Bone Morphogenetic Proteins (BMP) 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
, whether this is sufficient for pattern formation
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 cells 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 the study describes efficient methods to generate 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.
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.
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IJS, NUK, SBMB, UL, UM, UPUK
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
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► An ESC-specific splicing switch in FOXP1 transcripts produces the FOXP1-ES isoform ► FOXP1-ES has distinct DNA-binding properties compared to the canonical FOXP1 isoform ► FOXP1-ES stimulates key pluripotency genes and represses many differentiation genes ► FOXP1-ES is required for ESC pluripotency and efficient iPSC reprogramming
Alternative splicing produces an ESC-specific isoform of FOXP1 that represses genes responsible for differentiation and directly stimulates production of pluripotency genes, including
Oct4 and
Nanog
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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•A gene expression switch powered by genomic integration•Accelerated readout of additive transgenesis with one or multiple vectors•Faithful lineage tracing and mosaic analysis by somatic transfection•Near-universal applicability in cultured cells and animal models
Kumamoto et al. introduce iOn, a genetic switch that conditions exogenous transgene expression to integration in the host cell genome by DNA transposition. This system radically simplifies stable transgenesis with one or multiple plasmid vectors, opening new options to genetically manipulate cells in cultured systems and model organisms.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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 (elF4E). Nonionic detergents and RNase treatments failed to detach elF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and elF4E 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 elF4E 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 elF4E, indicating that several homeoproteins may modulate elF4E functions in the developing and adult nervous system.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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