A somitic contribution to the apical ectodermal ridge is essential for fin formation.

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.

Aurora kinase B regulates axonal outgrowth and regeneration in the spinal motor neurons of developing zebrafish.

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

Calpain Inhibition Is Protective in Machado-Joseph Disease Zebrafish Due to Induction of Autophagy.

The neurodegenerative disease Machado-Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (Danio rerio) model of MJD by expressing human ataxin-3 protein containing either 23 glutamines (23Q, wild-type) or 84Q (MJD-causing) within neurons. Phenotypic characterization of the zebrafish (male and female) revealed that the ataxin-3-84Q zebrafish have decreased survival compared with ataxin-3-23Q and develop ataxin-3 neuropathology, ataxin-3 cleavage fragments and motor impairment. Ataxin-3-84Q zebrafish swim shorter distances than ataxin-3-23Q zebrafish as early as 6 days old, even if expression of the human ataxin-3 protein is limited to motor neurons. This swimming phenotype provides a valuable readout for drug treatment studies. Treating the EGFP-ataxin-3-84Q zebrafish with the calpain inhibitor compound calpeptin decreased levels of ataxin-3 cleavage fragments, but also removed all human ataxin-3 protein (confirmed by ELISA) and prevented the early MJD zebrafish motor phenotype. We identified that this clearance of ataxin-3 protein by calpeptin treatment resulted from an increase in autophagic flux (indicated by decreased p62 levels and increased LC3II). Cotreatment with the autophagy inhibitor chloroquine blocked the decrease in human ataxin-3 levels and the improved movement produced by calpeptin treatment. This study demonstrates that this first transgenic zebrafish model of MJD is a valuable tool for testing potential treatments for MJD. Calpeptin treatment is protective in this model of MJD and removal of human ataxin-3 through macro-autophagy plays an important role in this beneficial effect.SIGNIFICANCE STATEMENT We have established the first transgenic zebrafish model of the neurodegenerative disease MJD, and identified relevant disease phenotypes, including impaired movement from an early age, which can be used in rapid drug testing studies. We have found that treating the MJD zebrafish with the calpain inhibitor compound calpeptin produces complete removal of human ataxin-3 protein, due to induction of the autophagy quality control pathway. This improves the movement of the MJD zebrafish. Artificially blocking the autophagy pathway prevents the removal of human ataxin-3 and improved movement produced by calpeptin treatment. These findings indicate that induction of autophagy, and removal of ataxin-3 protein, plays an important role in the protective effects of calpain inhibition for the treatment of MJD.

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons.

Transactivating DNA-binding protein-43 (TDP-43) deposits represent a typical finding in almost all ALS patients, more than half of FTLD patients and patients with several other neurodegenerative disorders. It appears that perturbation of nucleo-cytoplasmic transport is an important event in these conditions but the mechanistic role and the fate of TDP-43 during neuronal degeneration remain elusive. We have developed an experimental system for visualising the perturbed nucleocytoplasmic transport of neuronal TDP-43 at the single-cell level in vivo using zebrafish spinal cord. This approach enabled us to image TDP-43-expressing motor neurons before and after experimental initiation of cell death. We report the formation of mobile TDP-43 deposits within degenerating motor neurons, which are normally phagocytosed by microglia. However, when microglial cells were depleted, injury-induced motor neuron degeneration follows a characteristic process that includes TDP-43 redistribution into the cytoplasm, axon and extracellular space. This is the first demonstration of perturbed TDP-43 nucleocytoplasmic transport in vivo, and suggests that impairment in microglial phagocytosis of dying neurons may contribute towards the formation of pathological TDP-43 presentations in ALS and FTLD.

Development and evolution of the muscles of the pelvic fin.

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.

Stac3 is required for myotube formation and myogenic differentiation in vertebrate skeletal muscle.

Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

The evolutionary history of the development of the pelvic fin/hindlimb.

The arms and legs of man are evolutionarily derived from the paired fins of primitive jawed fish. Few evolutionary changes have attracted as much attention as the origin of tetrapod limbs from the paired fins of ancestral fish. The hindlimbs of tetrapods are derived from the pelvic fins of ancestral fish. These evolutionary origins can be seen in the examination of shared gene and protein expression patterns during the development of pelvic fins and tetrapod hindlimbs. The pelvic fins of fish express key limb positioning, limb bud induction and limb outgrowth genes in a similar manner to that seen in hindlimb development of higher vertebrates. We are now at a point where many of the key players in the development of pelvic fins and vertebrate hindlimbs have been identified and we can now readily examine and compare mechanisms between species. This is yielding fascinating insights into how the developmental programme has altered during evolution and how that relates to anatomical change. The role of pelvic fins has also drastically changed over evolutionary history, from playing a minor role during swimming to developing into robust weight-bearing limbs. In addition, the pelvic fins/hindlimbs have been lost repeatedly in diverse species over evolutionary time. Here we review the evolution of pelvic fins and hindlimbs within the context of the changes in anatomical structure and the molecular mechanisms involved.

Whole-somite rotation generates muscle progenitor cell compartments in the developing zebrafish embryo.

