Cytoplasmic pool of U1 spliceosome protein SNRNP70 shapes the axonal transcriptome and regulates motor connectivity.

Regulation of pre-mRNA splicing and polyadenylation plays a profound role in neurons by diversifying the proteome and modulating gene expression in response to physiological cues. Although most of the pre-mRNA processing is thought to occur in the nucleus, numerous splicing regulators are also found in neurites. Here, we show that U1-70K/SNRNP70, a component of the major spliceosome, localizes in RNA-associated granules in zebrafish axons. We identify the extra-nuclear SNRNP70 as an important regulator of motor axonal growth, nerve-dependent acetylcholine receptor (AChR) clustering, and neuromuscular synaptogenesis. This cytoplasmic pool has a protective role for a limited number of transcripts regulating their abundance and trafficking inside axons. Moreover, non-nuclear SNRNP70 regulates splice variants of transcripts such as agrin, thereby controlling synapse formation. Our results point to an unexpected, yet essential, function of non-nuclear SNRNP70 in axonal development, indicating a role of spliceosome proteins in cytoplasmic RNA metabolism during neuronal connectivity.

Identification of a novel interaction of FUS and syntaphilin may explain synaptic and mitochondrial abnormalities caused by ALS mutations.

Aberrantly expressed fused in sarcoma (FUS) is a hallmark of FUS-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Wildtype FUS localises to synapses and interacts with mitochondrial proteins while mutations have been shown to cause to pathological changes affecting mitochondria, synapses and the neuromuscular junction (NMJ). This indicates a crucial physiological role for FUS in regulating synaptic and mitochondrial function that is currently poorly understood. In this paper we provide evidence that mislocalised cytoplasmic FUS causes mitochondrial and synaptic changes and that FUS plays a vital role in maintaining neuronal health in vitro and in vivo. Overexpressing mutant FUS altered synaptic numbers and neuronal complexity in both primary neurons and zebrafish models. The degree to which FUS was mislocalised led to differences in the synaptic changes which was mirrored by changes in mitochondrial numbers and transport. Furthermore, we showed that FUS co-localises with the mitochondrial tethering protein Syntaphilin (SNPH), and that mutations in FUS affect this relationship. Finally, we demonstrated mutant FUS led to changes in global protein translation. This localisation between FUS and SNPH could explain the synaptic and mitochondrial defects observed leading to global protein translation defects. Importantly, our results support the 'gain-of-function' hypothesis for disease pathogenesis in FUS-related ALS.

Lamination Speeds the Functional Development of Visual Circuits.

A common feature of the brain is the arrangement of synapses in layers. To examine the significance of this organizational feature, we studied the functional development of direction-selective (DS) circuits in the tectum of astray mutant zebrafish in which lamination of retinal ganglion cell (RGC) axons is lost. We show that although never laminar, the tuning of DS-RGC axons targeting the mutant tectum is normal. Analysis of mutant tectal neurons at late developmental stages reveals that directional tuning is indistinguishable from wild-type larvae. Furthermore, we show that structural plasticity of tectal dendrites and RGC axons compensates for the loss of lamination, establishing connectivity between DS-RGCs and their normal tectal targets. However, tectal direction selectivity is severely perturbed at earlier developmental stages. Thus, the formation of synaptic laminae is ultimately dispensable for the correct wiring of direction-selective tectal circuits, but it is crucial for the rapid assembly of these networks.

Neurobiology: imaging prey capture circuits in zebrafish.

Two recent studies used a virtual hunting assay and functional imaging to identify prey-capture circuits in zebrafish. Together they show that the optic tectum and a pretectal region are two retinorecipient areas important for the recognition and capture of prey.

Teneurin-3 specifies morphological and functional connectivity of retinal ganglion cells in the vertebrate visual system.

A striking feature of the CNS is the precise wiring of its neuronal connections. During vertebrate visual system development, different subtypes of retinal ganglion cells (RGCs) form specific connections with their corresponding synaptic partners. However, the underlying molecular mechanisms remain to be fully elucidated. Here, we report that the cell-adhesive transmembrane protein Teneurin-3 (Tenm3) is required by zebrafish RGCs for acquisition of their correct morphological and functional connectivity in vivo. Teneurin-3 is expressed by RGCs and their presynaptic amacrine and postsynaptic tectal cell targets. Knockdown of Teneurin-3 leads to RGC dendrite stratification defects within the inner plexiform layer, as well as mistargeting of dendritic processes into outer portions of the retina. Moreover, a subset of RGC axons exhibits tectal laminar arborization errors. Finally, functional analysis of RGCs targeting the tectum reveals a selective deficit in the development of orientation selectivity after Teneurin-3 knockdown. These results suggest that Teneurin-3 plays an instructive role in the functional wiring of the vertebrate visual system.

