Roof Plate Cells Research Articles

Recent advances in understanding cell type transitions during dorsal neural tube development.

The vertebrate neural tube is a representative example of a morphogen-patterned tissue that generates different cell types with spatial and temporal precision. More specifically, the development of the dorsal region of the neural tube is of particular interest because of its highly dynamic behavior. First, early premigratory neural crest progenitors undergo an epithelial-to-mesenchymal transition, exit the neural primordium, and generate, among many derivatives, most of the peripheral nervous system. Subsequently, the dorsal neural tube becomes populated by definitive roof plate cells that constitute an organizing center for dorsal interneurons and guide axonal patterning. In turn, roof plate cells transform into dorsal radial glia that contributes to and shapes the formation of the dorsal ependyma of the central nervous system.To form a normal functional spinal cord, these extraordinary transitions should be tightly regulated in time and space. Thus far, the underlying cellular changes and molecular mechanisms are only beginning to be uncovered. In this review, we discuss recent results that shed light on the end of neural crest production and delamination, the early formation of the definitive roof plate, and its further maturation into radial glia. The last of these processes culminate in the formation of the dorsal ependyma, a component of the stem cell niche of the central nervous system. We highlight how similar mechanisms operate throughout these transitions, which may serve to reveal common design principles applicable to the ontogeny of epithelial tissues.

Morphological and Functional Changes of Roof Plate Cells in Spinal Cord Development.

The most dorsal region, or roof plate, is the dorsal organizing center of developing spinal cord. This region is also involved in development of neural crest cells, which are the source of migratory neural crest cells. During early development of the spinal cord, roof plate cells secrete signaling molecules, such as Wnt and BMP family proteins, which regulate development of neural crest cells and dorsal spinal cord. After the dorso-ventral pattern is established, spinal cord dynamically changes its morphology. With this morphological transformation, the lumen of the spinal cord gradually shrinks to form the central canal, a cavity filled with cerebrospinal fluid that is connected to the ventricular system of the brain. The dorsal half of the spinal cord is separated by a glial structure called the dorsal (or posterior) median septum. However, underlying mechanisms of such morphological transformation are just beginning to be understood. Recent studies reveal that roof plate cells dramatically stretch along the dorso-ventral axis, accompanied by reduction of the spinal cord lumen. During this stretching process, the tips of roof plate cells maintain contact with cells surrounding the shrinking lumen, eventually exposed to the inner surface of the central canal. Interestingly, Wnt expression remains in stretched roof plate cells and activates Wnt/β-catenin signaling in ependymal cells surrounding the central canal. Wnt/β-catenin signaling in ependymal cells promotes proliferation of neural progenitor and stem cells in embryonic and adult spinal cord. In this review, we focus on the role of the roof plate, especially that of Wnt ligands secreted by roof plate cells, in morphological changes occurring in the spinal cord.

Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord.

Cell morphology changes dynamically during embryogenesis, and these changes create new interactions with surrounding cells, some of which are presumably mediated by intercellular signaling. However, the effects of morphological changes on intercellular signaling remain to be fully elucidated. In this study, we examined the effect of morphological changes in Wnt-producing cells on intercellular signaling in the spinal cord. After mid-gestation, roof-plate cells stretched along the dorsoventral axis in the mouse spinal cord, resulting in new contact at their tips with the ependymal cells that surround the central canal. Wnt1 and Wnt3a were produced by the stretched roof-plate cells and delivered to the cell process tip. Whereas Wnt signaling was activated in developing ependymal cells, Wnt activation in dorsal ependymal cells, which were close to the stretched roof plate, was significantly suppressed in embryos with roof plate-specific conditional knockout of Wls, which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in Wls conditional knockout mice during development and after induced spinal cord injury in adults. Therefore, morphological changes in Wnt-producing cells appear to generate new Wnt signal targets.

Xenopus laevis nucleotide binding protein 1 (xNubp1) is important for convergent extension movements and controls ciliogenesis via regulation of the actin cytoskeleton

Nucleotide binding protein 1 (Nubp1) is a highly conserved phosphate loop (P-loop) ATPase involved in diverse processes including iron–sulfur protein assembly, centrosome duplication and lung development. Here, we report the cloning, expression and functional characterization of Xenopus laevis Nubp1. We show that xNubp1 is expressed maternally, displays elevated expression in neural tissues and is required for convergent extension movements and neural tube closure. In addition, xNubp1knockdown leads to defective ciliogenesis of the multi-ciliated cells of the epidermis as well as the monociliated cells of the gastrocoel roof plate. Specifically, xNubp1 is required for basal body migration, spacing and docking in multi-ciliated cells and basal body positioning and axoneme elongation in monociliated gastrocoel roof plate cells. Live imaging of the different pools of actin and basal body migration during the process of ciliated cell intercalation revealed that two independent pools of actin are present from the onset of cell intercalation; an internal network surrounding the basal bodies, anchoring them to the cell cortex and an apical pool of punctate actin which eventually matures into the characteristic apical actin network. We show that xNubp1 colocalizes with the apical actin network of multiciliated cells and that problems in basal body transport in xNubp1 morphants are associated with defects of the internal network of actin, while spacing and polarity issues are due to a failure of the apical and sub-apical actin pools to mature into a network. Effects of xNubp1 knockdown on the actin cytoskeleton are independent of RhoA localization and activation, suggesting that xNubp1 may have a direct role in the regulation of the actin cytoskeleton.

