Annexin A11 mutations are associated with nuclear envelope dysfunction in vivo and in human tissue.

Annexin A11 mutations are a rare cause of amyotrophic lateral sclerosis (ALS), wherein replicated protein variants P36R, G38R, D40G and D40Y are located in a small-alpha helix within the long, disordered N-terminus. To elucidate disease mechanisms, we characterised the phenotypes induced by a genetic loss of function (LoF) and by misexpression of G38R and D40G in vivo. Loss of Annexin A11 results in a low-penetrant behavioural phenotype and aberrant axonal morphology in zebrafish homozygous knockout larvae, which is rescued by human WT Annexin A11. Both Annexin A11 knockout/down and ALS variants trigger nuclear dysfunction characterised by Lamin B2 mis-localisation. The Lamin B2 signature also presented in anterior horn, spinal cord neurons from post-mortem ALS+/-FTD patient tissue possessing G38R and D40G protein variants. These findings suggest mutant Annexin A11 acts as a dominant negative, revealing a potential early nucleopathy highlighting nuclear envelope abnormalities preceding behavioural abnormality in animal models.

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.

Prematurely terminated intron-retaining mRNAs invade axons in SFPQ null-driven neurodegeneration and are a hallmark of ALS.

Loss of SFPQ is a hallmark of motor degeneration in ALS and prevents maturation of motor neurons when occurring during embryogenesis. Here, we show that in zebrafish, developing motor neurons lacking SFPQ exhibit axon extension, branching and synaptogenesis defects, prior to degeneration. Subcellular transcriptomics reveals that loss of SFPQ in neurons produces a complex set of aberrant intron-retaining (IR) transcripts coding for neuron-specific proteins that accumulate in neurites. Some of these local IR mRNAs are prematurely terminated within the retained intron (PreT-IR). PreT-IR mRNAs undergo intronic polyadenylation, nuclear export, and localise to neurites in vitro and in vivo. We find these IR and PreT-IR mRNAs enriched in RNAseq datasets of tissue from patients with familial and sporadic ALS. This shared signature, between SFPQ-depleted neurons and ALS, functionally implicates SFPQ with the disease and suggests that neurite-centred perturbation of alternatively spliced isoforms drives the neurodegenerative process.

Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish.

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.

The zinc finger/RING domain protein Unkempt regulates cognitive flexibility.

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.

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.

A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons.

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.

Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear ß-Catenin Pools.

Dickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on ß-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of ß-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic ß-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits ß-catenin-dependent transcriptional output.

Pineal progenitors originate from a non-neural territory limited by FGF signalling.

The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.

Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits.

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte (ele) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mapping and cloning of the ele mutation showed that the affected gene is slbp, which encodes a conserved RNA stem-loop binding protein involved in replication dependent histone mRNA metabolism. Cells throughout the central nervous system remained in the cell cycle in ele mutant embryos at stages when, and locations where, post-mitotic cells have differentiated in wild-type siblings. Indeed, RNAseq analysis showed down-regulation of many genes associated with neuronal differentiation. This was coincident with changes in the levels and spatial localisation of expression of various genes implicated, for instance, in axon guidance, that likely underlie specific ele phenotypes. These results suggest that many of the cell and tissue specific phenotypes in ele mutant embryos are secondary to altered expression of modules of developmental regulatory genes that characterise, or promote transitions in, cell state and require the correct function of Slbp-dependent histone and chromatin regulatory genes.

BMP- and neuropilin 1-mediated motor axon navigation relies on spastin alternative translation.

Functional analyses of genes responsible for neurodegenerative disorders have unveiled crucial links between neurodegenerative processes and key developmental signalling pathways. Mutations in SPG4-encoding spastin cause hereditary spastic paraplegia (HSP). Spastin is involved in diverse cellular processes that couple microtubule severing to membrane remodelling. Two main spastin isoforms are synthesised from alternative translational start sites (M1 and M87). However, their specific roles in neuronal development and homeostasis remain largely unknown. To selectively unravel their neuronal function, we blocked spastin synthesis from each initiation codon during zebrafish development and performed rescue analyses. The knockdown of each isoform led to different motor neuron and locomotion defects, which were not rescued by the selective expression of the other isoform. Notably, both morphant neuronal phenotypes were observed in a CRISPR/Cas9 spastin mutant. We next showed that M1 spastin, together with HSP proteins atlastin 1 and NIPA1, drives motor axon targeting by repressing BMP signalling, whereas M87 spastin acts downstream of neuropilin 1 to control motor neuron migration. Our data therefore suggest that defective BMP and neuropilin 1 signalling may contribute to the motor phenotype in a vertebrate model of spastin depletion.

The Birth of the Eye Vesicle: When Fate Decision Equals Morphogenesis.

As the embryonic ectoderm is induced to form the neural plate, cells inside this epithelium acquire restricted identities that will dictate their behavior and progressive differentiation. The first behavior adopted by most neural plate cells is called neurulation, a morphogenetic movement shaping the neuroepithelium into a tube. One cell population is not adopting this movement: the eye field. Giving eye identity to a defined population inside the neural plate is therefore a key neural fate decision. While all other neural population undergo neurulation similarly, converging toward the midline, the eye field moves outwards, away from the rest of the forming neural tube, to form vesicles. Thus, while delay in acquisition of most other fates would not have significant morphogenetic consequences, defect in the establishment of the eye field would dramatically impact the formation of the eye. Yet, very little is understood of the molecular and cellular mechanisms driving them. Here, we summarize what is known across vertebrate species and propose a model highlighting what is required to form the essential vesicles that initiate the vertebrate eyes.

