ABSTRACT
Epithelial-Mesenchymal Transition (EMT) is a key process in physiological and pathological settings. EMT is often presented as a linear sequence with (i) disassembly of cell-cell junctions, (ii) loss of epithelial polarity and (iii) reorganization of the cytoskeleton leading to basal extrusion from the epithelium. Once out, cells can adopt a migratory phenotype with a front-rear polarity. While this sequence can occur, in vivo observations have challenged it. It is now accepted that multiple EMT scenarios coexist in heterogeneous cell populations. However, the relative importance of each step as well as that of variability and heterogeneity on the efficiency of cell extrusion has not been assessed. Here we used computational modelling to simulate multiple EMT-like scenarios and confronted these data to the EMT of neural crest cells. Overall, our data point to a key role of nuclear positioning and protrusive activity to generate timely basal extrusion.
Subject(s)
Cell Nucleus , Epithelial-Mesenchymal Transition , Cell Nucleus/metabolism , Animals , Models, Biological , Computer Simulation , Neural Crest/cytology , Cell Movement/physiology , Humans , Cell Polarity/physiology , Cytoskeleton/metabolism , Epithelial Cells/metabolism , Epithelial Cells/cytologyABSTRACT
The neural tube is the precursor of the central nervous system. Its early formation and growth are known to be extremely biased along the anteroposterior (AP) axis. Several mechanisms including addition of cells from the tail bud, lateral pressure from surrounding tissues and oriented cell divisions have been proposed to contribute to this biased growth. Here we show that, contrary to what has been found in posterior regions encompassing the tail bud region, the growth of the anterior trunk neural tube is slower along the AP direction than in the other axes. We found that this is due to anchorage of the neural tube to the matrix which favors apicobasal elongation at the expense of AP growth. In addition, as the neural tube develops, we found a moderate slowdown of cell proliferation that could account for the overall reduction of the pace of 3D growth in the same time window. However, as we found no preferred orientation of cell division, changes in cell cycle pace are unlikely to directly contribute to the observed AP-hindered growth of neural tube. Overall, these data indicate that neural tube growth is not intrinsically positively biased along the AP axis. Rather it switches from AP-favored to AP-hindered regimes between the most posterior and anterior trunk neural tube regions.
Subject(s)
Chickens , Neural Tube , Animals , Cell Division , Central Nervous System , Mesoderm , Neural Tube/metabolismABSTRACT
Sulf2a belongs to the Sulf family of extracellular sulfatases which selectively remove 6-O-sulfate groups from heparan sulfates, a critical regulation level for their role in modulating the activity of signalling molecules. Data presented here define Sulf2a as a novel player in the control of Sonic Hedgehog (Shh)-mediated cell type specification during spinal cord development. We show that Sulf2a depletion in zebrafish results in overproduction of V3 interneurons at the expense of motor neurons and also impedes generation of oligodendrocyte precursor cells (OPCs), three cell types that depend on Shh for their generation. We provide evidence that Sulf2a, expressed in a spatially restricted progenitor domain, acts by maintaining the correct patterning and specification of ventral progenitors. More specifically, Sulf2a prevents Olig2 progenitors to activate high-threshold Shh response and, thereby, to adopt a V3 interneuron fate, thus ensuring proper production of motor neurons and OPCs. We propose a model in which Sulf2a reduces Shh signalling levels in responding cells by decreasing their sensitivity to the morphogen factor. More generally, our work, revealing that, in contrast to its paralog Sulf1, Sulf2a regulates neural fate specification in Shh target cells, provides direct evidence of non-redundant functions of Sulfs in the developing spinal cord.
