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Increased insulin demand resulting from insulin resistance and/or overnutrition induces a compensatory increase in β-cell mass. The physiological factors responsible for the compensation have not been fully characterized. In zebrafish, overnutrition rapidly induces compensatory β-cell differentiation through triggering the release of a paracrine signal from persistently activated β-cells. We identified Fgf1 signaling as a key component of the overnutrition-induced β-cell differentiation signal in a small molecule screen. Fgf1 was confirmed as the overnutrition-induced β-cell differentiation signal, as inactivation of fgf1 abolished the compensatory β-cell differentiation. Furthermore, expression of human FGF1 solely in β-cells in fgf1(-/-) animals rescued the compensatory response, indicating that β-cells can be the source of FGF1. Additionally, constitutive secretion of FGF1 with an exogenous signal peptide increased β-cell number in the absence of overnutrition. These results demonstrate that fgf1 is necessary and FGF1 expression in β-cells is sufficient for the compensatory β-cell differentiation. We further show that FGF1 is secreted during prolonged activation of cultured mammalian β-cells and that endoplasmic reticulum stress acts upstream of FGF1 release. Thus, the recently discovered antidiabetes function of FGF1 may act partially through increasing β-cell differentiation.
© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3'UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.
Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been accepted widely as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain-of-function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen, which induced an Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase 4 (PDE4), leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy, and identifies a unique probe of downstream-of-Smo Hh modulation.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children and adults. While ADHD patients often display circadian abnormalities, the underlying mechanisms are unclear. Here we found that the zebrafish mutant for the circadian gene period1b (per1b) displays hyperactive, impulsive-like, and attention deficit-like behaviors and low levels of dopamine, reminiscent of human ADHD patients. We found that the circadian clock directly regulates dopamine-related genes monoamine oxidase and dopamine β hydroxylase, and acts via genes important for the development or maintenance of dopaminergic neurons to regulate their number and organization in the ventral diencephalic posterior tuberculum. We then found that Per1 knock-out mice also display ADHD-like symptoms and reduced levels of dopamine, thereby showing highly conserved roles of the circadian clock in ADHD. Our studies demonstrate that disruption of a circadian clock gene elicits ADHD-like syndrome. The circadian model for attention deficiency and hyperactive behavior sheds light on ADHD pathogenesis and opens avenues for exploring novel targets for diagnosis and therapy for this common psychiatric disorder.
Copyright © 2015 the authors 0270-6474/15/352572-16$15.00/0.
Insulin resistance creates an environment that promotes β-cell failure and development of diabetes. Understanding the events that lead from insulin resistance to diabetes is necessary for development of effective preventional and interventional strategies, and model systems that reflect the pathophysiology of disease progression are an important component toward this end. We have confirmed that insulin enhances glucose uptake in zebrafish skeletal muscle and have developed a zebrafish model of skeletal muscle insulin resistance using a dominant-negative IGF-IR. These zebrafish exhibit blunted insulin signaling and glucose uptake in the skeletal muscle, confirming insulin resistance. In young animals, we observed an increase in the number of β-cells and normal glucose tolerance that was indicative of compensation for insulin resistance. In older animals, the β-cell mass was reduced to that of control with the appearance of impaired glucose clearance but no elevation in fasting blood glucose. Combined with overnutrition, the insulin-resistant animals have an increased fasting blood glucose compared with the control animals, demonstrating that the β-cells in the insulin-resistant fish are in a vulnerable state. The relatively slow progression from insulin resistance to glucose intolerance in this model system has the potential in the future to test cooperating genes or metabolic conditions that may accelerate the development of diabetes and provide new therapeutic targets.
Copyright © 2015 the American Physiological Society.
Precise regulation of Notch signaling is essential for normal vertebrate development. Mind bomb (Mib) is a ubiquitin ligase that is required for activation of Notch by Notch׳s ligand, Delta. Sorting Nexin 5 (SNX5) co-localizes with Mib and Delta complexes and has been shown to directly bind to Mib. We show that microRNA-216a (miR-216a) is expressed in the retina during early development and regulates snx5 to precisely regulate Notch signaling. miR-216a and snx5 have complementary expression patterns. Knocking down miR-216a and/or overexpression of snx5 resulted in increased Notch activation. Conversely, knocking down snx5 and/or miR-216a overexpression caused a decrease in Notch activation. We propose a model in which SNX5, precisely controlled by miR-216a, is a vital partner of Mib in promoting endocytosis of Delta and subsequent activation of Notch signaling.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
The habenular nuclei of the limbic system regulate responses, such as anxiety, to aversive stimuli in the environment. The habenulae receive inputs from the telencephalon via elaborate dendrites that form in the center of the nuclei. The kinase Ulk2 positively regulates dendritogenesis on habenular neurons, and in turn is negatively regulated by the cytoplasmic protein Kctd12. Given that the habenulae are a nexus in the aversive response circuit, we suspected that incomplete habenular dendritogenesis would have profound implications for behavior. We find that Ulk2, which interacts with Kctd12 proteins via a small proline-serine rich domain, promotes branching and elaboration of dendrites. Loss of Kctd12 results in increased branching/elaboration and decreased anxiety. We conclude that fine-tuning of habenular dendritogenesis during development is essential for appropriate behavioral responses to negative stimuli.
BACKGROUND - Adult zebrafish spontaneously regenerate their retinas after damage. Although a number of genes and signaling pathways involved in regeneration have been identified, the exact mechanisms regulating various aspects of regeneration are unclear. microRNAs (miRNAs) were examined for their potential roles in regulating zebrafish retinal regeneration.
RESULTS - To investigate the requirement of miRNAs during zebrafish retinal regeneration, we knocked down the expression of Dicer in retinas prior to light-induced damage. Reduced Dicer expression significantly decreased the number of proliferating Müller glia-derived neuronal progenitor cells during regeneration. To identify individual miRNAs with roles in neuronal progenitor cell proliferation, we collected retinas at different stages of light damage and performed small RNA high-throughput sequencing. We identified subsets of miRNAs that were differentially expressed during active regeneration but returned to basal levels once regeneration was completed. We then knocked down five different miRNAs that increased in expression and assessed the effects on retinal regeneration. Reduction of miR-142b and miR-146a expression significantly reduced INL proliferation at 51 h of light treatment, while knockdown of miR-7a, miR-27c, and miR-31 expression significantly reduced INL proliferation at 72 h of constant light.
CONCLUSIONS - miRNAs exhibit dynamic expression profiles during retinal regeneration and are necessary for neuronal progenitor cell proliferation.
© 2014 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4(T804M) mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis.