Project description:Microglia are brain-resident macrophages that function as the first line of defense in brain. Embryonic microglial precursors originate in peripheral mesoderm and migrate into the brain during development. However, the mechanism by which they colonize the brain is incompletely understood. The retina is one of the first brain regions to accommodate microglia. In zebrafish, embryonic microglial precursors use intraocular hyaloid blood vessels as a pathway to migrate into the optic cup via the choroid fissure. Once retinal progenitor cells exit the cell cycle, microglial precursors associated with hyaloid blood vessels start to infiltrate the retina preferentially through neurogenic regions, suggesting that colonization of retinal tissue depends upon the neurogenic state. Along with blood vessels and retinal neurogenesis, IL34 also participates in microglial precursor colonization of the retina. Altogether, CSF receptor signaling, blood vessels, and neuronal differentiation function as cues to create an essential path for microglial migration into developing retina.
Project description:The mature vertebrate retina is a highly ordered neuronal network of cell bodies and synaptic neuropils arranged in distinct layers. Little, however, is known about the emergence of this spatial arrangement. Here, we investigate how the three main types of retinal inhibitory neuron (RIN)--horizontal cells (HCs), inner nuclear layer amacrine cells (iACs) and displaced amacrine cells (dACs)--reach their specific laminar positions during development. Using in vivo time-lapse imaging of zebrafish retinas, we show that RINs undergo distinct phases of migration. The first phase, common to all RINs, is bipolar migration directed towards the apicobasal centre of the retina. All RINs then transition to a less directionally persistent multipolar phase of migration. Finally, HCs, iACs and dACs each undergo cell type-specific migration. In contrast to current hypotheses, we find that most dACs send processes into the forming inner plexiform layer (IPL) before migrating through it and inverting their polarity. By imaging and quantifying the dynamics of HCs, iACs and dACs from birth to final position, this study thus provides evidence for distinct and new migration patterns during retinal lamination and insights into the initiation of IPL formation.
Project description:AimTo investigate the impact of titanium dioxide nanoparticles (TiO2 NPs) on embryonic development and retinal neurogenesis.MethodsThe agglomeration and sedimentation of TiO2 NPs solutions at different dilutions were observed, and the ultraviolet-visible spectra of their supernatants were measured. Zebrafish embryos were experimentally exposed to TiO2 NPs until 72h postfertilization (hpf). The retinal neurogenesis and distribution of the microglia were analyzed by immunohistochemistry and whole mount in situ hybridization.ResultsThe 1 mg/L was determined to be an appropriate exposure dose. Embryos exposed to TiO2 NPs had a normal phenotype. The neurogenesis was initiated on time, and ganglion cells, cones and rods were well differentiated at 72 hpf. The expression of fms mRNA and the 4C4 antibody, which were specific to microglia in the central nervous system (CNS), closely resembled their endogenous profile.ConclusionThese data demonstrate that short-term exposure to TiO2 NPs at a low dose does not lead to delayed embryonic development or retinal neurotoxicity.
Project description:Larval zebrafish (Danio rerio) are an ideal organism for studying color vision, as their retina possesses four types of cone photoreceptors, covering most of the visible range and into the UV.1,2 Additionally, their eye and nervous systems are accessible to imaging, given that they are naturally transparent.3-5 Recent studies have found that, through a set of wavelength-range-specific horizontal, bipolar, and retinal ganglion cells (RGCs),6-9 the eye relays tetrachromatic information to several retinorecipient areas (RAs).10-13 The main RA is the optic tectum, receiving 97% of the RGC axons via the neuropil mass termed arborization field 10 (AF10).14,15 Here, we aim to understand the processing of chromatic signals at the interface between RGCs and their major brain targets. We used 2-photon calcium imaging to separately measure the responses of RGCs and neurons in the brain to four different chromatic stimuli in awake animals. We find that chromatic information is widespread throughout the brain, with a large variety of responses among RGCs, and an even greater diversity in their targets. Specific combinations of response types are enriched in specific nuclei, but there is no single color processing structure. In the main interface in this pathway, the connection between AF10 and tectum, we observe key elements of neural processing, such as enhanced signal decorrelation and improved chromatic decoding.16,17 A richer stimulus set revealed that these enhancements occur in the context of a more distributed code in tectum, facilitating chromatic signal association in this small vertebrate brain.