Motor axon navigation relies on Fidgetin-like 1-driven microtubule plus end dynamics.

During neural circuit assembly, extrinsic signals are integrated into changes in growth cone (GC) cytoskeleton underlying axon guidance decisions. Microtubules (MTs) were shown to play an instructive role in GC steering. However, the numerous actors required for MT remodeling during axon navigation and their precise mode of action are far from being deciphered. Using loss- and gain-of-function analyses during zebrafish development, we identify in this study the meiotic clade adenosine triphosphatase Fidgetin-like 1 (Fignl1) as a key GC-enriched MT-interacting protein in motor circuit wiring and larval locomotion. We show that Fignl1 controls GC morphology and behavior at intermediate targets by regulating MT plus end dynamics and growth directionality. We further reveal that alternative translation of Fignl1 transcript is a sophisticated mechanism modulating MT dynamics: a full-length isoform regulates MT plus end-tracking protein binding at plus ends, whereas shorter isoforms promote their depolymerization beneath the cell cortex. Our study thus pinpoints Fignl1 as a multifaceted key player in MT remodeling underlying motor circuit connectivity.

Life-Long Neurogenic Activity of Individual Neural Stem Cells and Continuous Growth Establish an Outside-In Architecture in the Teleost Pallium.

Spatiotemporal variations of neurogenesis are thought to account for the evolution of brain shape. In the dorsal telencephalon (pallium) of vertebrates, it remains unresolved which ancestral neurogenesis mode prefigures the highly divergent cytoarchitectures that are seen in extant species. To gain insight into this question, we developed genetic tools to generate here the first 4-dimensional (3D + birthdating time) map of pallium construction in the adult teleost zebrafish. Using a Tet-On-based genetic birthdating strategy, we identify a "sequential stacking" construction mode where neurons derived from the zebrafish pallial germinal zone arrange in outside-in, age-related layers from a central core generated during embryogenesis. We obtained no evidence for overt radial or tangential neuronal migrations. Cre-lox-mediated tracing, which included following Brainbow clones, further demonstrates that this process is sustained by the persistent neurogenic activity of individual pallial neural stem cells (NSCs) from embryo to adult. Together, these data demonstrate that the spatiotemporal control of NSC activity is an important driver of the macroarchitecture of the zebrafish adult pallium. This simple mode of pallium construction shares distinct traits with pallial genesis in mammals and non-mammalian amniotes such as birds or reptiles, suggesting that it may exemplify the basal layout from which vertebrate pallial architectures were elaborated.

Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development.

Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process. VIDEO ABSTRACT.

Global identification of Smad2 and Eomesodermin targets in zebrafish identifies a conserved transcriptional network in mesendoderm and a novel role for Eomesodermin in repression of ectodermal gene expression.

BACKGROUND: Nodal signalling is an absolute requirement for normal mesoderm and endoderm formation in vertebrate embryos, yet the transcriptional networks acting directly downstream of Nodal and the extent to which they are conserved is largely unexplored, particularly in vivo. Eomesodermin also plays a role in patterning mesoderm and endoderm in vertebrates, but its mechanisms of action, and how it interacts with the Nodal signalling pathway are still unclear. RESULTS: Using a combination of ChIP-seq and expression analysis we identify direct targets of Smad2, the effector of Nodal signalling in blastula stage zebrafish embryos, including many novel target genes. Through comparison of these data with published ChIP-seq data in human, mouse and Xenopus we show that the transcriptional network driven by Smad2 in mesoderm and endoderm is conserved in these vertebrate species. We also show that Smad2 and zebrafish Eomesodermin a (Eomesa) bind common genomic regions proximal to genes involved in mesoderm and endoderm formation, suggesting Eomesa forms a general component of the Smad2 signalling complex in zebrafish. Combinatorial perturbation of Eomesa and Smad2-interacting factor Foxh1 results in loss of both mesoderm and endoderm markers, confirming the role of Eomesa in endoderm formation and its functional interaction with Foxh1 for correct Nodal signalling. Finally, we uncover a novel, role for Eomesa in repressing ectodermal genes in the early blastula. CONCLUSION: Our data demonstrate that evolutionarily conserved developmental functions of Nodal signalling occur through maintenance of the transcriptional network directed by Smad2. This network is modulated by Eomesa in zebrafish which acts to promote mesoderm and endoderm formation in combination with Nodal signalling, whilst Eomesa also opposes ectoderm gene expression. Eomesa therefore regulates the formation of all three germ layers in the early zebrafish embryo.

Spatial regionalization and heterochrony in the formation of adult pallial neural stem cells.

Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing, activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging, coupled with gene expression analyses and Notch invalidations, to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary, the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof, activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments.

The Wnt cries many: Wnt regulation of neurogenesis through tissue patterning, proliferation, and asymmetric cell division.

During early development Wnt signaling has a key role in patterning the prospective nervous system by regulation of cell fate specification, cell polarity, and cell migration. Wnt also coordinates the formation of neural circuits on multiple levels such as transcription, cell cycle, and asymmetric cell division. Here we review the latest findings addressing the role of canonical Wnt/ß-catenin signaling during developmental and adult neurogenesis; exploring the connection of in vivo data to the recently described Wnt-driven asymmetric stem cell division in vitro. Understanding how Wnt orchestrates these processes in a spatiotemporal manner during corticogenesis will be of crucial importance for the development of new strategies to regenerate neuronal circuits.

Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.