Folding makes an imprint.

Imprinted gene clusters are confined genomic regions containing genes with parent-of-origin-dependent transcriptional activity. In this issue of Genes & Development, Loftus and colleagues (pp. 829-843) made use of an insightful combination of descriptive approaches, genetic manipulations, and epigenome-editing approaches to show that differences in nuclear topology precede the onset of imprinted expression at the Peg13-Kcnk9 locus. Furthermore, the investigators provide data in line with a model suggesting that parent-of-origin-specific topological differences could be responsible for parent-of-origin-specific enhancer activity and thus imprinted expression.

Allelic chromatin structure precedes imprinted expression of Kcnk9 during neurogenesis.

Differences in chromatin state inherited from the parental gametes influence the regulation of maternal and paternal alleles in offspring. This phenomenon, known as genomic imprinting, results in genes preferentially transcribed from one parental allele. While local epigenetic factors such as DNA methylation are known to be important for the establishment of imprinted gene expression, less is known about the mechanisms by which differentially methylated regions (DMRs) lead to differences in allelic expression across broad stretches of chromatin. Allele-specific higher-order chromatin structure has been observed at multiple imprinted loci, consistent with the observation of allelic binding of the chromatin-organizing factor CTCF at multiple DMRs. However, whether allelic chromatin structure impacts allelic gene expression is not known for most imprinted loci. Here we characterize the mechanisms underlying brain-specific imprinted expression of the Peg13-Kcnk9 locus, an imprinted region associated with intellectual disability. We performed region capture Hi-C on mouse brains from reciprocal hybrid crosses and found imprinted higher-order chromatin structure caused by the allelic binding of CTCF to the Peg13 DMR. Using an in vitro neuron differentiation system, we showed that imprinted chromatin structure precedes imprinted expression at the locus. Additionally, activation of a distal enhancer induced imprinted expression of Kcnk9 in an allelic chromatin structure-dependent manner. This work provides a high-resolution map of imprinted chromatin structure and demonstrates that chromatin state established in early development can promote imprinted expression upon differentiation.

Imprinted Maternally Expressed microRNAs Antagonize Paternally Driven Gene Programs in Neurons.

Imprinted genes with parental-biased allelic expression are frequently co-regulated and enriched in common biological pathways. Here, we functionally characterize a large cluster of microRNAs (miRNAs) expressed from the maternally inherited allele ("maternally expressed") to explore the molecular and cellular consequences of imprinted miRNA activity. Using an induced neuron (iN) culture system, we show that maternally expressed miRNAs from the miR-379/410 cluster direct the RNA-induced silencing complex (RISC) to transcriptional and developmental regulators, including paternally expressed transcripts like Plagl1. Maternal deletion of this imprinted miRNA cluster resulted in increased protein levels of several targets and upregulation of a broader transcriptional program regulating synaptic transmission and neuronal function. A subset of the transcriptional changes resulting from miR-379/410 deletion can be attributed to de-repression of Plagl1. These data suggest maternally expressed miRNAs antagonize paternally driven gene programs in neurons.

A multifunctional locus controls motor neuron differentiation through short and long noncoding RNAs.

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.

HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA.

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons.

Human induced pluripotent stem cells (iPSCs) can be differentiated into neurons, providing living human neurons to model brain diseases. However, it is unclear how different types of molecules work together to regulate stem cell and neuron biology in healthy and disease states. In this study, we conducted integrated proteomics, lipidomics, and metabolomics analyses with confident identification, accurate quantification, and reproducible measurements to compare the molecular profiles of human iPSCs and iPSC-derived neurons. Proteins, lipids, and metabolites related to mitosis, DNA replication, pluripotency, glycosphingolipids, and energy metabolism were highly enriched in iPSCs, whereas synaptic proteins, neurotransmitters, polyunsaturated fatty acids, cardiolipins, and axon guidance pathways were highly enriched in neurons. Mutations in the GRN gene lead to the deficiency of the progranulin (PGRN) protein, which has been associated with various neurodegenerative diseases. Using this multiomics platform, we evaluated the impact of PGRN deficiency on iPSCs and neurons at the whole-cell level. Proteomics, lipidomics, and metabolomics analyses implicated PGRN's roles in neuroinflammation, purine metabolism, and neurite outgrowth, revealing commonly altered pathways related to neuron projection, synaptic dysfunction, and brain metabolism. Multiomics data sets also pointed toward the same hypothesis that neurons seem to be more susceptible to PGRN loss compared to iPSCs, consistent with the neurological symptoms and cognitive impairment from patients carrying inherited GRN mutations.

PBX1 acts as terminal selector for olfactory bulb dopaminergic neurons.

