ABSTRACT
Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of non-coding RNAs (ncRNAs), remain incompletely understood. Here, we establish the transcriptomic landscape and alternative splicing patterns of circular RNAs (circRNAs) at key developmental timepoints in mouse mDA neurons in vivo using fluorescence-activated cell sorting followed by short- and long-read RNA sequencing. In situ hybridisation shows expression of several circRNAs during early mDA neuron development and post-transcriptional silencing unveils roles for different circRNAs in regulating mDA neuron morphology. Finally, in utero electroporation and time-lapse imaging implicate circRmst, a circRNA with widespread morphological effects, in the migration of developing mDA neurons in vivo. Together, these data for the first time suggest a functional role for circRNAs in developing mDA neurons and characterise poorly defined aspects of mDA neuron development.
Subject(s)
Cell Movement , Dopaminergic Neurons , Gene Expression Regulation, Developmental , Mesencephalon , RNA, Circular , Animals , RNA, Circular/genetics , RNA, Circular/metabolism , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/cytology , Mesencephalon/metabolism , Mesencephalon/cytology , Mesencephalon/embryology , Mice , Cell Movement/genetics , Neurogenesis/genetics , Female , Alternative Splicing , Mice, Inbred C57BL , TranscriptomeABSTRACT
Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos.
Subject(s)
Gene Expression Regulation, Developmental , PAX2 Transcription Factor , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/embryology , Zebrafish/metabolism , PAX2 Transcription Factor/metabolism , PAX2 Transcription Factor/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Gastrulation/genetics , Animals, Genetically Modified , Wnt1 Protein/genetics , Wnt1 Protein/metabolism , Embryo, Nonmammalian/metabolism , POU Domain Factors/genetics , POU Domain Factors/metabolism , Octamer Transcription Factor-3/metabolism , Octamer Transcription Factor-3/genetics , Embryonic Development/genetics , Mesencephalon/metabolism , Mesencephalon/embryology , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Somites/metabolism , Somites/embryology , Fibroblast Growth FactorsABSTRACT
OBJECTIVES: To investigate midbrain growth, including corpus callusum (CC) and cerebellar vermis (CV) and cortical development in late fetal growth restricted (FGR) subclassified according to the umbilical vein blood flow (UVBF) values. METHODS: This was a prospective study on singleton fetuses late FGR with abnormal placental cerebral ratio (PCR). FGR fetuses were further subdivided into normal (≥fifth centile) and abnormal (Subject(s)
Fetal Growth Retardation
, Mesencephalon
, Ultrasonography, Prenatal
, Umbilical Veins
, Humans
, Female
, Fetal Growth Retardation/diagnostic imaging
, Fetal Growth Retardation/physiopathology
, Pregnancy
, Prospective Studies
, Cross-Sectional Studies
, Umbilical Veins/diagnostic imaging
, Adult
, Ultrasonography, Prenatal/methods
, Mesencephalon/diagnostic imaging
, Mesencephalon/blood supply
, Mesencephalon/embryology
, Fetal Development/physiology
, Cerebral Cortex/diagnostic imaging
, Cerebral Cortex/blood supply
, Cerebral Cortex/embryology
ABSTRACT
Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1-5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6-11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.
Subject(s)
Embryo, Mammalian , Gastrulation , Models, Biological , Neurulation , Organogenesis , Animals , Cell Lineage , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryonic Stem Cells/cytology , Endoderm/cytology , Endoderm/embryology , Heart/embryology , Mesencephalon/embryology , Mice , Neural Tube/embryology , PAX6 Transcription Factor/deficiency , PAX6 Transcription Factor/genetics , Prosencephalon/embryology , Somites/embryologyABSTRACT
Wnt/ß-catenin signaling contributes to patterning, proliferation, and differentiation throughout vertebrate neural development. Wnt/ß-catenin signaling is important for mammalian midbrain dopaminergic neurogenesis, while little is known about its role in ventral forebrain dopaminergic development. Here, we focus on the A11-like, Otp-dependent diencephalospinal dopaminergic system in zebrafish. We show that Wnt ligands, receptors and extracellular antagonist genes are expressed in the vicinity of developing Otp-dependent dopaminergic neurons. Using transgenic Wnt/ß-catenin-reporters, we found that Wnt/ß-catenin signaling activity is absent from these dopaminergic neurons, but detected Wnt/ß-catenin activity in cells adjacent to the caudal DC5/6 clusters of Otp-dependent dopaminergic neurons. Pharmacological manipulations of Wnt/ß-catenin signaling activity, as well as heat-shock driven overexpression of Wnt agonists and antagonists, interfere with the development of DC5/6 dopaminergic neurons, such that Wnt/ß-catenin activity positively correlates with their number. Wnt/ß-catenin activity promoted dopaminergic development specifically at stages when DC5/6 dopaminergic progenitors are in a proliferative state. Our data suggest that Wnt/ß-catenin signaling acts in a spatially and temporally restricted manner on proliferative dopaminergic progenitors in the hypothalamus to positively regulate the size of the dopaminergic neuron groups DC5 and DC6.