Stretching Morphogenesis of the Roof Plate and Formation of the Central Canal

BackgroundNeurulation is driven by apical constriction of actomyosin cytoskeleton resulting in conversion of the primitive lumen into the central canal in a mechanism driven by F-actin constriction, cell overcrowding and buildup of axonal tracts. The roof plate of the neural tube acts as the dorsal morphogenetic center and boundary preventing midline crossing by neural cells and axons.Methodology/Principal FindingsThe roof plate zebrafish transgenics expressing cytosolic GFP were used to study and describe development of this structure in vivo for a first time ever. The conversion of the primitive lumen into the central canal causes significant morphogenetic changes of neuroepithelial cells in the dorsal neural tube. We demonstrated that the roof plate cells stretch along the D–V axis in parallel with conversion of the primitive lumen into central canal and its ventral displacement. Importantly, the stretching of the roof plate is well-coordinated along the whole spinal cord and the roof plate cells extend 3× in length to cover 2/3 of the neural tube diameter. This process involves the visco-elastic extension of the roof place cytoskeleton and depends on activity of Zic6 and the Rho-associated kinase (Rock). In contrast, stretching of the floor plate is much less extensive.Conclusions/SignificanceThe extension of the roof plate requires its attachment to the apical complex of proteins at the surface of the central canal, which depends on activity of Zic6 and Rock. The D–V extension of the roof plate may change a range and distribution of morphogens it produces. The resistance of the roof plate cytoskeleton attenuates ventral displacement of the central canal in illustration of the novel mechanical role of the roof plate during development of the body axis.

Widespread Contribution of Gdf7 Lineage to Cerebellar Cell Types and Implications for Hedgehog-Driven Medulloblastoma Formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling.

Enforced expression of the transcription factor HOXD3 under the control of the Wnt1 regulatory element modulates cell adhesion properties in the developing mouse neural tube

HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.

The dorsal neural tube: A dynamic setting for cell fate decisions

The dorsal neural tube first generates neural crest cells that exit the neural primordium following an epithelial-to-mesenchymal conversion to become sympathetic ganglia, Schwann cells, dorsal root sensory ganglia, and melanocytes of the skin. Following the end of crest emigration, the dorsal midline of the neural tube becomes the roof plate, a signaling center for the organization of dorsal neuronal cell types. Recent lineage analysis performed before the onset of crest delamination revealed that the dorsal tube is a highly dynamic region sequentially traversed by fate-restricted crest progenitors. Furthermore, prospective roof plate cells were shown to originate ventral to presumptive crest and to progressively relocate dorsalward to occupy their definitive midline position following crest delamination. These data raise important questions regarding the mechanisms of cell emigration in relation to fate acquisition, and suggest the possibility that spatial and/or temporal information in the dorsal neural tube determines initial segregation of neural crest cells into their derivatives. In addition, they emphasize the need to address what controls the end of neural crest production and consequent roof plate formation, a fundamental issue for understanding the separation between central and peripheral lineages during development of the nervous system.

Wnt1 regulates neurogenesis and mediates lateral inhibition of boundary cell specification in the zebrafish hindbrain

The formation of localised signalling centres is essential for patterning of a number of tissues during development. Previous work has revealed that a distinct population of boundary cells forms at the interface of segments in the vertebrate hindbrain, but the role of these cells is not known. We have investigated the function of the Wnt1 signalling molecule that is expressed by boundary and roof plate cells in the zebrafish hindbrain. Knockdown of wnt1 or of tcf3b, a mediator of Wnt signalling, leads to ectopic expression of boundary cell markers, rfng and foxb1.2, in non-boundary regions of the hindbrain. Ectopic boundary marker expression also occurs following knockdown of rfng, a modulator of Notch signalling required for wnt1 expression at hindbrain boundaries. We show that the boundary and roof plate expression of wnt1 each contribute to upregulation of proneural and delta gene expression and neurogenesis in non-boundary regions, which in turn blocks ectopic boundary marker expression. Boundary cells therefore play a key role in the regulation of cell differentiation in the zebrafish hindbrain. The network of genes underlying the regulation of neurogenesis and lateral inhibition of boundary cell formation by Wnt1 has a striking similarity to mechanisms at the dorsoventral boundary in the Drosophila wing imaginal disc.

Control of roof plate formation by Lmx1a in the developing spinal cord.

Numerous studies have identified the roof plate as an embryonic signaling center critical for dorsal central nervous system patterning, but little is known about mechanisms that control its formation and its separation from clonally related neural crest cells and dI1 sensory interneurons. We demonstrate that the LIM homeodomain transcription factor, Lmx1a, mutated in the dreher mouse, acts to withdraw dorsal spinal cord progenitors from the cell cycle and simultaneously direct their differentiation into functional roof plate cells. Lmx1a cell-autonomously represses the dI1 progenitor fate, distinguishing the roof plate and dI1 interneuron programs, two major developmental programs of the dorsal neural tube. Lmx1a is not directly involved in neural crest development. We establish that Bmp signaling from epidermal ectoderm is necessary and sufficient for inducing Lmx1a and other co-factors that also regulate the extent of roof plate induction. We conclude that Lmx1a controls multiple aspects of dorsal midline patterning and is a major mediator of early Bmp signaling in the developing spinal cord.

Distinct activities of Msx1 and Msx3 in dorsal neural tube development.

Patterning of the dorsal neural tube involves Bmp signaling, which results in activation of multiple pathways leading to the formation of neural crest, roof plate and dorsal interneuron cell types. We show that constitutive activation of Bmp signaling at early stages (HH10-12) of chick neural tube development induces roof-plate cell fate, accompanied by an increase of programmed cell death and a repression of neuronal differentiation. These activities are mimicked by the overexpression of the homeodomain transcription factor Msx1, a factor known to be induced by Bmp signaling. By contrast, the closely related factor, Msx3, does not have these activities. At later stages of neural tube development (HH14-16), dorsal progenitor cells lose their competence to generate roof-plate cells in response to Bmp signaling and instead generate dorsal interneurons. This aspect of Bmp signaling is phenocopied by the overexpression of Msx3 but not Msx1. Taken together, these results suggest that these two different Msx family members can mediate distinct aspects of Bmp signaling during neural tube development.

A Role for BMP Heterodimers in Roof Plate-Mediated Repulsion of Commissural Axons

During spinal cord development, commissural neurons extend their axons ventrally, away from the roof plate. The roof plate is the source of a diffusible repellent that orients commissural axons in vitro and, thus, may regulate the trajectory of commissural axons in vivo. Of three Bmps expressed in the roof plate, BMP7, but not BMP6 or GDF7, mimics the roof plate activity in vitro. We show here that expression of both Bmp7 and Gdf7 by roof plate cells is required for the fidelity of commissural axon growth in vivo. We also demonstrate that BMP7 and GDF7 heterodimerize in vitro and that, under these conditions, GDF7 enhances the axon-orienting activity of BMP7. Our findings suggest that a GDF7:BMP7 heterodimer functions as a roof plate-derived repellent that establishes the initial ventral trajectory of commissural axons.

The specification of dorsal cell fates in the vertebrate central nervous system.

The generation of distinct classes of neurons at defined positions within the developing vertebrate nervous system depends on inductive signals provided by local cell groups that act as organizing centers. Genetic and embryological studies have begun to elucidate the processes that control the pattern and identity of neuronal cell types. Here we discuss the cellular interactions and molecular mechanisms that direct neuronal cell fates in the dorsal half of the vertebrate central nervous system. The specification of dorsal neuronal cell fates appears to depend on a cascade of inductive signals initiated by cells of the epidermal ectoderm that flank the neural plate and propagated by roof plate cells within the neural tube. Members of the transforming growth factor-beta (TGF beta) family of secreted proteins have a prominent role in mediating these dorsalizing signals. Additional signals involving members of the Wnt and fibroblast growth factor (FGF) families may also contribute to the proliferation and differentiation of dorsal neuronal cell types.

Neuronal patterning by BMPs: a requirement for GDF7 in the generation of a discrete class of commissural interneurons in the mouse spinal cord.

Inductive factors are known to direct the regional differentiation of the vertebrate central nervous system (CNS) but their role in the specification of individual neuronal cell types is less clear. We have examined the function of GDF7, a BMP family member expressed selectively by roof plate cells, in the generation of neuronal cell types in the dorsal spinal cord. We find that GDF7 can promote the differentiation in vitro of two dorsal sensory interneuron classes, D1A and D1B neurons. In Gdf7-null mutant embryos, the generation of D1A neurons is eliminated but D1B neurons and other identified dorsal interneurons are unaffected. These findings show that GDF7 is an inductive signal from the roof plate required for the specification of neuronal identity in the dorsal spinal cord and that GDF7 and other BMP family members expressed by the roof plate have non-redundant functions in vivo. More generally, these results suggest that BMP signaling may have a prominent role in the assignment of neuronal identity within the mammalian CNS.