Subject(s)
Hedgehog Proteins/metabolism , Spinal Cord/growth & development , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Gene Expression Regulation, Developmental , Hedgehog Proteins/genetics , Heparitin Sulfate/metabolism , Interneurons/metabolism , Oligodendrocyte Precursor Cells/metabolism , Oligodendroglia/metabolism , Signal Transduction , Spinal Cord/metabolism , Sulfatases/genetics , Sulfatases/metabolism , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/geneticsABSTRACT
Generation of glial cell diversity in the developing spinal cord is known to depend on spatio-temporal patterning programs. In particular, expression of the transcription factor Olig2 in neural progenitors of the pMN domain is recognized as critical to their fate choice decision to form oligodendrocyte precursor cells (OPCs) instead of astrocyte precursors (APs). However, generating some confusion, lineage-tracing studies of Olig2 progenitors in the spinal cord provided evidence that these progenitors also generate some astrocytes. Here, we addressed the role of the heparan sulfate-editing enzyme Sulf2 in the control of gliogenesis and found an unanticipated function for this enzyme. At initiation of gliogenesis in mouse, Sulf2 is expressed in ventral neural progenitors of the embryonic spinal cord, including in Olig2-expressing cells of the pMN domain. We found that sulf2 deletion, while not affecting OPC production, impairs generation of a previously unknown Olig2-expressing pMN-derived cell subtype that, in contrast to OPCs, does not upregulate Sox10, PDGFRα or Olig1. Instead, these cells activate expression of AP identity genes, including aldh1L1 and fgfr3 and, of note, retain Olig2 expression as they populate the spinal parenchyma at embryonic stages but also as they differentiate into mature astrocytes at postnatal stages. Thus, our study, by revealing the existence of Olig2-expressing APs that segregate early from pMN cells under the influence of Sulf2, supports the existence of a common source of APs and OPCs in the ventral spinal cord and highlights divergent regulatory mechanism for the development of pMN-derived OPCs and APs.
Subject(s)
Astrocytes/enzymology , Oligodendrocyte Transcription Factor 2/metabolism , Spinal Cord/enzymology , Sulfatases/metabolism , Animals , Astrocytes/cytology , Gray Matter/cytology , Gray Matter/enzymology , Gray Matter/growth & development , Mice, Transgenic , Neural Stem Cells/cytology , Neural Stem Cells/enzymology , Neurogenesis/physiology , Receptor, Fibroblast Growth Factor, Type 3/metabolism , SOXE Transcription Factors/metabolism , Spinal Cord/cytology , Spinal Cord/growth & development , Sulfatases/geneticsABSTRACT
BACKGROUND: Most oligodendrocytes of the spinal cord originate from ventral progenitor cells of the pMN domain, characterized by expression of the transcription factor Olig2. A minority of oligodendrocytes is also recognized to emerge from dorsal progenitors during fetal development. The prevailing view is that generation of ventral oligodendrocytes depends on Sonic hedgehog (Shh) while dorsal oligodendrocytes develop under the influence of Fibroblast Growth Factors (FGFs). RESULTS: Using the well-established model of the chicken embryo, we show that ventral spinal progenitor cells activate FGF signaling at the onset of oligodendrocyte precursor cell (OPC) generation. Inhibition of FGF receptors at that time appears sufficient to prevent generation of ventral OPCs, highlighting that, in addition to Shh, FGF signaling is required also for generation of ventral OPCs. We further reveal an unsuspected interplay between Shh and FGF signaling by showing that FGFs serve dual essential functions in ventral OPC specification. FGFs are responsible for timely induction of a secondary Shh signaling center, the lateral floor plate, a crucial step to create the burst of Shh required for OPC specification. At the same time, FGFs prevent down-regulation of Olig2 in pMN progenitor cells as these cells receive higher threshold of the Shh signal. Finally, we bring arguments favoring a key role of newly differentiated neurons acting as providers of the FGF signal required to trigger OPC generation in the ventral spinal cord. CONCLUSION: Altogether our data reveal that the FGF signaling pathway is activated and required for OPC commitment in the ventral spinal cord. More generally, our data may prove important in defining strategies to produce large populations of determined oligodendrocyte precursor cells from undetermined neural progenitors, including stem cells. In the long run, these new data could be useful in attempts to stimulate the oligodendrocyte fate in residing neural stem cells.
