ABSTRACT
Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.
Subject(s)
Axons/metabolism , Ephrins/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Animals , Chick Embryo , Embryo, Mammalian/metabolism , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Mice , Motor Neurons/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-ß precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.
Subject(s)
Axons/metabolism , Neurogenesis , Presenilins/metabolism , Animals , Chick Embryo , Glycoproteins/metabolism , Mice , Mice, Transgenic , Mutation , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Netrin-1 , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Presenilins/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Immunologic/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Roundabout ProteinsABSTRACT
Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.
Subject(s)
Neuralgia , Nociceptors , Animals , Gene Transfer Techniques , Mice , Neuralgia/etiology , Neuralgia/therapy , Posterior Horn Cells , Spinal Cord , Spinal Cord Dorsal Horn , SwineABSTRACT
Selective neuronal loss is a hallmark of neurodegenerative diseases, which, counterintuitively, are often caused by mutations in widely expressed genes. Charcot-Marie-Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies. A subtype of these diseases--CMT type 2D (CMT2D)--is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-transfer RNA (tRNA) synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons, leading to deficits in distal motor function. How mutations in GlyRS (GlyRS(CMT2D)) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRS(CMT2D) acquires a neomorphic binding activity that directly antagonizes an essential signalling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRS(CMT2D) to bind the neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRS(CMT2D) that antagonizes the VEGF-Nrp1 interaction, and indicate that the VEGF-Nrp1 signalling axis is an actionable target for treating CMT2D.
Subject(s)
Binding, Competitive , Charcot-Marie-Tooth Disease/metabolism , Glycine-tRNA Ligase/metabolism , Animals , Axons/enzymology , Axons/metabolism , Axons/pathology , Cell Line , Cell Survival , Charcot-Marie-Tooth Disease/drug therapy , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Female , Glycine-tRNA Ligase/chemistry , Glycine-tRNA Ligase/genetics , Ligands , Male , Mice , Models, Molecular , Motor Neurons/enzymology , Motor Neurons/metabolism , Motor Neurons/pathology , Motor Skills/drug effects , Mutation/genetics , Neuropilin-1/deficiency , Neuropilin-1/genetics , Neuropilin-1/metabolism , Protein Binding , Protein Multimerization , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor A/therapeutic useABSTRACT
Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.
Subject(s)
Brain/growth & development , Gene Expression Regulation, Developmental , MicroRNAs/metabolism , RNA Stability , Trans-Activators/metabolism , Transcriptional Activation , Animals , Brain/metabolism , Chick Embryo , Exons , HEK293 Cells , HeLa Cells , Humans , Mice , MicroRNAs/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neurogenesis/genetics , Neurons/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , RNA Helicases , RNA-Binding Proteins , Rats , Trans-Activators/genetics , Xenopus laevisABSTRACT
Endogenous retroviruses (ERVs) constitute a substantial portion of mammalian genomes, and their retrotransposition activity helped to drive genetic variation, yet their expression is tightly regulated to prevent unchecked amplification. We generated a series of mouse mutants and embryonic stem (ES) cell lines carrying "deletable" and "rescuable" alleles of the lysine-specific demethylase LSD1/KDM1A. In the absence of KDM1A, the murine endogenous retrovirus MuERV-L/MERVL becomes overexpressed and embryonic development arrests at gastrulation. A number of cellular genes normally restricted to the zygotic genome activation (ZGA) period also become up-regulated in Kdm1a mutants. Strikingly, many of these cellular genes are flanked by MERVL sequences or have cryptic LTRs as promoters that are targets of KDM1A repression. Using genome-wide epigenetic profiling of Kdm1a mutant ES cells, we demonstrate that this subset of ZGA genes and MERVL elements displays increased methylation of histone H3K4, increased acetylation of H3K27, and decreased methylation of H3K9. As a consequence, Kdm1a mutant ES cells exhibit an unusual propensity to generate extraembryonic tissues. Our findings suggest that ancient retroviral insertions were used to co-opt regulatory sequences targeted by KDM1A for epigenetic silencing of cell fate genes during early mammalian embryonic development.