Subject(s)
Dopaminergic Neurons/metabolism , Neurogenesis , Wnt Signaling Pathway , Zebrafish/embryology , beta Catenin/metabolism , Animals , Animals, Genetically Modified , Cell Differentiation , Gene Expression Regulation, Developmental , Mesencephalon/embryology , Mesencephalon/metabolism , Zebrafish/geneticsABSTRACT
Advances in tissue clearing enable analysis of complex migratory patterns of developing neurons in whole intact tissue. Here, we implemented a modified version of 3DISCO to study migration of midbrain dopamine (DA) neurons. We provide a detailed protocol starting from whole-brain immunostaining, tissue clearing, and ultramicroscopic imaging to post-acquisition quantification and analysis. This protocol enables precise quantification of DA neuron migration but can also be applied more generally for analyzing neuron migration throughout the nervous system. For complete details on the use and execution of this protocol, please refer to Brignani et al. (2020).
Subject(s)
Dopaminergic Neurons , Imaging, Three-Dimensional/methods , Mesencephalon/cytology , Mesencephalon/embryology , Microscopy/methods , Animals , Female , Mesencephalon/metabolism , Mice, Transgenic , Microscopy/instrumentation , PregnancyABSTRACT
The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.
Subject(s)
Brain/embryology , Cell Adhesion Molecules/metabolism , Promoter Regions, Genetic/genetics , Animals , Brain/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Hippocampus/embryology , Hippocampus/metabolism , Immunohistochemistry , In Situ Hybridization , Male , Mesencephalon/embryology , Mesencephalon/metabolism , Mice , Mice, Inbred C57BL , Prosencephalon/embryology , Prosencephalon/metabolism , Spinal Cord/embryology , Spinal Cord/metabolismABSTRACT
Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.
Subject(s)
Autism Spectrum Disorder/metabolism , Dopamine/metabolism , Heparitin Sulfate/metabolism , Lysosomal Storage Diseases/metabolism , Animals , Autism Spectrum Disorder/drug therapy , Autism Spectrum Disorder/pathology , Benzazepines/therapeutic use , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Dopamine Antagonists/therapeutic use , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Heparitin Sulfate/pharmacology , Lysosomal Storage Diseases/drug therapy , Lysosomal Storage Diseases/pathology , Mesencephalon/drug effects , Mesencephalon/embryology , Mesencephalon/pathology , Mice , Mucopolysaccharidosis III/drug therapy , Mucopolysaccharidosis III/metabolism , Mucopolysaccharidosis III/pathology , Receptors, Dopamine D1/antagonists & inhibitors , Receptors, Dopamine D1/metabolismABSTRACT
BMP signaling plays iterative roles during vertebrate neural crest development from induction through craniofacial morphogenesis. However, far less is known about the role of BMP activity in cranial neural crest epithelial-to-mesenchymal transition and delamination. By measuring canonical BMP signaling activity as a function of time from specification through early migration of avian midbrain neural crest cells, we found elevated BMP signaling during delamination stages. Moreover, inhibition of canonical BMP activity via a dominant negative mutant Type I BMP receptor showed that BMP signaling is required for neural crest migration from the midbrain, independent from an effect on EMT and delamination. Transcriptome profiling on control compared to BMP-inhibited cranial neural crest cells identified novel BMP targets during neural crest delamination and early migration including targets of the Notch pathway that are upregulated following BMP inhibition. These results suggest potential crosstalk between the BMP and Notch pathways in early migrating cranial neural crest and provide novel insight into mechanisms regulated by BMP signaling during early craniofacial development.
Subject(s)
Bone Morphogenetic Proteins/physiology , Mesencephalon/embryology , Neural Crest/metabolism , Signal Transduction , Animals , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Proteins/metabolism , Chick Embryo , Embryonic Development/genetics , Gene Expression Regulation, Developmental , Mesencephalon/metabolism , Neural Crest/embryology , Skull/embryology , Skull/metabolism , Tissue Culture TechniquesABSTRACT
Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.