Subject(s)
Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental/physiology , Hedgehog Proteins/metabolism , Oligodendroglia/metabolism , Signal Transduction/physiology , Spinal Cord/cytology , Animals , Chick Embryo , Electroporation , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation, Developmental/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hedgehog Proteins/genetics , In Vitro Techniques , Nerve Tissue Proteins , Oligodendrocyte Transcription Factor 2/metabolism , Organ Culture Techniques , Spinal Cord/embryology , Stem Cells/physiologyABSTRACT
A substantial amount of data has highlighted the crucial influence of Shh signalling on the generation of diverse classes of neurons and glial cells throughout the developing central nervous system. A critical step leading to this diversity is the establishment of distinct neural progenitor cell domains during the process of pattern formation. The forming spinal cord, in particular, has served as an excellent model to unravel how progenitor cells respond to Shh to produce the appropriate pattern. In recent years, considerable advances have been made in our understanding of important parameters that control the temporal and spatial interpretation of the morphogen signal at the level of Shh-receiving progenitor cells. Although less studied, the identity and position of Shh source cells also undergo significant changes over time, raising the question of how moving the Shh source contributes to cell diversification in response to the morphogen. Here, we focus on the dynamics of Shh-producing cells and discuss specific roles for these time-variant Shh sources with regard to the temporal events occurring in the receiving field.
ABSTRACT
In the ventral spinal cord, generation of neuronal and glial cell subtypes is controlled by Sonic hedgehog (Shh). This morphogen contributes to cell diversity by regulating spatial and temporal sequences of gene expression during development. Here, we report that establishing Shh source cells is not sufficient to induce the high-threshold response required to specify sequential generation of ventral interneurons and oligodendroglial cells at the right time and place in zebrafish. Instead, we show that Shh-producing cells must repeatedly upregulate the secreted enzyme Sulfatase1 (Sulf1) at two critical time points of development to reach their full inductive capacity. We provide evidence that Sulf1 triggers Shh signaling activity to establish and, later on, modify the spatial arrangement of gene expression in ventral neural progenitors. We further present arguments in favor of Sulf1 controlling Shh temporal activity by stimulating production of active forms of Shh from its source. Our work, by pointing out the key role of Sulf1 in regulating Shh-dependent neural cell diversity, highlights a novel level of regulation, which involves temporal evolution of Shh source properties.
Subject(s)
Hedgehog Proteins/metabolism , Spinal Cord/embryology , Spinal Cord/metabolism , Sulfatases/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Body Patterning/genetics , Body Patterning/physiology , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Knockdown Techniques , Hedgehog Proteins/deficiency , Hedgehog Proteins/genetics , Mice , Neural Stem Cells/classification , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurogenesis/physiology , Signal Transduction , Spinal Cord/cytology , Sulfatases/genetics , Sulfotransferases/genetics , Sulfotransferases/metabolism , Zebrafish/genetics , Zebrafish Proteins/deficiency , Zebrafish Proteins/geneticsABSTRACT
In the developing ventral spinal cord, motor neurons (MNs) and oligodendrocyte precursor cells (OPCs) are sequentially generated from a common pool of neural progenitors included in the so-called pMN domain characterized by Olig2 expression. Here, we establish that the secreted Sulfatase 1 (Sulf1) is a major component of the mechanism that causes these progenitors to stop producing MNs and change their fate to generate OPCs. We show that specification of OPCs is severely affected in sulf1-deficient mouse embryos. This defect does not rely on abnormal patterning of the spinal cord or failure in maintenance of pMN progenitors at the onset of OPC specification. Instead, the efficiency of OPC induction is reduced, only few Olig2 progenitors are recruited to generate OPCs, meanwhile they continue to produce MNs beyond the normal timing of the neuroglial switch. Using the chicken embryo, we show that Sulf1 activity is required precisely at the stage of the MN-to-OPC fate switch. Finally, we bring arguments supporting the view that Sulf1 controls the level of Sonic Hedgehog (Shh) signaling activity, behaving as an enhancer rather than an obligatory component in the Shh pathway. Our study provides additional insights into the temporal control of Olig2 progenitor cell fate change by the identification of Sulf1 as an extracellular timing signal in the ventral spinal cord.
