598 research outputs found
Important role of endocannabinoid signaling in the development of functional vision and locomotion in zebrafish
No full textThe developmental role of the endocannabinoid system still remains to be fully understood. Here, we report the presence of a complete endocannabinoid system during zebrafish development and show that the genes that code for enzymes that catalyze the anabolism and catabolism (mgll and dagla) of the endocannabinoid, 2-AG (2-arachidonoylglycerol), as well as 2-AG main receptor in the brain, cannabinoid receptor type 1, are coexpressed in defined regions of axonal growth. By using morpholino-induced transient knockdown of the zebrafish Daglα homolog and its pharmacologic rescue, we suggest that synthesis of 2-AG is implicated in the control of axon formation in the midbrain-hindbrain region and that animals that lack Daglα display abnormal physiological behaviors in tests that measure stereotyped movement and motion perception. Our results suggest that the well-established role for 2-AG in axonal outgrowth has implications for the control of vision and movement in zebrafish and, thus, is likely common to all vertebrates.-Martella, A., Sepe, R. M., Silvestri, C., Zang, J., Fasano, G., Carnevali, O., De Girolamo, P., Neuhauss, S. C. F., Sordino, P., Di Marzo, V. Important role of endocannabinoid signaling in the development of functional vision and locomotion in zebrafish
miR-181a/b control the assembly of visual circuitry by regulating retinal axon specification and growth
No full textConnectivity and function of neuronal circuitry require the correct specification and growth of axons and dendrites. Here, we identify the microRNAs miR-181a and miR-181b as key regulators of retinal axon specification and growth. Loss of miR-181a/b in medaka fish (Oryzias latipes) failed to consolidate amacrine cell processes into axons and delayed the growth of retinal ganglion cell (RGC) axons. These alterations were accompanied by defects in visual connectivity and function. We demonstrated that miR-181a/b exert these actions through negative modulation of MAPK/ERK signaling that in turn leads to RhoA reduction and proper neuritogenesis in both amacrine cells and RGCs via local cytoskeletal rearrangement. Our results identify a new pathway for axon specification and growth unraveling a crucial role of miR-181a/b in the proper establishment of visual system connectivity and function
Evolution of visual guanylyl cyclases and their activating proteins with respect to clade and species-specific visual system adaptation
Get PDFMembrane guanylyl cyclase receptors are important regulators of local cGMP production, critically influencing cell growth and differentiation as well as ion transport, blood pressure and calcium feedback of vertebrate phototransduction. Currently, seven different subtypes of membrane guanylyl cyclase receptors have been characterized. These receptors have tissue specific expression and are activated either by small extracellular ligands, changing CO concentrations or, in the case of visual guanylyl cyclases, intracellularly interacting Ca-dependent activating proteins. In this report, we focus on the visual guanylyl cyclase receptors (GCs) GC-E (gucy2d/e) and GC-F (gucy2f) and their activating proteins (GCAP1/2/3; guca1a/b/c). While gucy2d/e has been detected in all analyzed vertebrates, GC-F receptors are missing in several clades (reptiles, birds, and marsupials) and/or individual species. Interestingly, the absence of GC-F in highly visual sauropsida species with up to 4 different cone-opsins is compensated by an increased number of guanylyl cyclase activating proteins, whereas in nocturnal or visually impaired species with reduced spectral sensitivity it is consolidated by the parallel inactivation of these activators. In mammals, the presence of GC-E and GC-F is accompanied by the expression of one to three GCAPs, whereas in lizards and birds, up to five different GCAPs are regulating the activity of the single GC-E visual membrane receptor. In several nearly blind species, a single GC-E enzyme is often accompanied by a single variant of GCAP, suggesting that one cyclase and one activating protein are both sufficient and required for conferring the basic detection of light
Vestibular deficits do not underlie looping behavior in achiasmatic fish
Get PDFZebrafish belladonna (bel) mutants carry a mutation in the lhx2 gene that encodes a Lim domain homeobox transcription factor, leading to a defect in the retinotectal axon pathfinding. As a result, a large fraction of homozygous bel mutants is achiasmatic. Achiasmatic bel mutants display ocular motor instabilities, both reserved optokinetic response (OKR) and spontaneous eye oscillations, and an unstable swimming behavior, described as looping. All these unstable behaviors have been linked to the underlying optic nerve projection defect. Looping has been investigated under different visual stimuli and shown to be vision dependent and contrast sensitive. In addition, looping correlates perfectly with reversed OKR and the spontaneous oscillations of the eyes. Hence, it has been hypothesized that looping is a compensatory response to the perception of self-motion induced by the spontaneous eye oscillations. However, both ocular and postural instabilities could also be caused by a yet unidentified vestibular deficit. Here, we performed a preliminary test of the vestibular function in achiasmatic bel larval mutants in order to clarify the potential role of a vestibular deficit in looping. We found that the vestibular ocular reflex (VOR) is normally directed in both bel mutants and wild types and therefore exclude the possibility that nystagmus and looping in reverse to the rotating optokinetic drum can be attributed to an underlying vestibular deficit
VisioTracker, an innovative automated approach to oculomotor analysis
Get PDFInvestigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications. Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time. Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity. Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance
Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor.
