Blunted first-phase insulin secretion and insulin deficiency are indicators of beta cell dysfunction and diabetes manifestation. Therefore, insights into molecular mechanisms that regulate insulin homeostasis might provide entry sites to replenish insulin content and restore beta cell function. Here, we identify the insulin inhibitory receptor (inceptor; encoded by the gene IIR/ELAPOR1) as an insulin-binding receptor that regulates insulin stores by lysosomal degradation. Using human induced pluripotent stem cell (SC)-derived islets, we show that IIR knockout (KO) results in enhanced SC beta cell differentiation and survival. Strikingly, extended in vitro culture of IIR KO SC beta cells leads to greatly increased insulin content and glucose-stimulated insulin secretion (GSIS). We find that inceptor localizes to clathrin-coated vesicles close to the plasma membrane and in the trans-Golgi network as well as in secretory granules, where it acts as a sorting receptor to direct proinsulin and insulin towards lysosomal degradation. Targeting inceptor using a monoclonal antibody increases proinsulin and insulin content and improves SC beta cell GSIS. Altogether, our findings reveal the basic mechanisms of beta cell insulin turnover and identify inceptor as an insulin degradation receptor.

Decreased insulin action and insulin receptor signalling contribute to the pathology of diabetes. Liu et al. uncover a role for the Ephrin type-B receptor 4 in insulin receptor degradation regulating liver and systemic insulin sensitivity.

[This corrects the article DOI: 10.1371/journal.pbio.3000708.].

Regulation of quiescence and cell cycle entry is pivotal for the maintenance of stem cell populations. Regulatory mechanisms, however, are poorly understood. In particular, it is unclear how the activity of single stem cells is coordinated within the population or if cells divide in a purely random fashion. We addressed this issue by analyzing division events in an adult neural stem cell (NSC) population of the zebrafish telencephalon. Spatial statistics and mathematical modeling of over 80,000 NSCs in 36 brain hemispheres revealed weakly aggregated, nonrandom division patterns in space and time. Analyzing divisions at 2 time points allowed us to infer cell cycle and S-phase lengths computationally. Interestingly, we observed rapid cell cycle reentries in roughly 15% of newly born NSCs. In agent-based simulations of NSC populations, this redividing activity sufficed to induce aggregated spatiotemporal division patterns that matched the ones observed experimentally. In contrast, omitting redivisions leads to a random spatiotemporal distribution of dividing cells. Spatiotemporal aggregation of dividing stem cells can thus emerge solely from the cells’ history.

The regulation of quiescence and cell cycle entry is pivotal for the maintenance of stem cell populations. Regulatory mechanisms however are poorly understood. In particular it is unclear how the activity of single stem cells is coordinated within the population, or if cells divide in a purely random fashion. We addressed this issue by analyzing division events in an adult neural stem cell (NSC) population of the zebrafish telencephalon. Spatial statistics and mathematical modeling of over 80,000 NSCs in 36 brains revealed weakly aggregated, non-random division patterns in space and time. Analyzing divisions at two timepoints allowed us to infer cell cycle and S-phase lengths computationally. Interestingly, we observed rapid cell cycle re-entries in roughly 15% of newly born NSCs. In agent based simulations of NSC populations, this re-dividing activity sufficed to induce aggregated spatio-temporal division patterns that matched the ones observed experimentally. In contrast, omitting re-divisions lead to a random spatio-temporal distribution of dividing cells. Spatio-temporal aggregation of dividing stem cells can thus emerge from the cell’s history, regardless of possible feedback mechanisms in the population.

In adult stem cell populations, recruitment into division is parsimonious and most cells maintain a quiescent state. How individual cells decide to enter the cell cycle and how they coordinate their activity remains an essential problem to be resolved. It is thus important to develop methods to elucidate the mechanisms of cell communication and recruitment into the cell cycle. We made use of the advantageous architecture of the adult zebrafish telencephalon to isolate the surface proteins of an intact neural stem cell (NSC) population. We identified the proteome of NSCs in young and old brains. The data revealed a group of proteins involved in filopodia, which we validated by a morphological analysis of single cells, showing apically located cellular extensions. We further identified an age-related decrease in insulin-like growth factor (IGF) receptors. Expressing IGF2b induced divisions in young brains but resulted in incomplete divisions in old brains, stressing the role of cell-intrinsic processes in stem cell behavior.

Proliferating stem cells in the adult body are the source of constant regeneration. In the brain, neural stem cells (NSCs) divide to maintain the stem cell population and generate neural progenitor cells that eventually replenish mature neurons and glial cells. How much spatial coordination of NSC division and differentiation is present in a functional brain is an open question. To quantify the patterns of stem cell divisions, one has to (i) identify the pool of NSCs that have the ability to divide, (ii) determine NSCs that divide within a given time window, and (iii) analyze the degree of spatial coordination. Here, we present a bioimage informatics pipeline that automatically identifies GFP expressing NSCs in three-dimensional image stacks of zebrafish brain from whole-mount preparations. We exploit the fact that NSCs in the zebrafish hemispheres are located on a two-dimensional surface and identify between 1,500 and 2,500 NSCs in six brain hemispheres. We then determine the position of dividing NSCs in the hemisphere by EdU incorporation into cells undergoing S-phase and calculate all pairwise NSC distances with three alternative metrics. Finally, we fit a probabilistic model to the observed spatial patterns that accounts for the non-homogeneous distribution of NSCs. We find a weak positive coordination between dividing NSCs irrespective of the metric and conclude that neither strong inhibitory nor strong attractive signals drive NSC divisions in the adult zebrafish brain. © 2017 International Society for Advancement of Cytometry.

Conventionally, neuronal development is regarded to follow a stereotypic sequence of neurogenesis, migration, and differentiation. We demonstrate that this notion is not a general principle of neuronal development by documenting the timing of mitosis in relation to multiple differentiation events for bipolar cells (BCs) in the zebrafish retina using in vivo imaging. We found that BC progenitors undergo terminal neurogenic divisions while in markedly disparate stages of neuronal differentiation. Remarkably, the differentiation state of individual BC progenitors at mitosis is not arbitrary but matches the differentiation state of post‐mitotic BCs in their surround. By experimentally shifting the relative timing of progenitor division and differentiation, we provide evidence that neurogenesis and differentiation can occur independently of each other. We propose that the uncoupling of neurogenesis and differentiation could provide neurogenic programs with flexibility, while allowing for synchronous neuronal development within a continuously expanding cell pool.

Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1–4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.

The zebrafish has recently become a source of new data on the mechanisms of neural stem cell (NSC) maintenance and ongoing neurogenesis in adult brains. In this vertebrate, neurogenesis occurs at high levels in all ventricular regions of the brain, and brain injuries recover successfully, owing to the recruitment of radial glia, which function as NSCs. This new vertebrate model of adult neurogenesis is thus advancing our knowledge of the molecular cues in use for the activation of NSCs and fate of their progeny. Because the regenerative potential of somatic stem cells generally weakens with increasing age, it is important to assess the extent to which zebrafish NSC potential decreases or remains unaltered with age. We found that neurogenesis in the ventricular zone, in the olfactory bulb, and in a newly identified parenchymal zone of the telencephalon indeed declines as the fish ages and that oligodendrogenesis also declines. In the ventricular zone, the radial glial cell population remains largely unaltered morphologically but enters less frequently into the cell cycle and hence produces fewer neuroblasts. The neuroblasts themselves do not change their behavior with age and produce the same number of postmitotic neurons. Thus, decreased neurogenesis in the physiologically aging zebrafish brain is correlated with an increasing quiescence of radial glia. After injuries, radial glia in aged brains are reactivated, and the percentage of cell cycle entry is increased in the radial glia population. However, this reaction is far less pronounced than in younger animals, pointing to irreversible changes in aging zebrafish radial glia.

All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts.

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia-like cells, actively proliferating cells, and label-retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow-dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100beta), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA-NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow-cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA-positive cells in fast domains further display a morphology distinct from radial glia and co-express PSA-NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA-NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ.

For more than a decade, the zebrafish has proven to be an excellent model organism to investigate the mechanisms of neurogenesis during development. The often cited advantages, namely external development, genetic, and optical accessibility, have permitted direct examination and experimental manipulations of neurogenesis during development. Recent studies have begun to investigate adult neurogenesis, taking advantage of its widespread occurrence in the mature zebrafish brain to investigate the mechanisms underlying neural stem cell maintenance and recruitment. Here we provide a comprehensive overview of the tools and techniques available to study neurogenesis in zebrafish both during development and in adulthood. As useful resources, we provide tables of available molecular markers, transgenic, and mutant lines. We further provide optimized protocols for studying neurogenesis in the adult zebrafish brain, including in situ hybridization, immunohistochemistry, in vivo lipofection and electroporation methods to deliver expression constructs, administration of bromodeoxyuridine (BrdU), and finally slice cultures. These currently available tools have put zebrafish on par with other model organisms used to investigate neurogenesis.