Subject(s)
Embryonic Stem Cells/metabolism , Embryonic Stem Cells/virology , Gene Expression Regulation, Developmental , Oxidoreductases, N-Demethylating/metabolism , Animals , Cell Line , Cells, Cultured , Histone Deacetylases/metabolism , Histone Demethylases , Histones/metabolism , Mice , Mice, Inbred C57BL , Mice, Nude , Mutation , Nuclear Proteins/metabolism , Oxidoreductases, N-Demethylating/genetics , Promoter Regions, Genetic/genetics , Repressor Proteins/metabolism , Retroviridae , Tripartite Motif-Containing Protein 28 , Virus Activation/geneticsABSTRACT
Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.
Subject(s)
Cell Dedifferentiation/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endogenous Retroviruses/genetics , Pluripotent Stem Cells/cytology , Totipotent Stem Cells/cytology , Totipotent Stem Cells/metabolism , Animals , Cell Dedifferentiation/physiology , Cell Lineage/genetics , Chimera/embryology , Chromatin/genetics , Chromatin/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryo, Mammalian/virology , Embryonic Stem Cells/virology , Epigenesis, Genetic , Female , Gene Expression Regulation, Developmental , Genes, Reporter/genetics , Histones/chemistry , Histones/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Lysine/chemistry , Lysine/metabolism , Methylation , Mice , Phenotype , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/virology , Terminal Repeat Sequences/genetics , Totipotent Stem Cells/virology , Transcriptome/geneticsABSTRACT
In gnathostome vertebrates, including fish, birds and mammals, peripheral nerves link nervous system, body and immediate environment by integrating efferent pathways controlling movement apparatus or organ function and afferent pathways underlying somatosensation. Several lines of evidence suggest that peripheral nerve assembly involves instructive interactions between efferent and afferent axon types, but conflicting findings challenge this view. Using genetic modeling in zebrafish, chick and mouse we uncover here a conserved hierarchy of axon type-dependent extension and selective fasciculation events that govern peripheral nerve assembly, which recapitulates the successive phylogenetic emergence of peripheral axon types and circuits in the vertebrate lineage.
Subject(s)
Axons/physiology , Peripheral Nerves/embryology , Animals , Chick Embryo , Chickens , Dermis/innervation , Mice , Motor Neurons/physiology , Neurons, Afferent/physiology , Neurons, Efferent/physiology , Peripheral Nerves/physiology , Zebrafish/embryologyABSTRACT
TRIM28 is critical for the silencing of endogenous retroviruses (ERVs) in embryonic stem (ES) cells. Here, we reveal that an essential impact of this process is the protection of cellular gene expression in early embryos from perturbation by cis-acting activators contained within these retroelements. In TRIM28-depleted ES cells, repressive chromatin marks at ERVs are replaced by histone modifications typical of active enhancers, stimulating transcription of nearby cellular genes, notably those harboring bivalent promoters. Correspondingly, ERV-derived sequences can repress or enhance expression from an adjacent promoter in transgenic embryos depending on their TRIM28 sensitivity in ES cells. TRIM28-mediated control of ERVs is therefore crucial not just to prevent retrotransposition, but more broadly to safeguard the transcriptional dynamics of early embryos.
Subject(s)
Embryonic Stem Cells/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Retroelements , Transcription, Genetic , Animals , Chromatin/genetics , Chromatin/metabolism , Chromosome Mapping , DNA Methylation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/virology , Endogenous Retroviruses/genetics , Gene Deletion , Gene Expression Regulation, Developmental , Gene Silencing , Genetic Loci , Histones/genetics , Histones/metabolism , Mice , Nuclear Proteins/genetics , Promoter Regions, Genetic , Repressor Proteins/genetics , Sequence Analysis, RNA , Tripartite Motif-Containing Protein 28 , Up-RegulationABSTRACT
There is immense cellular and molecular heterogeneity in biological systems. Here, we demonstrate the utility of integrating an inverted light microscope with an ambient ionization source, nanospray electrospray desorption ionization, attached to a high-resolution mass spectrometer to characterize the molecular composition of mouse spinal cords. We detected a broad range of molecules, including peptides and proteins, as well as metabolites such as lipids, sugars, and other small molecules, including S-adenosyl methionine and glutathione, through top-down MS. Top-down analysis revealed variation in the expression of Hb, including the transition from fetal to adult Hb and heterogeneity in Hb subunits consistent with the genetic diversity of the mouse models. Similarly, temporal changes to actin-sequestering proteins ß-thymosins during development were observed. These results demonstrate that interfacing microscopy with ambient ionization provides the means to perform targeted in situ ambient top-down mass spectral analysis to study the pattern of proteins, lipids, and sugars in biologically heterogeneous samples.
