Elucidation of how the Mir-23-27-24 cluster regulates development and aging.

MicroRNAs (miRNAs) are pivotal regulators of gene expression and are involved in biological processes spanning from early developmental stages to the intricate process of aging. Extensive research has underscored the fundamental role of miRNAs in orchestrating eukaryotic development, with disruptions in miRNA biogenesis resulting in early lethality. Moreover, perturbations in miRNA function have been implicated in the aging process, particularly in model organisms such as nematodes and flies. miRNAs tend to be clustered in vertebrate genomes, finely modulating an array of biological pathways through clustering within a single transcript. Although extensive research of their developmental roles has been conducted, the potential implications of miRNA clusters in regulating aging remain largely unclear. In this review, we use the Mir-23-27-24 cluster as a paradigm, shedding light on the nuanced physiological functions of miRNA clusters during embryonic development and exploring their potential involvement in the aging process. Moreover, we advocate further research into the intricate interplay among miRNA clusters, particularly the Mir-23-27-24 cluster, in shaping the regulatory landscape of aging.

LncRNA Litchi is a regulator for harmonizing maturity and resilient functionality in spinal motor neurons.

Long noncoding RNAs (lncRNAs) play pivotal roles in modulating gene expression during development and disease. Despite their high expression in the central nervous system (CNS), understanding the precise physiological functions of CNS-associated lncRNAs has been challenging, largely due to the in vitro-centric nature of studies in this field. Here, utilizing mouse embryonic stem cell (ESC)-derived motor neurons (MNs), we identified an unexplored MN-specific lncRNA, Litchi (Long Intergenic RNAs in Chat Intron). By employing an "exon-only" deletion strategy in ESCs and a mouse model, we reveal that Litchi deletion profoundly impacts MN dendritic complexity, axonal growth, and altered action potential patterns. Mechanistically, voltage-gated channels and neurite growth-related genes exhibited heightened sensitivity to Litchi deletion. Our Litchi-knockout mouse model displayed compromised motor behaviors and reduced muscle strength, highlighting Litchi's critical role in motor function. This study unveils an underappreciated function of lncRNAs in orchestrating MN maturation and maintaining robust electrophysiological properties.

MiR34 contributes to spinal muscular atrophy and AAV9-mediated delivery of MiR34a ameliorates the motor deficits in SMA mice.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by the selective loss of spinal motor neurons (MNs) and concomitant muscle weakness. Mutation of SMN1 is known to cause SMA, and restoring SMN protein levels via antisense oligonucleotide treatment is effective for ameliorating symptoms. However, this approach is hindered by exorbitant costs, invasive procedures, and poor treatment responses of some patients. Here, we seek to circumvent these hurdles by identifying reliable biomarkers that could predict treatment efficacy. We uncovered that MiR34 exhibits consistent downregulation during SMA progression in both human and rodent contexts. Importantly, Mir34 family-knockout mice display axon swelling and reduced neuromuscular junction (NMJ) endplates, recapitulating SMA pathology. Introducing MiR34a via scAAV9 improved the motor ability of SMNΔ7 mice, possibly by restoring NMJ endplate size. Finally, we observed a consistent decreasing trend in MiR34 family expression in the cerebrospinal fluid (CSF) of type I SMA patients during the loading phase of nusinersen treatment. Baseline CSF MiR34 levels before nusinersen injection proved predictive of patient motor skills 1 year later. Thus, we propose that MiR34 may serve as a biomarker of SMA since it is associated with the pathology and can help evaluate the therapeutic effects of nusinersen.

Single-cell transcriptomic analysis reveals diversity within mammalian spinal motor neurons.

Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate movements. In vertebrates, the molecular identities of the cardinal MN types such as those innervating limb versus trunk muscles are well elucidated. Yet the identities of finer subtypes within these cell populations that innervate individual muscle groups remain enigmatic. Here we investigate heterogeneity in mouse MNs using single-cell transcriptomics. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. Additionally, we uncover that axial MNs are subdivided into three molecularly distinct subtypes, defined by mediolaterally-biased Satb2, Nr2f2 or Bcl11b expression patterns with different axon guidance signatures. These three subtypes are present in chicken and human embryos, suggesting a conserved axial MN expression pattern across higher vertebrates. Overall, our study provides a molecular resource of spinal MN types and paves the way towards deciphering how neuronal subtypes evolved to accommodate vertebrate motor behaviors.

Inducible lncRNA transgenic mice reveal continual role of HOTAIR in promoting breast cancer metastasis.

HOTAIR is a 2.2-kb long noncoding RNA (lncRNA) whose dysregulation has been linked to oncogenesis, defects in pattern formation during early development, and irregularities during the process of epithelial-to-mesenchymal transition (EMT). However, the oncogenic transformation determined by HOTAIR in vivo and its impact on chromatin dynamics are incompletely understood. Here, we generate a transgenic mouse model with doxycycline-inducible expression of human HOTAIR in the context of the MMTV-PyMT breast cancer-prone background to systematically interrogate the cellular mechanisms by which human HOTAIR lncRNA acts to promote breast cancer progression. We show that sustained high levels of HOTAIR over time increased breast metastatic capacity and invasiveness in breast cancer cells, promoting migration and subsequent metastasis to the lung. Subsequent withdrawal of HOTAIR overexpression reverted the metastatic phenotype, indicating oncogenic lncRNA addiction. Furthermore, HOTAIR overexpression altered both the cellular transcriptome and chromatin accessibility landscape of multiple metastasis-associated genes and promoted EMT. These alterations are abrogated within several cell cycles after HOTAIR expression is reverted to basal levels, indicating an erasable lncRNA-associated epigenetic memory. These results suggest that a continual role for HOTAIR in programming a metastatic gene regulatory program. Targeting HOTAIR lncRNA may potentially serve as a therapeutic strategy to ameliorate breast cancer progression.

The m6A epitranscriptome on neural development and degeneration.

N6-methyladenosine (m6A) is the most prevalent, conserved, and abundant RNA modification of the mRNAs of most eukaryotes, including mammals. Similar to epigenetic DNA modifications, m6A has been proposed to function as a critical regulator for gene expression. This modification is installed by m6A methylation "writers" (Mettl3/Mettl14 methyltransferase complex), and it can be reversed by demethylase "erasers" (Fto and Alkbh5). Furthermore, m6A can be recognized by "readers" (Ythdf and Ythdc families), which may be interpreted to affect mRNA splicing, stability, translation or localization. Levels of m6A methylation appear to be highest in the brain, where it plays important functions during embryonic stem cell differentiation, brain development, and neurodevelopmental disorders. Depletion of the m6A methylation writer Mettl14 from mouse embryonic nervous systems prolongs cell cycle progression of radial glia and extends cortical neurogenesis into postnatal stages. Recent studies further imply that dysregulated m6A methylation may be significantly correlated with neurodegenerative diseases. In this review, we give an overview of m6A modifications during neural development and associated disorders, and provide perspectives for studying m6A methylation.

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.

MicroRNAs mediate precise control of spinal interneuron populations to exert delicate sensory-to-motor outputs.

Although the function of microRNAs (miRNAs) during embryonic development has been intensively studied in recent years, their postnatal physiological functions remain largely unexplored due to inherent difficulties with the presence of redundant paralogs of the same seed. Thus, it is particularly challenging to uncover miRNA functions at neural circuit level since animal behaviors would need to be assessed upon complete loss of miRNA family functions. Here, we focused on the neural functions of MiR34/449 that manifests a dynamic expression pattern in the spinal cord from embryonic to postnatal stages. Our behavioral assays reveal that the loss of MiR34/449 miRNAs perturb thermally induced pain response thresholds and compromised delicate motor output in mice. Mechanistically, MiR34/449 directly target Satb1 and Satb2 to fine-tune the precise number of a sub-population of motor synergy encoder (MSE) neurons. Thus, MiR34/449 fine-tunes optimal development of Satb1/2on interneurons in the spinal cord, thereby refining explicit sensory-to-motor circuit outputs.