Subject(s)
Embryonic Development/genetics , Enhancer Elements, Genetic/genetics , Fibroblast Growth Factor 8/genetics , Gene Expression Regulation, Developmental , Genetic Engineering/methods , Animals , CRISPR-Cas Systems/genetics , Ectoderm/embryology , Embryo, Mammalian , Extremities/embryology , Feasibility Studies , Female , Fibroblast Growth Factor 8/metabolism , Gene Regulatory Networks , Male , Mesencephalon/embryology , Mice , Mice, Transgenic , Rhombencephalon/embryologyABSTRACT
Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.
Subject(s)
Apoptosis , Diencephalon/embryology , Mesencephalon/embryology , Neural Tube/embryology , Animals , Apoptotic Protease-Activating Factor 1/genetics , Apoptotic Protease-Activating Factor 1/metabolism , Mice , Mice, Transgenic , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
A subset of adult ventral tegmental area dopamine (DA) neurons expresses vesicular glutamate transporter 2 (VGluT2) and releases glutamate as a second neurotransmitter in the striatum, while only few adult substantia nigra DA neurons have this capacity. Recent work showed that cellular stress created by neurotoxins such as MPTP and 6-hydroxydopamine can upregulate VGluT2 in surviving DA neurons, suggesting the possibility of a role in cell survival, although a high level of overexpression could be toxic to DA neurons. Here we examined the level of VGluT2 upregulation in response to neurotoxins and its impact on postlesional plasticity. We first took advantage of an in vitro neurotoxin model of Parkinson's disease and found that this caused an average 2.5-fold enhancement of Vglut2 mRNA in DA neurons. This could represent a reactivation of a developmental phenotype because using an intersectional genetic lineage-mapping approach, we find that >98% of DA neurons have a VGluT2+ lineage. Expression of VGluT2 was detectable in most DA neurons at embryonic day 11.5 and was localized in developing axons. Finally, compatible with the possibility that enhanced VGluT2 expression in DA neurons promotes axonal outgrowth and reinnervation in the postlesional brain, we observed that DA neurons in female and male mice in which VGluT2 was conditionally removed established fewer striatal connections 7 weeks after a neurotoxin lesion. Thus, we propose here that the developmental expression of VGluT2 in DA neurons can be reactivated at postnatal stages, contributing to postlesional plasticity of dopaminergic axons.SIGNIFICANCE STATEMENT A small subset of dopamine neurons in the adult, healthy brain expresses vesicular glutamate transporter 2 (VGluT2) and thus releases glutamate as a second neurotransmitter in the striatum. This neurochemical phenotype appears to be plastic as exposure to neurotoxins, such as 6-OHDA or MPTP, that model certain aspects of Parkinson's disease pathophysiology, boosts VGluT2 expression in surviving dopamine neurons. Here we show that this enhanced VGluT2 expression in dopamine neurons drives axonal outgrowth and contributes to dopamine neuron axonal plasticity in the postlesional brain. A better understanding of the neurochemical changes that occur during the progression of Parkinson's disease pathology will aid the development of novel therapeutic strategies for this disease.
Subject(s)
Corpus Striatum/physiology , Dopaminergic Neurons/metabolism , Vesicular Glutamate Transport Protein 2/biosynthesis , Animals , Animals, Newborn , Axons/physiology , Cell Lineage/genetics , Cell Survival/genetics , Corpus Striatum/embryology , Corpus Striatum/growth & development , Female , MPTP Poisoning/genetics , MPTP Poisoning/metabolism , Mesencephalon/embryology , Mesencephalon/growth & development , Mesencephalon/physiology , Mice , Mice, Knockout , Neural Pathways/embryology , Neural Pathways/growth & development , Neural Pathways/physiology , Neurotoxins/toxicity , Pregnancy , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , Vesicular Glutamate Transport Protein 2/geneticsABSTRACT
Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here, we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. We show that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.
