ABSTRACT
Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.
Subject(s)
Alternative Splicing , Cellular Reprogramming , Embryonic Stem Cells/metabolism , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Pluripotent Stem Cells/metabolism , Repressor Proteins/metabolism , Animals , DNA/metabolism , Embryonic Stem Cells/cytology , Genes, Homeobox , Humans , Mice , Pluripotent Stem Cells/cytology , Protein Isoforms/metabolismABSTRACT
Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications.This article has an associated 'The people behind the papers' interview.
Subject(s)
Circadian Clocks , Homeodomain Proteins/metabolism , Motor Neurons/metabolism , Pluripotent Stem Cells/metabolism , Benzamides/pharmacology , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation , Circadian Clocks/drug effects , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Development , Fibroblast Growth Factors/antagonists & inhibitors , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation, Developmental , Growth Differentiation Factors/genetics , Growth Differentiation Factors/metabolism , Growth Differentiation Factors/pharmacology , Homeodomain Proteins/genetics , Humans , Motor Neurons/cytology , Pluripotent Stem Cells/cytology , Pyrimidines/pharmacology , Signal Transduction/drug effects , Spinal Cord/metabolismABSTRACT
Bone morphogenetic proteins (BMPs) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity in vivo, whether this is sufficient for pattern formation in vitro is unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cell-derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types, and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species-specific tempo of neural differentiation. Together, this study describes efficient methods for generating patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.
Subject(s)
Bone Morphogenetic Protein 4/physiology , Cell Differentiation/genetics , Organoids/physiology , Smad Proteins/metabolism , Spine/cytology , Animals , Cell Lineage/genetics , Cells, Cultured , Embryo, Mammalian , Gene Expression Regulation, Developmental , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Interneurons/cytology , Interneurons/physiology , Mice , Neural Crest/cytology , Neural Crest/physiology , Neural Tube/cytology , Neural Tube/embryology , Neurons/cytology , Neurons/physiology , Organoids/cytology , Signal Transduction/genetics , Smad Proteins/geneticsABSTRACT
Formation of functional motor circuits relies on the ability of distinct spinal motor neuron subtypes to project their axons with high precision to appropriate muscle targets. While guidance cues contributing to motor axon pathfinding have been identified, the intracellular pathways underlying subtype-specific responses to these cues remain poorly understood. In particular, it remains controversial whether responses to axon guidance cues depend on axonal protein synthesis. Using a growth cone collapse assay, we demonstrate that mouse embryonic stem cell-derived spinal motor neurons (ES-MNs) respond to ephrin-A5, Sema3f, and Sema3a in a concentration-dependent manner. At low doses, ES-MNs exhibit segmental or subtype-specific responses, while this selectivity is lost at higher concentrations. Response to high doses of semaphorins and to all doses of ephrin-A5 is protein synthesis independent. In contrast, using microfluidic devices and stripe assays, we show that growth cone collapse and guidance at low concentrations of semaphorins rely on local protein synthesis in the axonal compartment. Similar bimodal response to low and high concentrations of guidance cues is observed in human ES-MNs, pointing to a general mechanism by which neurons increase their repertoire of responses to the limited set of guidance cues involved in neural circuit formation.
Subject(s)
Axons/physiology , Cues , Motor Neurons/physiology , Protein Biosynthesis/physiology , Animals , Axons/metabolism , Cells, Cultured , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Ephrin-A5/administration & dosage , Ephrin-A5/physiology , Growth Cones/pathology , Growth Cones/physiology , Humans , Male , Membrane Proteins/administration & dosage , Membrane Proteins/physiology , Mice , Motor Neurons/classification , Nerve Tissue Proteins/administration & dosage , Nerve Tissue Proteins/physiology , Semaphorin-3A , Signal Transduction/physiology , Spinal Cord/cytology , Spinal Cord/physiologyABSTRACT
Neurulation is a critical step in early embryonic development, giving rise to the neural tube, the primordium of the central nervous system in amniotes. Understanding this complex, multi-scale, multi-tissue morphogenetic process is essential to provide insights into normal development and the etiology of neural tube defects. Innovations in tissue engineering have fostered the generation of pluripotent stem cell-based in vitro models, including organoids, that are emerging as unique tools for delving into neurulation mechanisms, especially in the context of human development. Each model captures specific aspects of neural tube morphogenesis, from epithelialization to neural tissue elongation, folding and cavitation. In particular, the recent models of human and mouse trunk morphogenesis, such as gastruloids, that form a spinal neural plate-like or neural tube-like structure are opening new avenues to study normal and pathological neurulation. Here, we review the morphogenetic events generating the neural tube in the mammalian embryo and questions that remain unanswered. We discuss the advantages and limitations of existing in vitro models of neurulation and possible future technical developments.