Somites are transient, mesodermally derived structures that give rise to a number of different cell types within the vertebrate embryo. To achieve this, somitic cells are partitioned into lineage-restricted domains, whose fates are determined by signals secreted from adjacent tissues. While the molecular nature of many of the inductive signals that trigger formation of different cell fates within the nascent somite has been identified, less is known about the processes that coordinate the formation of the subsomitic compartments from which these cells arise. Utilizing a combination of vital dye-staining and lineage-tracking techniques, we describe a previously uncharacterized, lineage-restricted compartment of the zebrafish somite that generates muscle progenitor cells for the growth of appendicular, hypaxial, and axial muscles during development. We also show that formation of this compartment occurs via whole-somite rotation, a process that requires the action of the Sdf family of secreted cytokines.

Sodium valproate increases activity of the sirtuin pathway resulting in beneficial effects for spinocerebellar ataxia-3 in vivo.

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia type 3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (valproate) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with valproate improved the swimming of the MJD zebrafish, affected levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that valproate treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in valproate treated MJD zebrafish and HEK293 cells expressing ataxin-3 84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with valproate alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by valproate and the beneficial effects of valproate on the movement in the MJD zebrafish, supporting that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.

In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood. Bimolecular fluorescence complementation (BiFC) takes advantage of the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation-prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize, and visualize the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC. Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos.

Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.

Kynurenine 3-Monooxygenase Activity in Human Primary Neurons and Effect on Cellular Bioenergetics Identifies New Neurotoxic Mechanisms.

Upregulation of the kynurenine pathway (KP) of tryptophan metabolism is commonly observed in neurodegenerative disease. When activated, L-kynurenine (KYN) increases in the periphery and central nervous system where it is further metabolised to other neuroactive metabolites including 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA) and quinolinic acid (QUIN). Particularly biologically relevant metabolites are 3-HK and QUIN, formed downstream of the enzyme kynurenine 3-monooxygenase (KMO) which plays a pivotal role in maintaining KP homeostasis. Indeed, excessive production of 3-HK and QUIN has been described in neurodegenerative disease including Alzheimer's disease and Huntington's disease. In this study, we characterise KMO activity in human primary neurons and identified new mechanisms by which KMO activation mediates neurotoxicity. We show that while transient activation of the KP promotes synthesis of the essential co-enzyme nicotinamide adenine dinucleotide (NAD+), allowing cells to meet short-term increased energy demands, chronic KMO activation induces production of reactive oxygen species (ROS), mitochondrial damage and decreases spare-respiratory capacity (SRC). We further found that these events generate a vicious-cycle, as mitochondrial dysfunction further shunts the KP towards the KMO branch of the KP to presumably enhance QUIN production. These mechanisms may be especially relevant in neurodegenerative disease as neurons are highly sensitive to oxidative stress and mitochondrial impairment.

Motor Neuron Abnormalities Correlate with Impaired Movement in Zebrafish that Express Mutant Superoxide Dismutase 1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p < 0.002) and traveled a significantly shorter distance during behavioral testing (p < 0.001) when compared with WT SOD1 and noninjected larvae. Furthermore, there was a positive correlation between distance traveled and motor axon length (R2 = 0.357, p < 0.001). These data represent the first correlative investigation of motor axonopathies and impaired movement in SOD1-expressing zebrafish, confirming functional relevance and validating movement as a disease phenotype for the testing of disease treatments for ALS.

FishNet: an online database of zebrafish anatomy.

BACKGROUND: Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish. RESULTS: To overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D) models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood. CONCLUSION: FishNet contains more than 36,000 images of larval zebrafish, with more than 1,500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D.

Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo.

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Neuronal cell culture from transgenic zebrafish models of neurodegenerative disease.

We describe a protocol for culturing neurons from transgenic zebrafish embryos to investigate the subcellular distribution and protein aggregation status of neurodegenerative disease-causing proteins. The utility of the protocol was demonstrated on cell cultures from zebrafish that transgenically express disease-causing variants of human fused in sarcoma (FUS) and ataxin-3 proteins, in order to study amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-3 (SCA3), respectively. A mixture of neuronal subtypes, including motor neurons, exhibited differentiation and neurite outgrowth in the cultures. As reported previously, mutant human FUS was found to be mislocalized from nuclei to the cytosol, mimicking the pathology seen in human ALS and the zebrafish FUS model. In contrast, neurons cultured from zebrafish expressing human ataxin-3 with disease-associated expanded polyQ repeats did not accumulate within nuclei in a manner often reported to occur in SCA3. Despite this, the subcellular localization of the human ataxin-3 protein seen in cell cultures was similar to that found in the SCA3 zebrafish themselves. The finding of similar protein localization and aggregation status in the neuronal cultures and corresponding transgenic zebrafish models confirms that this cell culture model is a useful tool for investigating the cell biology and proteinopathy signatures of mutant proteins for the study of neurodegenerative disease.

Morphological diversity: taking the spine out of three-spine stickleback.

A gene already known to play a crucial developmental role in chick and mouse embryos has been fingered as a candidate for naturally occurring variation in three-spine stickleback anatomy.