Subject(s)
Microscopy/methods , Spectrometry, Mass, Electrospray Ionization/methods , Spinal Cord/growth & development , Spinal Cord/metabolism , Amino Acid Sequence , Animals , Body Patterning , Carbohydrate Metabolism , Female , Hemoglobins/genetics , Hemoglobins/metabolism , Lipid Metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Mice, Transgenic , Microscopy/instrumentation , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Pregnancy , Spectrometry, Mass, Electrospray Ionization/instrumentation , Spinal Cord/embryology , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/methods , Thymosin/genetics , Thymosin/metabolismABSTRACT
Implantable electrode arrays are powerful tools for directly interrogating neural circuitry in the brain, but implementing this technology in the spinal cord in behaving animals has been challenging due to the spinal cord's significant motion with respect to the vertebral column during behavior. Consequently, the individual and ensemble activity of spinal neurons processing motor commands remains poorly understood. Here, we demonstrate that custom ultraflexible 1-µm-thick polyimide nanoelectronic threads can conduct laminar recordings of many neuronal units within the lumbar spinal cord of unrestrained, freely moving mice. The extracellular action potentials have high signal-to-noise ratio, exhibit well-isolated feature clusters, and reveal diverse patterns of activity during locomotion. Furthermore, chronic recordings demonstrate the stable tracking of single units and their functional tuning over multiple days. This technology provides a path for elucidating how spinal circuits compute motor actions.
Subject(s)
Electrodes, Implanted , Spinal Cord , Animals , Spinal Cord/physiology , Mice , Action Potentials/physiology , Motor Activity/physiology , Neurons/physiology , Locomotion/physiology , Mice, Inbred C57BL , MaleABSTRACT
Innate and goal-directed movements require a high-degree of trunk and appendicular muscle coordination to preserve body stability while ensuring the correct execution of the motor action. The spinal neural circuits underlying motor execution and postural stability are finely modulated by propriospinal, sensory and descending feedback, yet how distinct spinal neuron populations cooperate to control body stability and limb coordination remains unclear. Here, we identified a spinal microcircuit composed of V2 lineage-derived excitatory (V2a) and inhibitory (V2b) neurons that together coordinate ipsilateral body movements during locomotion. Inactivation of the entire V2 neuron lineage does not impair intralimb coordination but destabilizes body balance and ipsilateral limb coupling, causing mice to adopt a compensatory festinating gait and be unable to execute skilled locomotor tasks. Taken together our data suggest that during locomotion the excitatory V2a and inhibitory V2b neurons act antagonistically to control intralimb coordination, and synergistically to coordinate forelimb and hindlimb movements. Thus, we suggest a new circuit architecture, by which neurons with distinct neurotransmitter identities employ a dual-mode of operation, exerting either synergistic or opposing functions to control different facets of the same motor behavior.
ABSTRACT
Schwann cells respond to acute axon damage by transiently transdifferentiating into specialized repair cells that restore sensorimotor function. However, the molecular systems controlling repair cell formation and function are not well defined, and consequently, it is unclear whether this form of cellular plasticity has a role in peripheral neuropathies. Here, we identify Mitf as a transcriptional sensor of axon damage under the control of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling. Mitf regulates a core transcriptional program for generating functional repair Schwann cells following injury and during peripheral neuropathies caused by CMT4J and CMT4D. In the absence of Mitf, core genes for epithelial-to-mesenchymal transition, metabolism, and dedifferentiation are misexpressed, and nerve repair is disrupted. Our findings demonstrate that Schwann cells monitor axonal health using a phosphoinositide signaling system that controls Mitf nuclear localization, which is critical for activating cellular plasticity and counteracting neural disease.