Subject(s)
Evolution, Molecular , Gene Regulatory Networks , Mesencephalon/physiology , Animals , Behavior, Animal , Brain/embryology , Brain/metabolism , Brain/physiology , Drosophila , Fibroblast Growth Factor 8/genetics , Fibroblast Growth Factor 8/metabolism , Gene Expression Regulation, Developmental , Humans , Mesencephalon/embryology , Mesencephalon/metabolism , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Pathways , Paired Box Transcription Factors/genetics , Paired Box Transcription Factors/metabolism , Regulatory Sequences, Nucleic Acid , Rhombencephalon/embryology , Rhombencephalon/metabolism , Rhombencephalon/physiology , Signal TransductionABSTRACT
The mesodiencephalic dopaminergic (mdDA) group of neurons comprises molecularly distinct subgroups, of which the substantia nigra (SN) and ventral tegmental area (VTA) are the best known, due to the selective degeneration of the SN during Parkinson's disease. However, although significant research has been conducted on the molecular build-up of these subsets, much is still unknown about how these subsets develop and which factors are involved in this process. In this review, we aim to describe the life of an mdDA neuron, from specification in the floor plate to differentiation into the different subsets. All mdDA neurons are born in the mesodiencephalic floor plate under the influence of both SHH-signaling, important for floor plate patterning, and WNT-signaling, involved in establishing the progenitor pool and the start of the specification of mdDA neurons. Furthermore, transcription factors, like Ngn2, Ascl1, Lmx1a, and En1, and epigenetic factors, like Ezh2, are important in the correct specification of dopamine (DA) progenitors. Later during development, mdDA neurons are further subdivided into different molecular subsets by, amongst others, Otx2, involved in the specification of subsets in the VTA, and En1, Pitx3, Lmx1a, and WNT-signaling, involved in the specification of subsets in the SN. Interestingly, factors involved in early specification in the floor plate can serve a dual function and can also be involved in subset specification. Besides the mdDA group of neurons, other systems in the embryo contain different subsets, like the immune system. Interestingly, many factors involved in the development of mdDA neurons are similarly involved in immune system development and vice versa. This indicates that similar mechanisms are used in the development of these systems, and that knowledge about the development of the immune system may hold clues for the factors involved in the development of mdDA neurons, which may be used in culture protocols for cell replacement therapies.
Subject(s)
Dopaminergic Neurons/metabolism , Mesencephalon/cytology , Mesencephalon/embryology , Animals , Cell Differentiation/genetics , Dopamine/metabolism , Dopaminergic Neurons/physiology , Embryo, Mammalian/cytology , Gene Expression Regulation, Developmental/genetics , Humans , Mesencephalon/metabolism , Mesencephalon/physiology , Substantia Nigra/embryology , Substantia Nigra/metabolism , Transcription Factors/genetics , Ventral Tegmental Area/embryology , Ventral Tegmental Area/metabolismABSTRACT
Nuclear receptor-related 1 (Nurr1) protein has been identified as an obligatory transcription factor in midbrain dopaminergic neurogenesis, but the global set of human NURR1 target genes remains unexplored. Here, we identified direct gene targets of NURR1 by analyzing genome-wide differential expression of NURR1 together with NURR1 consensus sites in three human neural stem cell (hNSC) lines. Microarray data were validated by quantitative PCR in hNSCs and mouse embryonic brains and through comparison to published human data, including genome-wide association study hits and the BioGPS gene expression atlas. Our analysis identified ~40 NURR1 direct target genes, many of them involved in essential protein modules such as synapse formation, neuronal cell migration during brain development, and cell cycle progression and DNA replication. Specifically, expression of genes related to synapse formation and neuronal cell migration correlated tightly with NURR1 expression, whereas cell cycle progression correlated negatively with it, precisely recapitulating midbrain dopaminergic development. Overall, this systematic examination of NURR1-controlled regulatory networks provides important insights into this protein's biological functions in dopamine-based neurogenesis.
Subject(s)
Cell Cycle Checkpoints/genetics , Gene Regulatory Networks , Genome-Wide Association Study , Neural Stem Cells/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Synapses/metabolism , Animals , Base Sequence , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation/genetics , Cell Line , Cell Lineage/genetics , Cell Movement/genetics , Gene Ontology , Humans , Mesencephalon/embryology , Mice , Neural Stem Cells/cytology , Neurogenesis/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Parkinson Disease/genetics , Promoter Regions, Genetic/genetics , Reproducibility of Results , Transcriptional Activation/geneticsABSTRACT
Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6ß2-containing (α6ß2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6ß2* agonist exhibiting functional selectivity toward other nAChRs, including α4ß2, α3ß4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6ß2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6ß2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.