Neuronal specification is a protracted process that begins with the commitment of progenitor cells and culminates with the generation of mature neurons. Many transcription factors are continuously expressed during this process but it is presently unclear how these factors modify their targets as cells transition through different stages of specification. In olfactory bulb adult neurogenesis, the transcription factor PBX1 controls neurogenesis in progenitor cells and the survival of migrating neuroblasts. Here, we show that, at later differentiation stages, PBX1 also acts as a terminal selector for the dopaminergic neuron fate. PBX1 is also required for the morphological maturation of dopaminergic neurons and to repress alternative interneuron fates, findings that expand the known repertoire of terminal-selector actions. Finally, we reveal that the temporal diversification of PBX1 functions in neuronal specification is achieved, at least in part, through the dynamic regulation of alternative splicing. In Caenorhabditis elegans, PBX/CEH-20 also acts as a dopaminergic neuron terminal selector, which suggests an ancient role for PBX factors in the regulation of terminal differentiation of dopaminergic neurons.

Enhancing structural plasticity of PC12 neurons during differentiation and neurite regeneration with a catalytically inactive mutant version of the zRICH protein.

BACKGROUND: Studies of the molecular mechanisms of nerve regeneration have led to the discovery of several proteins that are induced during successful nerve regeneration. RICH proteins were identified as proteins induced during the regeneration of the optic nerve of teleost fish. These proteins are 2',3'-cyclic nucleotide, 3'-phosphodiesterases that can bind to cellular membranes through a carboxy-terminal membrane localization domain. They interact with the tubulin cytoskeleton and are able to enhance neuronal structural plasticity by promoting the formation of neurite branches. RESULTS: PC12 stable transfectant cells expressing a fusion protein combining a red fluorescent protein with a catalytically inactive mutant version of zebrafish RICH protein were generated. These cells were used as a model to analyze effects of the protein on neuritogenesis. Differentiation experiments showed a 2.9 fold increase in formation of secondary neurites and a 2.4 fold increase in branching points. A 2.2 fold increase in formation of secondary neurites was observed in neurite regeneration assays. CONCLUSIONS: The use of a fluorescent fusion protein facilitated detection of expression levels. Two computer-assisted morphometric analysis methods indicated that the catalytically inactive RICH protein induced the formation of branching points and secondary neurites both during differentiation and neurite regeneration. A procedure based on analysis of random field images provided comparable results to classic neurite tracing methods.

MicroRNA governs bistable cell differentiation and lineage segregation via a noncanonical feedback.

Positive feedback driven by transcriptional regulation has long been considered a key mechanism underlying cell lineage segregation during embryogenesis. Using the developing spinal cord as a paradigm, we found that canonical, transcription-driven feedback cannot explain robust lineage segregation of motor neuron subtypes marked by two cardinal factors, Hoxa5 and Hoxc8. We propose a feedback mechanism involving elementary microRNA-mRNA reaction circuits that differ from known feedback loop-like structures. Strikingly, we show that a wide range of biologically plausible post-transcriptional regulatory parameters are sufficient to generate bistable switches, a hallmark of positive feedback. Through mathematical analysis, we explain intuitively the hidden source of this feedback. Using embryonic stem cell differentiation and mouse genetics, we corroborate that microRNA-mRNA circuits govern tissue boundaries and hysteresis upon motor neuron differentiation with respect to transient morphogen signals. Our findings reveal a previously underappreciated feedback mechanism that may have widespread functions in cell fate decisions and tissue patterning.

Ribosomal RACK1 Regulates the Dendritic Arborization by Repressing FMRP Activity.

FMRP is an RNA-binding protein that represses the translation of specific mRNAs. In neurons, its depletion determines the exaggerated translation of mRNAs leading to dendritic and axonal aberrant development, two peculiar features of Fragile X syndrome patients. However, how FMRP binds to translational machinery to regulate the translation of its mRNA targets is not yet fully understood. Here, we show that FMRP localizes on translational machinery by interacting with the ribosomal binding protein, Receptor for Activated C Kinase 1 (RACK1). The binding of FMRP to RACK1 removes the translational repressive activity of FMRP and promotes the translation of PSD-95 mRNA, one specific target of FMRP. This binding also results in a reduction in the level of FMRP phosphorylation. We also find that the morphological abnormalities induced by Fmr1 siRNA in cortical neurons are rescued by the overexpression of a mutant form of RACK1 that cannot bind ribosomes. Thus, these results provide a new mechanism underlying FMRP activity that contributes to altered development in FXS. Moreover, these data confirm the role of ribosomal RACK1 as a ribosomal scaffold for RNA binding proteins.

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons.

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.

Using Drosophila Motor Mutants to Teach Neurodevelopment in an Undergraduate Neurobiology Lab.

Most undergraduate neuroscience courses include a neurodevelopment component. Typically, the focus is on development of the mammalian central nervous system, including the concepts of neurulation, patterning of the neural tube, and differentiation of the various cells required to build a functional nervous system. However, it can be challenging to design an affordable undergraduate laboratory exercise to reinforce these concepts for students outside of lecture. Here we describe a laboratory exercise that takes advantage of the high level of conservation in neurodevelopmental pathways using Drosophila as a model organism to illuminate the connection between cell differentiation and nervous system function. Following a lesson discussing spinal cord development, students use Drosophila larvae to assess the effects of mutations in highly conserved motor neuron differentiation genes on motor behaviors such as crawling. As outcomes of this laboratory, students are able to master important neurodevelopmental concepts, connect neurodevelopment to nervous system function, and gain experience with experimental design and data analysis.