Subject(s)
Neural Tube Defects , Neurulation , Mice , Animals , Humans , Neurulation/physiology , Neural Tube , Neural Plate , Stem Cells , MammalsABSTRACT
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.
Subject(s)
Motor Neurons , Transcriptome , Animals , Mice , Humans , Transcriptome/genetics , Motor Neurons/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Muscle, Skeletal/metabolism , Embryo, Mammalian/metabolism , Spinal Cord/metabolism , Mammals/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Integrated in vitro models of human organogenesis are needed to elucidate the multi-systemic events underlying development and disease. Here we report the generation of human trunk-like structures that model the co-morphogenesis, patterning and differentiation of the human spine and spinal cord. We identified differentiation conditions for human pluripotent stem cells favoring the formation of an embryo-like extending antero-posterior (AP) axis. Single-cell and spatial transcriptomics show that somitic and spinal cord differentiation trajectories organize along this axis and can self-assemble into a neural tube surrounded by somites upon extracellular matrix addition. Morphogenesis is coupled with AP patterning mechanisms, which results, at later stages of organogenesis, in in vivo-like arrays of neural subtypes along a neural tube surrounded by spine and muscle progenitors contacted by neuronal projections. This integrated system of trunk development indicates that in vivo-like multi-tissue co-morphogenesis and topographic organization of terminal cell types can be achieved in human organoids, opening windows for the development of more complex models of organogenesis.
ABSTRACT
The connectivity patterns of neurons sustaining the functionality of spinal locomotor circuits rely on the specification of hundreds of motor neuron and interneuron subtypes precisely arrayed within the embryonic spinal cord. Knowledge acquired by developmental biologists on the molecular mechanisms underpinning this process in vivo has supported the development of 2D and 3D differentiation strategies to generate spinal neuronal diversity from mouse and human pluripotent stem cells (PSCs). Here, we review recent breakthroughs in this field and the perspectives opened up by models of in vitro embryogenesis to approach the mechanisms underlying neuronal diversification and the formation of functional mouse and human locomotor circuits. Beyond serving fundamental investigations, these new approaches should help engineering neuronal circuits differentially impacted in neuromuscular disorders, such as amyotrophic lateral sclerosis or spinal muscular atrophies, and thus open new avenues for disease modeling and drug screenings.
Subject(s)
Amyotrophic Lateral Sclerosis , Spinal Cord , Animals , Interneurons , Mammals , Mice , Motor NeuronsABSTRACT
Assembly of functional neural circuits relies on the ability of axons to navigate a complex landscape of guidance cues in the extracellular environment. In this report, we investigate localized cell signaling in response to these cues by combining a microfabricated compartmentalization chamber with multicomponent, protein-micropatterned surfaces; this system offers improved spatial resolution and new capabilities for targeted manipulation of neuronal axons. We illustrate the potential of this system by addressing the role of fibroblast growth factor receptor (FGFR) signaling in modulating axon guidance by N-cadherin. Motor neurons that were derived from embryonic stem cells extend axons from one compartment through a microchannel barrier and into a second compartment containing patterns of N-cadherin, against a background of laminin. N-cadherin was effective in both guiding and accelerating motor axon outgrowth. Using the chamber system to target the application of pharmacological agents to specific parts of the neuron, we demonstrate that FGFR signaling in the axon but not the cell body increases the rate of axon outgrowth while not affecting guidance along N-cadherin. These results demonstrate that cell signaling must take into account the spatial layout of the cell. This new platform provides a powerful tool for understanding such effects over a wide range of signaling systems.