The spinal cord is an information superhighway that connects the body with the brain. There, circuits of neurons process information from the brain before sending commands to muscles to generate movement. Each spinal cord circuit contains many types of neurons, whose identity is defined by the set of genes that are active or 'expressed' in each cell. When a gene is turned on, its DNA sequence is copied to produce a messenger RNA (mRNA), a type of molecule that the cell then uses as a template to produce a protein. MicroRNAs (or miRNAs), on the other hand, are tiny RNA molecules that help to regulate gene expression by binding to and 'deactivating' specific mRNAs, stopping them from being used to make proteins. Mammalian cells contain thousands of types of microRNAs, many of which have unknown roles: this includes MiR34/449, a group of six microRNAs found mainly within the nervous system. By using genetic technology to delete this family from the mouse genome, Chang et al. now show that MiR34/449 has a key role in regulating spinal cord circuits. The first clue came from discovering that mice without the MiR34/449 family had unusual posture and a tendency to walk on tiptoe. The animals were also more sensitive to heat, flicking their tails away from a heat source more readily than control mice. At a finer level, the spinal cords of the mutants contained greater numbers of cells in which two genes, Satb1 and Satb2, were turned on. Compared to their counterparts in control mice, the Satb1/2-positive neurons also showed differences in the rest of the genes they expressed. In essence, these neurons had a different genetic profile in MiR34/449 mutant mice, therefore disrupting the neural circuit they belong to. Based on these findings, Chang et al. propose that in wild-type mice, the MiR34/449 family fine-tunes the expression of Satb1/2 in the spinal cord during development. In doing so, it regulates the formation of the spinal cord circuits that help to control movement. More generally, these results provide clues about how miRNAs help to determine cell identities; further studies could then examine whether other miRNAs contribute to the development and maintenance of neuronal circuits.

Functional Roles of Long Non-coding RNAs in Motor Neuron Development and Disease.

Long non-coding RNAs (lncRNAs) have gained increasing attention as they exhibit highly tissue- and cell-type specific expression patterns. LncRNAs are highly expressed in the central nervous system and their roles in the brain have been studied intensively in recent years, but their roles in the spinal motor neurons (MNs) are largely unexplored. Spinal MN development is controlled by precise expression of a gene regulatory network mediated spatiotemporally by transcription factors, representing an elegant paradigm for deciphering the roles of lncRNAs during development. Moreover, many MN-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are associated with RNA metabolism, yet the link between MN-related diseases and lncRNAs remains obscure. In this review, we summarize lncRNAs known to be involved in MN development and disease, and discuss their potential future therapeutic applications.

Multifaceted roles of microRNAs: From motor neuron generation in embryos to degeneration in spinal muscular atrophy.

Two crucial questions in neuroscience are how neurons establish individual identity in the developing nervous system and why only specific neuron subtypes are vulnerable to neurodegenerative diseases. In the central nervous system, spinal motor neurons serve as one of the best-characterized cell types for addressing these two questions. In this review, we dissect these questions by evaluating the emerging role of regulatory microRNAs in motor neuron generation in developing embryos and their potential contributions to neurodegenerative diseases such as spinal muscular atrophy (SMA). Given recent promising results from novel microRNA-based medicines, we discuss the potential applications of microRNAs for clinical assessments of SMA disease progression and treatment.

Mir-17∼92 Confers Motor Neuron Subtype Differential Resistance to ALS-Associated Degeneration.

Progressive degeneration of motor neurons (MNs) is the hallmark of amyotrophic lateral sclerosis (ALS). Limb-innervating lateral motor column MNs (LMC-MNs) seem to be particularly vulnerable and are among the first MNs affected in ALS. Here, we report association of this differential susceptibility with reduced expression of the mir-17∼92 cluster in LMC-MNs prior to disease onset. Reduced mir-17∼92 is accompanied by elevated nuclear PTEN in spinal MNs of presymptomatic SOD1G93A mice. Selective dysregulation of the mir-17∼92/nuclear PTEN axis in degenerating SOD1G93A LMC-MNs was confirmed in a double-transgenic embryonic stem cell system and recapitulated in human SOD1+/L144F-induced pluripotent stem cell (iPSC)-derived MNs. We further show that overexpression of mir-17∼92 significantly rescues human SOD1+/L144F MNs, and intrathecal delivery of adeno-associated virus (AAV)9-mir-17∼92 improves motor deficits and survival in SOD1G93A mice. Thus, mir-17∼92 may have value as a prognostic marker of MN degeneration and is a candidate therapeutic target in SOD1-linked ALS. VIDEO ABSTRACT.

Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity.

The mammalian imprinted Dlk1-Dio3 locus produces multiple long non-coding RNAs (lncRNAs) from the maternally inherited allele, including Meg3 (i.e., Gtl2) in the mammalian genome. Although this locus has well-characterized functions in stem cell and tumor contexts, its role during neural development is unknown. By profiling cell types at each stage of embryonic stem cell-derived motor neurons (ESC~MNs) that recapitulate spinal cord development, we uncovered that lncRNAs expressed from the Dlk1-Dio3 locus are predominantly and gradually enriched in rostral motor neurons (MNs). Mechanistically, Meg3 and other Dlk1-Dio3 locus-derived lncRNAs facilitate Ezh2/Jarid2 interactions. Loss of these lncRNAs compromises the H3K27me3 landscape, leading to aberrant expression of progenitor and caudal Hox genes in postmitotic MNs. Our data thus illustrate that these lncRNAs in the Dlk1-Dio3 locus, particularly Meg3, play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes and they shape MN subtype identity by regulating Hox genes.

When a gene is active, its DNA sequence is 'transcribed' to form a molecule of RNA. Many of these RNAs act as templates for making proteins. But for some genes, the protein molecules are not their final destinations. Their RNA molecules instead help to control gene activity, which can alter the behaviour or the identity of a cell. For example, experiments performed in individual cells suggest that so-called long non-coding RNAs (or lncRNAs for short) guide how stem cells develop into different types of mature cells. However, it is not clear whether lncRNAs play the same critical role in embryos.Yen et al. used embryonic stem cells to model how motor neurons develop in the spinal cord of mouse embryos. This revealed that motor neurons produce large amounts of a specific group of lncRNAs, particularly one called Meg3. Further experiments showed that motor neurons in mouse embryos that lack Meg3 do not correctly silence a set of genes called the Hox genes, which are crucial for laying out the body plans of many different animal embryos. These neurons also incorrectly continue to express genes that are normally active in an early phase of the stem-like cells that make motor neurons.There is wide interest in how lncRNAs help to regulate embryonic development. With this new knowledge of how Meg3 regulates the activity of Hox genes in motor neurons, research could now be directed toward investigating whether lncRNAs help other tissues to develop in a similar way.

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Canonical mRNA is the exception, rather than the rule.

A report on the Second Aegean International Conference on the Long and the Short of Non-Coding RNAs, held in Heraklion, Greece, 9-14 June 2017.

MicroRNA filters Hox temporal transcription noise to confer boundary formation in the spinal cord.