The limited generation of neurons during adulthood is controlled by a balance between quiescence and recruitment of neural stem cells (NSCs). We use here the germinal zone of the zebrafish adult telencephalon to examine how the frequency of NSC divisions is regulated. We show, using several in vivo techniques, that progenitors transit back and forth between the quiescent and dividing state, according to varying levels of Notch activity: Notch induction drives progenitors into quiescence, whereas blocking Notch massively reinitiates NSC division and subsequent commitment toward becoming neurons. Notch activation appears predominantly triggered by newly recruited progenitors onto their neighbors, suggesting an involvement of Notch in a self-limiting mechanism, once neurogenesis is started. These results identify for the first time a lateral inhibition-like mechanism in the context of adult neurogenesis and suggest that the equilibrium between quiescence and neurogenesis in the adult brain is controlled by fluctuations of Notch activity, thereby regulating the amount of adult-born neurons.

The maintenance of progenitor cells is a crucial aspect of central nervous system development and maturation, and bHLH transcription factors of the E(Spl) subfamily are involved in this process in all vertebrates studied to date. In the zebrafish embryonic neural plate, a large number of E(Spl) genes (her genes) are at play. We review recent data on this point, and propose a model where distinct subsets of these genes define different progenitor subtypes. Analysis of her genes expression in the adult zebrafish brain suggests that part of the embryonic her cascade might also be reused to define progenitors during adulthood. Further, available evidence on orthologous genes in the mouse (Hes genes) point to different modes of Hes regulation depending on cell location within the embryonic neural tube, perhaps associated with distinct progenitor types in this species as well. Out of these comparisons emerges a simple model of neural stem cell maintenance applicable from embryonic development until adulthood as well as across species. This working model suggests the directions for future experiments.

Adult neurogenesis is an exciting and rapidly advancing field of research. It addresses basic biological questions, such as the how and why of de novo neuronal production during adulthood, as well as medically relevant issues, including the potential link between adult neural stem cells and psychiatric disorders, or how stem cell manipulation might be used as a strategy for neuronal replacement. Current research mainly focuses on rodents, but we review here recent examination of non‐mammalian vertebrates, which demonstrates that bona fide adult neural stem cells exist in these species. Importantly, especially in teleost fish, these cells can be abundant and located in various brain areas. Hence, non‐mammalian vertebrate species provide invaluable comparative material for extracting core mechanisms of adult neural stem cell maintenance and fate. BioEssays 29:745–757, 2007. © 2007 Wiley Periodicals, Inc.

Current models of vertebrate adult neural stem cells are largely restricted to the rodent forebrain. To extract the general mechanisms of neural stem cell biology, we sought to identify new adult stem cell populations, in other model systems and/or brain areas. The teleost zebrafish appears to be an ideal system, as cell proliferation in the adult zebrafish brain is found in many more niches than in the mammalian brain. As a starting point towards identifying stem cell populations in this system, we used an embryonic neural stem cell marker, the E(spl) bHLH transcription factor Her5. We demonstrate that her5 expression is not restricted to embryonic neural progenitors, but also defines in the adult zebrafish brain a new proliferation zone at the junction between the mid- and hindbrain. We show that adult her5-expressing cells proliferate slowly, self-renew and express neural stem cell markers. Finally, using in vivo lineage tracing in her5:gfp transgenic animals, we demonstrate that the her5-positive population is multipotent, giving rise in situ to differentiated neurons and glia that populate the basal midbrain. Our findings conclusively identify a new population of adult neural stem cells, as well as their fate and their endogenous environment, in the intact vertebrate brain. This cell population, located outside the forebrain, provides a powerful model to assess the general mechanisms of vertebrate neural stem cell biology. In addition, the first transcription factor characteristic of this cell population, Her5, points to the E(Spl) as a promising family of candidate adult neural stem cell regulators.

Our understanding of the cellular and molecular mechanisms underlying the adult neural stem cell state remains fragmentary. To provide new models on this issue, we searched for stem cells in the adult brain of the zebrafish. Using BrdU tracing and immunodetection of cell-type-specific markers, we demonstrate that the adult zebrafish telencephalon contains self-renewing progenitors, which show features of adult mammalian neural stem cells but distribute along the entire dorso-ventral extent of the telencephalic ventricular zone. These progenitors give rise to newborn neurons settling close to the ventricular zone within the telencephalon proper. They have no equivalent in mammals and therefore constitute a new model of adult telencephalic neural stem cells. In addition, progenitors from the ventral subpallium generate rapidly dividing progenitors and neuroblasts that reach the olfactory bulb (OB) via a rostral migratory stream and differentiate into GABAergic and TH-positive neurons. These ventral progenitors are comparable to the mammalian neural stem cells of the subependymal zone. Interestingly, dorsal and ventral progenitors in the adult telencephalon express a different combination of transcription factors than their embryonic counterparts. In the case of neurogenin1, this is due to the usage of different enhancer elements. Together, our results highlight the conserved and unique phylogenic and ontogenic features of adult neurogenesis in the zebrafish telencephalon and open the way to the identification of adult neural stem cell characters in cross-species comparative studies.