Subject(s)
Biological Assay/instrumentation , Cadherins/administration & dosage , Growth Cones/physiology , Microfluidic Analytical Techniques/instrumentation , Motor Neurons/physiology , Receptors, Fibroblast Growth Factor/chemistry , Receptors, Fibroblast Growth Factor/metabolism , Animals , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Flow Injection Analysis/instrumentation , Flow Injection Analysis/methods , Growth Cones/drug effects , Growth Cones/ultrastructure , Mice , Mice, Transgenic , Microfluidic Analytical Techniques/methods , Motor Neurons/drug effects , Motor Neurons/ultrastructure , Protein BindingABSTRACT
Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.
Subject(s)
Gene Expression , Gene Transfer Techniques , Neural Stem Cells , Transgenes , Animals , Cell Lineage , Genetic Vectors , Humans , MiceABSTRACT
A growing number of specific cell types have been successfully derived from embryonic stem cells (ES cells), including a variety of neural cells. In vitro generated cells need to be extensively characterized to establish functional equivalency with their in vivo counterparts. The ultimate test for the ability of ES cell-derived neurons to functionally integrate into neural networks is transplantation into the developing central nervous system, a challenging technique limited by the poor accessibility of mammalian embryos. Here we describe xenotransplantation of mouse embryonic stem cell-derived motor neurons into the developing chick neural tube as an alternative for testing the ability of in vitro generated neurons to survive, integrate, extend axons, and form appropriate synaptic contacts with functionally relevant targets in vivo. Similar methods can be adapted to study functionality of other mammalian cells, including derivatives of human ES cells.
Subject(s)
Embryonic Stem Cells/cytology , Motor Neurons/transplantation , Spinal Cord/cytology , Spinal Cord/embryology , Stem Cell Transplantation/methods , Transplantation, Heterologous/methods , Animals , Cell Differentiation , Cells, Cultured , Chickens , Mice , Neural Tube/cytologyABSTRACT
Specification of cell identity during development depends on exposure of cells to sequences of extrinsic cues delivered at precise times and concentrations. Identification of combinations of patterning molecules that control cell fate is essential for the effective use of human pluripotent stem cells (hPSCs) for basic and translational studies. Here we describe a scalable, automated approach to systematically test the combinatorial actions of small molecules for the targeted differentiation of hPSCs. Applied to the generation of neuronal subtypes, this analysis revealed an unappreciated role for canonical Wnt signaling in specifying motor neuron diversity from hPSCs and allowed us to define rapid (14 days), efficient procedures to generate spinal and cranial motor neurons as well as spinal interneurons and sensory neurons. Our systematic approach to improving hPSC-targeted differentiation should facilitate disease modeling studies and drug screening assays.
Subject(s)
Combinatorial Chemistry Techniques , Neurons/cytology , Pluripotent Stem Cells/cytology , Cell Differentiation , HumansABSTRACT
The fundamental inaccessibility of the human neural cell types affected by neurological disorders prevents their isolation for in vitro studies of disease mechanisms or for drug screening efforts. Pluripotent stem cells represent a new interesting way to generate models of human neurological disorders, explore the physiopathological mechanisms and develop new therapeutic strategies. Disease-specific human embryonic stem cells were the first source of material to be used to study certain disease states. The recent demonstration that human somatic cells, such as fibroblasts or blood cells, can be genetically converted to induced pluripotent stem cells (hiPSCs) together with the continuous improvement of methods to differentiate these cells into disease-affected neuronal subtypes opens new perspectives to model and understand a large number of human pathologies. This review focuses on the opportunities concerning the use disease-specific human pluripotent stem cells as well as the different challenges that still need to be overcome. We also discuss the recent improvements in the genetic manipulation of human pluripotent stem cells and the consequences of these on disease modeling and drug screening for neurological diseases.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells , Nervous System Diseases/therapy , Fibroblasts/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/transplantation , Nervous System Diseases/genetics , Nervous System Diseases/pathology , Neurons/cytology , Neurons/metabolismABSTRACT
Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation-sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type-specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.
Subject(s)
Cell Differentiation/physiology , Motor Neurons/physiology , Stem Cells/physiology , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryo, Mammalian , Embryonic Stem Cells , Gene Expression , Gene Expression Profiling , Homeodomain Proteins/metabolism , Ki-67 Antigen/metabolism , LIM-Homeodomain Proteins/metabolism , Mice , Motor Neurons/cytology , Nerve Tissue Proteins/metabolism , Oligodendrocyte Transcription Factor 2 , Protein Binding/drug effects , Protein Binding/genetics , Protein Structure, Tertiary , Spinal Cord/cytology , Stem Cells/cytology , Stem Cells/drug effects , Time Factors , Transcription Factors/geneticsABSTRACT
Engrailed1 (En1) is a homeoprotein transcription factor expressed throughout adulthood in several midbrain cells, including the dopaminergic neurons of the substantia nigra. Here we report the presence of Engrailed protein and En1 mRNA in proximal dendrites of these neurons and of En1 mRNA in ventral midbrain synaptoneurosomes. We show that the 3' untranslated region of En1 mRNA contains a functional cytoplasmic polyadenylation element (CPE), suggesting that its dendritic localization is regulated by CPE binding protein (CPEB). In order to evaluate activity-regulated translation, conditions were developed using primary midbrain neurons. With this in vitro model, En1 mRNA translation is increased by depolarization in a polyadenylation dependent manner. Furthermore, En1 translation is prevented by rapamycin, implicating the mTOR pathway, which is known to regulate dendritic translation. Together, these results suggest an activity-dependent role for Engrailed in midbrain dopaminergic neuron physiology.
Subject(s)
Dendrites/metabolism , Homeodomain Proteins/metabolism , Neurons/cytology , Animals , Cells, Cultured , Electrophoretic Mobility Shift Assay/methods , Embryo, Mammalian , Gene Expression/physiology , Homeodomain Proteins/genetics , In Situ Hybridization/methods , Mesencephalon/cytology , Mice , Protein Biosynthesis , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Synaptosomes/metabolismABSTRACT
The main organization and gross morphology of the mammalian olfactory primary pathway, from the olfactory epithelium to the olfactory bulb, has been initially characterized using classical anatomical and ultrastructural approaches. During the last fifteen years, essentially thanks to the cloning of the odorant receptor genes, and to the characterization of a number of molecules expressed by the olfactory sensory neuron axons and their environment, significant new insights have been gained into the understanding of the development and adult functioning of this system. In the course of these genetic, biochemical and neuroanatomical studies, however, several molecular and structural features were uncovered that appear somehow to be unique to these axons. For example, these axons express odorant receptors in their terminal segment, and transport several mRNA species and at least two transcription factors. In the present paper, we review these unusual structural and molecular features and speculate about their possible functions in the development and maintenance of the olfactory system.
Subject(s)
Axons/physiology , Axons/ultrastructure , Olfactory Mucosa/innervation , Olfactory Receptor Neurons/cytology , Olfactory Receptor Neurons/physiology , Animals , Axons/chemistry , Gene Expression Regulation , Olfactory Bulb/chemistry , Olfactory Bulb/cytology , Olfactory Bulb/physiology , Olfactory Pathways/chemistry , Olfactory Pathways/cytology , Olfactory Pathways/physiology , Olfactory Receptor Neurons/chemistry , Protein Biosynthesis , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/physiology , Receptors, Odorant/analysis , Receptors, Odorant/genetics , Receptors, Odorant/physiology , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
We report that Emx2 homeogene is expressed at the mRNA and protein levels in the adult mouse olfactory neuroepithelium. As expected for a transcription factor, Emx2 is present in the nucleus of immature and mature olfactory sensory neurons. However, the protein is also detected in the axonal compartment of these neurons, both in the olfactory mucosa axon bundles and in axon terminals within the olfactory bulb. Emx2 axonal staining is heterogeneous, suggesting an association with particles. Subcellular fractionations of olfactory bulb synaptosomes, combined with chemical lesions of olfactory neurons, confirm the presence of Emx2 in axon terminals. Significant amounts of Emx2 protein cosediment with high density synaptosomal subfractions containing eukaryotic translation initiation factor 4E (eIF4E). Nonionic detergents and RNase treatments failed to detach eIF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and eIF4E can be coimmunoprecipitated from olfactory mucosa and bulb extracts and interact directly, as demonstrated in pull-down experiments. Emx2 axonal localization, association with high-density particles and interaction with eIF4E strongly suggest that this transcription factor has new nonnuclear functions most probably related to the local control of protein translation in the olfactory sensory neuron axons. Finally, we show that two other brain-expressed homeoproteins, Otx2 and Engrailed 2, also bind eIF4E, indicating that several homeoproteins may modulate eIF4E functions in the developing and adult nervous system.