The initial rostrocaudal patterning of the neural tube leads to differential expression of Hox genes that contribute to the specification of motor neuron (MN) subtype identity. Although several 3' Hox mRNAs are expressed in progenitors in a noisy manner, these Hox proteins are not expressed in the progenitors and only become detectable in postmitotic MNs. MicroRNA biogenesis impairment leads to precocious expression and propagates the noise of Hoxa5 at the protein level, resulting in an imprecise Hoxa5-Hoxc8 boundary. Here we uncover, using in silico simulation, two feed-forward Hox-miRNA loops accounting for the precocious and noisy Hoxa5 expression, as well as an ill-defined boundary phenotype in Dicer mutants. Finally, we identify mir-27 as a major regulator coordinating the temporal delay and spatial boundary of Hox protein expression. Our results provide a novel trans Hox-miRNA circuit filtering transcription noise and controlling the timing of protein expression to confer robust individual MN identity.

Mir-17∼92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN.

Motor neurons (MNs) are unique because they project their axons outside of the CNS to innervate the peripheral muscles. Limb-innervating lateral motor column MNs (LMC-MNs) travel substantially to innervate distal limb mesenchyme. How LMC-MNs fine-tune the balance between survival and apoptosis while wiring the sensorimotor circuit en route remains unclear. Here, we show that the mir-17∼92 cluster is enriched in embryonic stem cell (ESC)-derived LMC-MNs and that conditional mir-17∼92 deletion in MNs results in the death of LMC-MNs in vitro and in vivo. mir-17∼92 overexpression rescues MNs from apoptosis, which occurs spontaneously during embryonic development. PTEN is a primary target of mir-17∼92 responsible for LMC-MN degeneration. Additionally, mir-17∼92 directly targets components of E3 ubiquitin ligases, affecting PTEN subcellular localization through monoubiquitination. This miRNA-mediated regulation modulates both target expression and target subcellular localization, providing LMC-MNs with an intricate defensive mechanism that controls their survival.

An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool.

MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.

Apoptosis of limb innervating motor neurons and erosion of motor pool identity upon lineage specific dicer inactivation.

Diversification of mammalian spinal motor neurons into hundreds of subtypes is critical for the maintenance of body posture and coordination of complex movements. Motor neuron differentiation is controlled by extrinsic signals that regulate intrinsic genetic programs specifying and consolidating motor neuron subtype identity. While transcription factors have been recognized as principal regulators of the intrinsic program, the role of posttranscriptional regulations has not been systematically tested. MicroRNAs produced by Dicer mediated cleavage of RNA hairpins contribute to gene regulation by posttranscriptional silencing. Here we used Olig2-cre conditional deletion of Dicer gene in motor neuron progenitors to examine effects of miRNA biogenesis disruption on postmitotic spinal motor neurons. We report that despite the initial increase in the number of motor neuron progenitors, disruption of Dicer function results in a loss of many limb- and sympathetic ganglia-innervating spinal motor neurons. Furthermore, it leads to defects in motor pool identity specification. Thus, our results indicate that miRNAs are an integral part of the genetic program controlling motor neuron survival and acquisition of subtype specific properties.

Mir-17-3p controls spinal neural progenitor patterning by regulating Olig2/Irx3 cross-repressive loop.

Neural patterning relies on transcriptional cross-repressive interactions that ensure unequivocal assignment of neural progenitor identity to proliferating cells. Progenitors of spinal motor neurons (pMN) and V2 interneurons (p2) are specified by a pair of cross-repressive transcription factors, Olig2 and Irx3. Lineage tracing revealed that many p2 progenitors transiently express the pMN marker Olig2 during spinal cord development. Here we demonstrate that the repression of Olig2 in p2 domain is controlled by mir-17-3p microRNA-mediated silencing of Olig2 mRNA. Mice lacking all microRNAs or just the mir-17∼92 cluster manifest a dorsal shift in pMN/p2 boundary and impairment in the production of V2 interneurons. Our findings suggest that microRNA-mediated repression of Olig2 mRNA plays a critical role during the patterning of ventral spinal progenitor domains by shifting the balance of cross-repressive interactions between Olig2 and Irx3 transcription factors.