Project description:BACKGROUND:The oculomotor integrator (OI) in the vertebrate hindbrain transforms eye velocity input into persistent position coding output, which plays a crucial role in retinal image stability. For a mechanistic understanding of the integrator function and eye position control, knowledge about the tuning of the OI and other oculomotor nuclei is needed. Zebrafish are increasingly used to study integrator function and sensorimotor circuits, yet the precise neuronal tuning to motor variables remains uncharacterized. RESULTS:Here, we recorded cellular calcium signals while evoking monocular and binocular optokinetic eye movements at different slow-phase eye velocities. Our analysis reveals the anatomical distributions of motoneurons and internuclear neurons in the nucleus abducens as well as those of oculomotor neurons in caudally adjacent hindbrain volumes. Each neuron is tuned to eye position and/or velocity to variable extents and is only activated after surpassing particular eye position and velocity thresholds. While the abducens (rhombomeres 5/6) mainly codes for eye position, in rhombomeres 7/8, a velocity-to-position coding gradient exists along the rostro-caudal axis, which likely corresponds to the oculomotor structures storing velocity and position, and is in agreement with a feedforward mechanism of persistent activity generation. Position encoding neurons are recruited at eye position thresholds distributed across the behaviourally relevant dynamic range, while velocity-encoding neurons have more centred firing thresholds for velocity. In the abducens, neurons coding exclusively for one eye intermingle with neurons coding for both eyes. Many of these binocular neurons are preferentially active during conjugate eye movements and less active during monocular eye movements. This differential recruitment during monocular versus conjugate tasks represents a functional diversification in the final common motor pathway. CONCLUSIONS:We localized and functionally characterized the repertoire of oculomotor neurons in the zebrafish hindbrain. Our findings provide evidence for a mixed but task-specific binocular code and suggest that generation of persistent activity is organized along the rostro-caudal axis in the hindbrain.
Project description:The ability to image cells live and in situ as they proliferate and differentiate has proved to be an invaluable asset to biologists investigating developmental processes. Here, we describe a Spectrum of Fates approach that allows the identification of all the major neuronal subtypes in the zebrafish retina simultaneously. Spectrum of Fates is based on the combinatorial expression of differently coloured fluorescent proteins driven by the promoters of transcription factors that are expressed in overlapping subsets of retinal neurons. Here, we show how a Spectrum of Fates approach can be used to assess various aspects of neural development, such as developmental waves of differentiation, neuropil development, lineage tracing and hierarchies of fates in the developing zebrafish retina.
Project description:BackgroundThe zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ) contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells) occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood.ResultsWe used genetic and pharmacological manipulation of the β-catenin/Wnt signaling pathway to show that it is required to maintain proliferation in the CMZ and that hyperstimulation of β-catenin/Wnt signaling inhibits normal retinal differentiation and expands the population of proliferative retinal progenitors. To test whether similar effects occur during regeneration, we developed a method for making rapid, selective photoreceptor ablations in larval zebrafish with intense light. We found that dephosphorylated β-catenin accumulates in Müller glia as they re-enter the cell cycle following injury, but not in Müller glia that remain quiescent. Activation of Wnt signaling is required for regenerative proliferation, and hyperstimulation results in loss of Müller glia from the INL as all proliferative cells move into the ONL.Conclusionsβ-catenin/Wnt signaling is thus required for the maintenance of retinal progenitors during both initial development and lesion-induced regeneration, and is sufficient to prevent differentiation of those progenitors and maintain them in a proliferative state. This suggests that the β-catenin/Wnt cascade is part of the shared molecular circuitry that maintains retinal stem cells for both homeostatic growth and epimorphic regeneration.
Project description:As a biomarker of cellular activities, the transcriptome of a specific tissue or cell type during development and disease is of great biomedical interest. We have generated and analyzed 10,000 expressed sequence tags (ESTs) from three mouse eye tissue cDNA libraries: embryonic day 15.5 (M15E) eye, postnatal day 2 (M2PN) eye and adult retina (MRA).Annotation of 8,633 non-mitochondrial and non-ribosomal high-quality ESTs revealed that 57% of the sequences represent known genes and 43% are unknown or novel ESTs, with M15E having the highest percentage of novel ESTs. Of these, 2,361 ESTs correspond to 747 unique genes and the remaining 6,272 are represented only once. Phototransduction genes are preferentially identified in MRA, whereas transcripts for cell structure and regulatory proteins are highly expressed in the developing eye. Map locations of human orthologs of known genes uncovered a high density of ocular genes on chromosome 17, and identified 277 genes in the critical regions of 37 retinal disease loci. In silico expression profiling identified 210 genes and/or ESTs over-expressed in the eye; of these, more than 26 are known to have vital retinal function. Comparisons between libraries provided a list of temporally regulated genes and/or ESTs. A few of these were validated by qRT-PCR analysis.Our studies present a large number of potentially interesting genes for biological investigation, and the annotated EST set provides a useful resource for microarray and functional genomic studies.