Subject(s)
Peripheral Nerve Injuries , Peripheral Nervous System Diseases , Humans , Peripheral Nervous System Diseases/metabolism , Schwann Cells/metabolism , Axons/metabolism , Signal Transduction/physiology , Cell Plasticity , Nerve Regeneration/physiology , Peripheral Nerve Injuries/genetics , Peripheral Nerve Injuries/metabolism , Sciatic Nerve/metabolismABSTRACT
One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.
Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells , Injections, Spinal , Neural Stem Cells , Stem Cell Transplantation , Adult , Animals , Humans , Rats , Cell Differentiation/physiology , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , Genetic Vectors/genetics , Graft Survival/physiology , Induced Pluripotent Stem Cells/physiology , Induced Pluripotent Stem Cells/transplantation , Injections, Spinal/adverse effects , Injections, Spinal/instrumentation , Injections, Spinal/methods , Neural Stem Cells/physiology , Neural Stem Cells/transplantation , Sendai virus , Specimen Handling/methods , Stem Cell Transplantation/adverse effects , Stem Cell Transplantation/instrumentation , Stem Cell Transplantation/methods , Swine , Tissue and Organ Harvesting/methods , Treatment Outcome , Brain , Spinal CordABSTRACT
LIM transcription factors bind to nuclear LIM interactor (Ldb/NLI/Clim) in specific ratios to form higher-order complexes that regulate gene expression. Here we examined how the dosage of LIM homeodomain proteins Isl1 and Isl2 and LIM-only protein Lmo4 influences the assembly and function of complexes involved in the generation of spinal motor neurons (MNs) and V2a interneurons (INs). Reducing the levels of Islet proteins using a graded series of mutations favored V2a IN differentiation at the expense of MN formation. Although LIM-only proteins (LMOs) are predicted to antagonize the function of Islet proteins, we found that the presence or absence of Lmo4 had little influence on MN or V2a IN specification. We did find, however, that the loss of MNs resulting from reduced Islet levels was rescued by eliminating Lmo4, unmasking a functional interaction between these proteins. Our findings demonstrate that MN and V2a IN fates are specified by distinct complexes that are sensitive to the relative stoichiometries of the constituent factors and we present a model to explain how LIM domain proteins modulate these complexes and, thereby, this binary-cell-fate decision.
Subject(s)
Homeodomain Proteins/metabolism , Interneurons/metabolism , Motor Neurons/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Adaptor Proteins, Signal Transducing , Animals , Cell Differentiation/physiology , Homeodomain Proteins/genetics , Humans , Interneurons/cytology , LIM Domain Proteins , LIM-Homeodomain Proteins , Mice , Mice, Knockout , Motor Neurons/cytology , Transcription Factors/geneticsABSTRACT
Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60-70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs.
Subject(s)
Motor Neurons/cytology , Pluripotent Stem Cells/pathology , Transcription Factors/metabolism , Cell Differentiation , Cell Line , Embryonic Stem Cells/cytology , Gene Expression Profiling , Humans , Motor Neurons/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/geneticsABSTRACT
The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.
Subject(s)
Embryonic Development/physiology , Homeodomain Proteins/metabolism , Motor Neurons/metabolism , Spinal Cord/metabolism , Animals , Axons/metabolism , Homeodomain Proteins/genetics , LIM-Homeodomain Proteins , Mice , Mice, Transgenic , Spinal Cord/embryology , Transcription FactorsABSTRACT
microRNAs (miRNAs) have unique gene regulatory effects in different neuronal subpopulations. Here, we describe a protocol to identify neuronal subtype-specific effects of a miRNA in murine motor neuron subpopulations. We detail the preparation of primary mouse spinal tissue for single cell RNA sequencing and bioinformatics analyses of pseudobulk expression data. This protocol applies differential gene expression testing approaches to identify miRNA target networks in heterogeneous neuronal subpopulations that cannot otherwise be captured by bulk RNA sequencing approaches. For complete details on the use and execution of this protocol, please refer to Amin et al. (2021).