express pronesfatin immunoreactivity. We found pronesfatin immunopositive cells exclusively in the pancreatic islets of both CD1 mice and Fischer 344 rats. Our novel results indicate that the insulin producing beta cells colocalize pronesfatin in the islets of both mice and rats. No colocalization of glucagon and pronesfatin was found in mice, while some glucagon positive cells were positive for pronesfatin in rat islets. The abundant presence of pronesfatin immunoreactivity and its colocalization with insulin suggests a potential role for pronesfatin-derived peptides in islet biology and glucose homeostasis in rodents.     

Role of GPR40 in fatty acid action on the beta cell line INS-1E

GPR40 is a G protein-coupled receptor expressed preferentially in beta cells, that has been implicated in mediating free fatty acid-stimulated insulin release. GPR40 RNAi impaired the ability of palmitic acid (PA) to increase both insulin secretion and intracellular calcium ([Ca2+]i). The PA-dependent [Ca2+]i increase was attenuated by inhibitors of Galphaq, PLC, and SERCA. Thus GPR40 activates the Galphaq pathway, leading to release of Ca2+ from the ER. Yet the GPR40-dependent [Ca2+]i rise was dependent on extracellular Ca2+ and elevated glucose, and was blocked by inhibition of L-type calcium channels (LTCC) or opening of the K(ATP) channel; this suggests that GPR40 promotes Ca2+ influx through up-regulation of LTCC pre-activated by glucose and membrane depolarization. Taken together, the data indicate that GPR40 mediates the increase in [Ca2+]i and insulin secretion through the Galphaq-PLC pathway, resulting in release of Ca2+ from the ER and leading to up-regulation of Ca2+ influx via LTCC.     

Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state.     

Unique and conserved aspects of gut development in zebrafish

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.     

Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear

Sox2 has been variously implicated in maintenance of pluripotent stem cells or, alternatively, early stages of cell differentiation, depending on context. In the developing inner ear, Sox2 initially marks all cells in the nascent sensory epithelium and, in mouse, is required for sensory epithelium formation. Sox2 is eventually downregulated in hair cells but is maintained in support cells, the functional significance of which is unknown. Here we describe regulation and function of sox2 in the zebrafish inner ear. Expression of sox2 begins after the onset of sensory epithelium development and is regulated by Atoh1a/b, Fgf and Notch. Knockdown of sox2 does not prevent hair cell production, but the rate of accumulation is reduced due to sporadic death of differentiated hair cells. We next tested the capacity for hair cell regeneration following laser ablation of mature brn3c:gfp-labeled hair cells. In control embryos, regeneration of lost hair cells begins by 12 h post-ablation and involves transdifferentiation of support cells rather than asymmetric cell division. In contrast, regeneration does not occur in sox2-depleted embryos. These data show that zebrafish sox2 is required for hair cell survival, as well as for transdifferentiation of support cells into hair cells during regeneration.     

Innocuousness and intracellular distribution of PKH67: A fluorescent probe for cell proliferation assessment

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.     

The zebrafish cerebellar rhombic lip is spatially patterned in producing granule cell populations of different functional compartments

The upper rhombic lip, a prominent germinal zone of the cerebellum, was recently demonstrated to generate different neuronal cell types over time from spatial subdomains. We have characterized the differentiation of the upper rhombic lip derived granule cell population in stable GFP-transgenic zebrafish in the context of zebrafish cerebellar morphogenesis. Time-lapse analysis followed by individual granule cell tracing demonstrates that the zebrafish upper rhombic lip is spatially patterned along its mediolateral axis producing different granule cell populations simultaneously. Time-lapse recordings of parallel fiber projections and retrograde labeling reveal that spatial patterning within the rhombic lip corresponds to granule cells of two different functional compartments of the mature cerebellum: the eminentia granularis and the corpus cerebelli. These cerebellar compartments in teleosts correspond to the mammalian vestibulocerebellar and non-vestibulocerebellar system serving balance and locomotion control, respectively. Given the high conservation of cerebellar development in vertebrates, spatial partitioning of the mammalian granule cell population and their corresponding earlier-produced deep nuclei by patterning within the rhombic lip may also delineate distinct functional compartments of the cerebellum. Thus, our findings offer an explanation for how specific functional cerebellar circuitries are laid down by spatio-temporal patterning of cerebellar germinal zones during early brain development.