Subject(s)
Cell Differentiation/physiology , Hedgehog Proteins/metabolism , Motor Neurons/cytology , Oligodendroglia/cytology , Spinal Cord/embryology , Sulfotransferases/metabolism , Animals , Electroporation , Fluorescent Antibody Technique , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Motor Neurons/enzymology , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Oligodendroglia/enzymology , Signal Transduction/physiology , Spinal Cord/metabolismABSTRACT
In the embryonic chick ventral spinal cord, the initial emergence of oligodendrocytes is a relatively late event that depends on prolonged Sonic hedgehog (Shh) signaling. In this report, we show that specification of oligodendrocyte precursors (OLPs) from ventral Nkx2.2-expressing neural progenitors occurs precisely when these progenitors stop generating neurons, indicating that the mechanism of the neuronal/oligodendroglial switch is a common feature of ventral OLP specification. We further show that an experimental early increase in the concentration of Shh is sufficient to induce premature specification of OLPs at the expense of neuronal genesis indicating that the relative doses of Shh received by ventral progenitors determine whether they become neurons or glia. Accordingly, we observe that the Shh protein accumulates at the apical surface of Nkx2.2-expressing cells just before OLP specification, providing direct evidence that these cells are subjected to a higher concentration of the morphogen when they switch to an oligodendroglial fate. Finally, we show that this abrupt change in Shh distribution is most likely attributable to the timely activity of Sulfatase 1 (Sulf1), a secreted enzym that modulates the sulfation state of heparan sulfate proteoglycans. Sulf1 is expressed in the ventral neuroepithelium just before OLP specification, and we show that its experimental overexpression leads to apical concentration of Shh on neuroepithelial cells, a decisive event for the switch of ventral neural progenitors toward an oligodendroglial fate.
Subject(s)
Oligodendroglia/cytology , Oligodendroglia/metabolism , Spinal Cord/cytology , Spinal Cord/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Sulfotransferases/metabolism , Trans-Activators/metabolism , Animals , Cell Differentiation , Cells, Cultured , Chick Embryo , Hedgehog Proteins , Homeobox Protein Nkx-2.2 , Homeodomain Proteins , Neurons/cytology , Neurons/metabolism , Nuclear Proteins , Signal Transduction/physiology , Transcription FactorsABSTRACT
In the developing spinal cord, oligodendrocyte progenitors (OLPs) originate from the ventral neuroepithelium and the specification of this lineage depends on the inductive activity of Sonic hedgehog (Shh) produced by ventral midline cells. On the other hand, it has been shown that OLP identity is acquired by the coexpression of the transcription factors olig2 and nkx2.2. Although initially expressed in adjacent nonoverlapping domains of the ventral neuroepithelium, these transcription factors become coexpressed in the pMN domain at the time of OLP specification through dorsal extension of the Nkx2.2 domain. Here we show that Shh is sufficient to promote the coexpression of Olig2 and Nkx2.2 in neuroepithelial cells. In addition, Shh activity is necessary for this coexpression since blocking Shh signalling totally abolishes Olig2 expression and impedes dorsal extension of Nkx2.2. Although Shh at these stages affects neuroepithelial cell proliferation, the dorsal extension of the Nkx2.2 domain is not due to progenitor proliferation but to repatterning of the ventral neuroepithelium. Finally, Shh not only stimulates OLP specification but also simultaneously restricts the ventral extension of the astrocyte progenitor (AP) domain and reduces astrocyte development. We propose that specification of distinct glial lineages is the result of a choice that depends on Shh signalling.
Subject(s)
Astrocytes/physiology , Gene Expression Regulation, Developmental , Oligodendroglia/physiology , Signal Transduction , Spinal Cord/embryology , Trans-Activators/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors , Bromodeoxyuridine , Chick Embryo , DNA Primers , Hedgehog Proteins , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , In Situ Nick-End Labeling , Nerve Tissue Proteins/metabolism , Nuclear Proteins , Trans-Activators/physiology , Transcription Factors/metabolism , Zebrafish ProteinsABSTRACT
To address the question of the origin of glial cells and the mechanisms leading to their specification, we have sought to identify novel genes expressed in glial progenitors. We adopted suppression subtractive hybridization (SSH) to establish a chick cDNA library enriched for genes specifically expressed at 6 days of incubation (E6) in the ventral neuroepithelium, a tissue previously shown to contain glial progenitors. Screens were then undertaken to select differentially expressed cDNAs, and out of 82 unique SSH clones, 21 were confirmed to display a regionalized expression along the dorsoventral axis of the E6 ventral neuroepithelium. Among these, we identified a transcript coding for the chick orthologue of Sulf1, a recently identified cell surface sulfatase, as a new, early marker of oligodendrocyte (OL) precursors in the chick embryonic spinal cord. This study provides groundwork for the further identification of genes involved in glial specification.