Growth/differentiation 5 promotes the differentiation of retinal stem cells into neurons via Atoh8.

Retinal diseases are characterized by the degeneration of retinal neural cells, and are the main cause of blindness. Although the development of stem cell including retinal stem cell therapies raises hope for retinal neuron replacement, currently, there is still no efficient method to regenerate retinal neurons. To realize the potential roles of the production of retinal neurons, neurotrophic factor direct the differentiation of retinal stem cells should be extensively identified. In this article, we characterized growth/differentiation 5 (GDF5), which caused the activation of Smad signaling, can induce neurogenesis and neurite outgrowth in retinal stem cell differentiation. Moreover, a bHLH transcription factor, Atoh8 modulates the effects stimulated by GDF5. These data suggested that GDF5 regulates neuron differentiation through mediating Atoh8 and help us to understand the pathophysiological function of GDF5 in retinal regeneration.

Postmitotic diversification of olfactory neuron types is mediated by differential activities of the HMG-box transcription factor SOX-2.

Diversification of neuron classes is essential for functions of the olfactory system, but the underlying mechanisms that generate individual olfactory neuron types are only beginning to be understood. Here we describe a role of the highly conserved HMG-box transcription factor SOX-2 in postmitotic specification and alternative differentiation of the Caenorhabditis elegans AWC and AWB olfactory neurons. We show that SOX-2 partners with different transcription factors to diversify postmitotic olfactory cell types. SOX-2 functions cooperatively with the OTX/OTD transcription factor CEH-36 to specify an AWC "ground state," and functions with the LIM homeodomain factor LIM-4 to suppress this ground state and drive an AWB identity instead. Our findings provide novel insights into combinatorial codes that drive terminal differentiation programs in the nervous system and reveal a biological function of the deeply conserved Sox2 protein that goes beyond its well-known role in stem cell biology.

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness.

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a "bottom-up" manner, from their biochemistry and protein-protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.

The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types.

Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.

Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

Novel LINS1 missense mutation in a family with non-syndromic intellectual disability.

Newer sequencing technologies decipher molecular variations and increase the knowledge of pathogenesis of complex diseases like intellectual disability (ID), affecting 2-3% of the population. We report a novel family with a missense mutation in LINS1 as a cause for non-syndromic ID. Clinical exome sequencing for ID related genes carried out for a male with dysmorphism, mutism, and cognitive delay was uninformative. Subsequently, "pathogenic" and "likely pathogenic" variants associated with other inherited disorders were searched for as secondary findings. Further, PCR-RFLP carried out in other family members confirmed the result. A novel missense variant (c.937G>A) in exon 5 of LINS1 was detected in the proband. His affected elder brother was homozygous and the parents were heterozygous respectively, for the mutation. No mutation was observed in his unaffected sister. Mutations in LINS1 were suspected in this non-syndromic ID case with mutism. LINS1 alterations affect ELAV1 expression and result in reduction in the commissural axonal growth, thus affecting peripheral and central neuronal function. LINS1 acts in association with ß-catenin to influence WNT1 signaling. It is hypothesized that mutations in LINS1 may alter HuR expression during neural differentiation, leading to ID in humans. © 2017 Wiley Periodicals, Inc.

The Order and Place of Neuronal Differentiation Establish the Topography of Sensory Projections and the Entry Points within the Hindbrain.

Establishing topographical maps of the external world is an important but still poorly understood feature of the vertebrate sensory system. To study the selective innervation of hindbrain regions by sensory afferents in the zebrafish embryo, we mapped the fine-grained topographical representation of sensory projections at the central level by specific photoconversion of sensory neurons. Sensory ganglia located anteriorly project more medially than do ganglia located posteriorly, and this relates to the order of sensory ganglion differentiation. By single-plane illumination microscopy (SPIM) in vivo imaging, we show that (1) the sequence of arrival of cranial ganglion inputs predicts the topography of central projections, and (2) delaminated neuroblasts differentiate in close contact with the neural tube, and they never loose contact with the neural ectoderm. Afferent entrance points are established by plasma membrane interactions between primary differentiated peripheral sensory neurons and neural tube border cells with the cooperation of neural crest cells. These first contacts remain during ensuing morphological growth to establish pioneer axons. Neural crest cells and repulsive slit1/robo2 signals then guide axons from later-differentiating neurons toward the neural tube. Thus, this study proposes a new model by which the topographical representation of cranial sensory ganglia is established by entrance order, with the entry points determined by cell contact between the sensory ganglion cell bodies and the hindbrain.