Subject(s)
Neuroprotective Agents/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/metabolism , Action Potentials/drug effects , Animals , Calcium Signaling/drug effects , Dopamine/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Male , Mesencephalon/drug effects , Mesencephalon/embryology , Mice , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacokinetics , Nicotinic Agonists/administration & dosage , Nicotinic Agonists/chemistry , Nicotinic Agonists/pharmacokinetics , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Protein Binding , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptors, Nicotinic/genetics , Synaptosomes/drug effects , Synaptosomes/metabolism , Xenopus laevisABSTRACT
Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Hedgehog Proteins/physiology , Nerve Tissue Proteins/physiology , Prosencephalon/embryology , Animals , CRISPR-Cas Systems , Eye Proteins/physiology , Gene Knockout Techniques , Genes, Reporter , Hedgehog Proteins/biosynthesis , Hedgehog Proteins/genetics , Holoprosencephaly/genetics , Homeodomain Proteins/physiology , Hypothalamus/abnormalities , Hypothalamus/embryology , Hypothalamus/metabolism , Lac Operon , Mesencephalon/embryology , Mesencephalon/metabolism , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Prosencephalon/abnormalities , Prosencephalon/metabolism , Signal Transduction , Transgenes , Homeobox Protein SIX3ABSTRACT
Although the regulation of stress granules has become an intensely studied topic, current investigations of stress granule assembly, disassembly and dynamics are mainly performed in cultured cells. Here, we report the establishment of a stress granule reporter to facilitate the real-time study of stress granules in vivo Using CRISPR/Cas9, we fused a green fluorescence protein (GFP) to endogenous G3BP1 in zebrafish. The GFP-G3BP1 reporter faithfully and robustly responded to heat stress in zebrafish embryos and larvae. The induction of stress granules varied by brain regions under the same stress condition, with the midbrain cells showing the highest efficiency and dynamics. Furthermore, pre-conditioning using lower heat stress significantly limited stress granule formation during subsequent higher heat stress. More interestingly, stress granule formation was much more robust in zebrafish embryos than in larvae and coincided with significantly elevated levels of phosphorylated eIF2α and enhanced heat resilience. Therefore, these findings have generated new insights into stress response in zebrafish during early development and demonstrated that the GFP-G3BP1 knock-in zebrafish could be a valuable tool for the investigation of stress granule biology.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Cytoplasmic Granules , Heat-Shock Response , Mesencephalon/embryology , Neurons/metabolism , RNA Helicases , Zebrafish , Animals , Animals, Genetically Modified/embryology , Animals, Genetically Modified/genetics , CRISPR-Cas Systems , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , RNA Helicases/genetics , RNA Helicases/metabolism , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
Development of the central nervous system requires coordination of the proliferation and differentiation of neural stem cells. Here, we show that laminin alpha 2 (lm-α2) is a component of the midbrain dopaminergic neuron (mDA) progenitor niche in the ventral midbrain (VM) and identify a concentration-dependent role for laminin α2ß1γ1 (lm211) in regulating mDA progenitor proliferation and survival via a distinct set of receptors. At high concentrations, lm211-rich environments maintain mDA progenitors in a proliferative state via integrins α6ß1 and α7ß1, whereas low concentrations of lm211 support mDA lineage survival via dystroglycan receptors. We confirmed our findings in vivo, demonstrating that the VM was smaller in the absence of lm-α2, with increased apoptosis; furthermore, the progenitor pool was depleted through premature differentiation, resulting in fewer mDA neurons. Examination of mDA neuron subtype composition showed a reduction in later-born mDA neurons of the ventral tegmental area, which control a range of cognitive behaviours. Our results identify a novel role for laminin in neural development and provide a possible mechanism for autism-like behaviours and the brainstem hypoplasia seen in some individuals with mutations of LAMA2.
Subject(s)
Dopaminergic Neurons/physiology , Laminin/physiology , Mesencephalon/embryology , Neurogenesis , Animals , Cell Line , Cell Proliferation , Cell Survival , Humans , Integrins/metabolism , Laminin/genetics , Mesencephalon/cytology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurogenesis/geneticsABSTRACT
Developmental control of long-range neuronal connections in the mammalian midbrain remains unclear. We explored the mechanisms regulating target selection of the developing superior colliculus (SC). The SC is a midbrain center that directs orienting behaviors and defense responses. We discovered that a transcription factor, Rorß, controls establishment of axonal projections from the SC to two thalamic nuclei: the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior nucleus (LP). A genetic strategy used to visualize SC circuits revealed that in control animals Rorß+ neurons abundantly innervate the dLGN but barely innervate the LP. The opposite phenotype was observed in global and conditional Rorb mutants: projections to the dLGN were strongly decreased, and projections to the LP were increased. Furthermore, overexpression of Rorb in the wild type showed increased projections to the dLGN and decreased projections to the LP. In summary, we identified Rorß as a key developmental mediator of colliculo-thalamic innervation. Such regulation could represent a general mechanism orchestrating long-range neuronal connections in the mammalian brain.