No full textFGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man
The zebrafish, brain-specific, aromatase cyp19a2 is neither expressed nor distributed in a sexually dimorphic manner during sexual differentiation.
Get PDFDifferential cyp19 aromatase expression during development leads to sexual dimorphisms in the mammalian brain. Whether this is also true for fish is unknown. The aim of the current study has been to follow the expression of the brain-specific aromatase cyp19a2 in the brains of sexually differentiating zebrafish. To assess the role of cyp19a2 in the zebrafish brain during gonadal differentiation, we used quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry to detect differences in the transcript or protein levels and/or expression pattern in juvenile fish, histology to monitor the gonadal status, and double immunofluorescence with neuronal or radial glial markers to characterize aromatase-positive cells. Our data show that cyp19a2 expression levels during zebrafish sexual differentiation cannot be assigned to a particular sex; the expression pattern in the brain is similar in both sexes and aromatase-positive cells appear to be mostly of radial glial nature. Developmental Dynamics 236:3155-3166, 2007. (c) 2007 Wiley-Liss, Inc
Catalytic P-H activation by Ti and Zr catalysts
No full textCatalytic dehydrocoupling of phosphines was investigated using the anionic zirconocene trihydride salts [Cp*Zr-2(mu-H)(3)Li](3) (1a) or [Cp*Zr-2(mu-H)(3)K(thf)(4)] (1b), and the metallocycles [CpTi(NPtBu3)(CH2)(4)] (6) and [Cp*M(NPtBu3)(CH2)(4)] (M = Ti 20, Zr 21) as catalyst precursors. Dehydrocoupling of primary phosphines RPH2 (R = Ph, C6H2Me3, Cy, C10H7) gave both dehydrocoupled dimers RP(H)P(H)R or cyclic oligophosphines (RP)(n) (n = 4, 5) while reaction of tBu(3)C(6)H(2)PH(2) gave the phosphaindoline tBu(2)(Me2CCH2)C6H2PH (9). Stoichiometric reactions of these catalyst precursors with primary phosphines afforded [Cp*Zr-2((PR)(2))H][K(thf)(4)] (R = Ph 2, Cy 3, C6H2Me3 4), [Cp*Zr-2((PPh)(3))H] [K(thf)(4)] (5), [CpTi(NPtBu3)(PPh)(3)] (7) and [CpTi(NPtBu3)(mu-PHPh)](2) (8), while reaction of 6 with (C(6)H(2)tBu3)PH2 in the presence of PMe3 afforded [CpTi(NPtBu3)(PMe3)(p(C(6)H(2)tBu(3))] (10). The secondary phosphines Ph2PH and (PhHPCH2)(2)CH2 also undergo dehydrocoupling affording (Ph2P)(2) and (PhPCH2)(2)CH2. The bisphosphines (CH2PH2)(2) and C6H4(PH2)(2) are dehydrocoupled to give (PCH2CH2PH)(2) (12) and (C6H4P(PH))(2) (13) while prolonged reaction of 13 gave (C6H4P2)(8) (14). The analogous bisphosphine Me2C6H4(PH)(2) (17) was prepared and dehydrocoupling catalysis afforded (Me2C6H2P(PH))(2) (18) and subsequently [(Me2C6H2P2)(2)(mu-Me2C6H2P2)](2) (19). Stoichiometric reactions with these bisphosphines gave [Cp*Zr-2(H)(PH)(2)C6H4] [Li(thf)(4)] (22), [Cp*Ti(NPtBu3)(PH)(2)C6H4](2) (23) and [Cp*Ti(NPtBu3)(PH)(2)C6H4] (24). 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Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model
Get PDFCLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease
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