A systems-based dissection of retinal inputs to the zebrafish tectum reveals different rules for different functional classes during development.

We have examined the form, diversity, and organization of three functional classes of retinal inputs to the zebrafish optic tectum during development. Our systems-based approach was to analyze data from populations of retinal ganglion cells labeled with a presynaptic targeted calcium indicator, synaptophysin GCaMP3 (SyGCaMP3). Collectively, our findings provide an insight as to the degree of visual encoding during retino-tectal development and how it dynamically evolves from a nascent and noisy presynaptic neural-scape to an increasingly complex and refined representation. We report five key features: (1) direction-selective inputs are developmentally invariant; (2) orientation-selective inputs exhibit highly dynamic properties over the same period, with changes in their functional characteristics and spatial organization; (3) inputs defined as anisotropic are an early dominant functional class, with heterogeneous response profiles, which progressively diminish in incidence and spatial extent; (4) dark rearing selectively affects the orientation-selective responses: both functional characteristics and relative spatial distributions; and (5) orientation-selective inputs exhibit four subtypes, two more than previously identified in any species. Our approach was to label RGC axon terminals with an indicator of activity and quantitatively characterize coherent response properties to different visual stimuli. Its application in the zebrafish, given its small size and the accessibility of the tectum, has enabled a quick yet robust assessment of multiple functional populations of responses.

Parametric functional maps of visual inputs to the tectum.

How features of the visual scene are encoded in the population activity of retinal ganglion cells (RGCs) targeting specific regions of the brain is not well understood. To address this, we have used a genetically encoded reporter of presynaptic function (SyGCaMP3) to record visually evoked activity in the population of RGC axons innervating the zebrafish tectum. Using unbiased voxel-wise analysis of SyGCaMP3 signals, we identify three subtypes of direction-selective and two subtypes of orientation-selective retinal input. Composite parametric functional maps generated across many larvae show laminar segregation of direction- and orientation-selective responses and unexpected retinotopic biases in the distribution of functional subtypes. These findings provide a systematic description of the form, organization, and dimensionality of visual inputs to the brain and will serve as a platform for understanding emergent properties in tectal circuits associated with visually driven behavior.

Imaging circuit formation in zebrafish.

The study of nervous system development has been greatly facilitated by recent advances in molecular biology and imaging techniques. These approaches are perfectly suited to young transparent zebrafish where they have allowed direct observation of neural circuit assembly in vivo. In this review we will highlight a number of key studies that have applied optical and genetic techniques in zebrafish to address questions relating to axonal and dendritic arbor development,synapse assembly and neural plasticity. These studies have revealed novel cellular phenomena and modes of growth that may reflect general principles governing the assembly of neural circuits.

Localization of Cadm2a and Cadm3 proteins during development of the zebrafish nervous system.

Members of the Cadm/SynCAM/Necl/IGSF/TSLC family of cell adhesion molecules are known to have diverse functions during development of the nervous system, but information regarding their role during central nervous system (CNS) development in vivo is scarce. The rapid development of a relatively simple nervous system in larval zebrafish makes them a highly tractable model organism for studying gene function during nervous system development. An essential prerequisite for functional studies is a description of protein localization. To address this we have generated subtype-specific antibodies to two members of the zebrafish cell adhesion molecule family: cadm2a and cadm3. Using these novel antibodies we show that cadm3 and cadm2a are expressed throughout the nervous system of larval stage zebrafish. Particularly striking, and largely nonoverlapping expression of cadm2a and cadm3 is observed in the developing retina and spinal cord. Using in vitro binding assays we show that cadm2a and cadm3 bind heterophilically and preferentially to cadm1 and cadm4, respectively. These binding preferences are very similar to those seen for tetrapod Cadms but our study of protein localization suggests novel and diverse functions of cadms during nervous system development.

Lunatic fringe promotes the lateral inhibition of neurogenesis.

Previous studies have identified roles of the modulation of Notch activation by Fringe homologues in boundary formation and in regulating the differentiation of vertebrate thymocytes and Drosophila glial cells. We have investigated the role of Lunatic fringe (Lfng) expression during neurogenesis in the vertebrate neural tube. We find that in the zebrafish hindbrain, Lfng is expressed by progenitors in neurogenic regions and downregulated in cells that have initiated neuronal differentiation. Lfng is required cell autonomously in neural epithelial cells to limit the amount of neurogenesis and to maintain progenitors. By contrast, Lfng is not required for the role of Notch in interneuronal fate choice, which we show is mediated by Notch1a. The expression of Lfng does not require Notch activity, but rather is regulated downstream of proneural genes that are widely expressed by neural progenitors. These findings suggest that Lfng acts in a feedback loop downstream of proneural genes, which, by promoting Notch activation, maintains the sensitivity of progenitors to lateral inhibition and thus limits further proneural upregulation.