A Role for the Roof Plate and Its Resident TGFβ-Related Proteins in Neuronal Patterning in the Dorsal Spinal Cord

Distinct neuronal cell types are generated at characteristic times and positions in the dorsal horn of the spinal cord. We provide evidence that the identity and pattern of generation of dorsal neurons depend initially on BMP-mediated signals that derive from the epidermal ectoderm and induce dorsal midline cells of the roof plate. Roof plate cells provide a secondary source of TGFβ-related signals that are required for the generation of distinct classes of dorsal interneurons. These inductive interactions involve both qualitative and quantitative differences in signaling by TGFβ-related factors and temporal changes in the response of neural progenitor cells.

Annexin IV is a marker of roof and floor plate development in the murine CNS

Midline structures, such as the notochord and floor plate, are crucial to the developing central nervous system (CNS). Previously, we demonstrated that annexin IV is an excellent marker of midline structures. In the present study, we explore the possible role of annexin IV in development of the CNS midline. Using immunocytochemistry with an antibody to annexin IV, we have elucidated the temporal and spatial expression of this molecule. Annexin IV is present in the notochord at embryonic day (E) 8.5, prior to its expression in any structures within the neural tube. Subsequently, annexin IV is expressed by floor plate cells at E9.5. Annexin IV is also expressed in the roof plate, but not until E10.5. To determine if normal morphogenesis of these midline structures is essential for annexin IV expression, we analyzed two strains of mutant mice that have defective formation of either the floor or the roof plate. In Danforth's short-tail mice, the floor plate is absent from the caudal spinal cord, and annexin IV immunopositivity disappears at the level where the floor plate is missing. In curly tail mutant mice, there can be a failure of the neural tube to close, and in these regions there is no annexin IV expression in presumptive roof plate cells. Finally, annexin IV immunolabeling is present from the caudal spinal cord, through the brainstem up to the diencephalon and lamina terminalis. Thus, annexin IV is an excellent marker for differentiated midline cells, is temporally and spatially correlated with development of the floor and roof plates, and is expressed in a rostral-caudal manner that supports the hypothesis that the floor plate extends the full length of the original neural tube.

Dorsal and Ventral Cell Types Can Arise from Common Neural Tube Progenitors

To challenge the developmental potential of dorsal neural tube cells and test whether single neuroepithelial cells can give rise to the full range of neural tube derivatives, we grafted a notochord lateral to the closing neural folds. This results in juxtaposition of dorsal and ventral cell types, by inducing floor plate cells and motor neurons dorsally. Clonal analysis with the vital dye lysinated rhodamine dextran showed that both “dorsal” and “ventral” neural tube derivatives can arise from a single precursor. Cells as diverse as sensory ganglion cells, presumptive pigment cells, roof plate cells, motor neurons, and floor plate cells were observed in the same clone. The presence of such diversity within single clones indicates that the responses to dorsal and ventral signals are not mutually exclusive; even in the early neural tube, neuroepithelial cells are not restricted to form only dorsal or ventral neural tube derivatives.

The developmental relationships of the neural tube and the notochord: short and long term effects of the notochord on the dorsal spinal cord

Patterning of the ventral half of the neural tube results from the inductive influence of the notochord and of the floor plate. We have studied here the effect of an ectopically grafted notochord on the development of the dorsalmost part of the neural tube i.e. roof plate and alar plates. We show that at their early stages, dorsal genes are repressed by the dorsal graft of a notochord, as shown previously in other studies. We found also that when the notochord is implanted in a mediodorsal position on top of the roof plate (and not laterally as previously performed in other studies) the genes specifics of the floor plate are not induced, and motoneurons do not differentiate. The notochord prevents the formation of the medial septum from roof plate cells and induces their active proliferation between E5 and E7. Roof and dorsal alar plates derived cells start to die from E7 onward leaving a dorsally truncated spinal cord. If the notochord is grafted at 20°–30° from the sagittal plane ventral genes and structures are induced and the roof plate differentiates normally. We conclude that roof plate cells exhibit a specific response to notochord signals, the short range effect of which is thus strikingly demonstrated.

Regional differentiation of the human fetal ependyma: immunocytochemical markers.

The development of the ependyma from 6 weeks (wk) gestation to term was studied in 26 human fetuses and infants for immunocytochemical differentiation using antibodies against vimentin, several cytokeratins, glial fibrillary acidic protein (GFAP) and S-100 protein. Acridine orange-RNA fluorescence was uniform in all differentiated ependymal cells. Marked differences were demonstrated among various anticytokeratin antibodies. Vimentin was demonstrated in undifferentiated cells, particularly during mitosis, and persisted as the ependyma matured. It was strong in floor plate cells and processes forming the ventral median septum. Vimentin and cytokeratin CK-904 coexisted with other immunoreactive proteins but disappeared in a caudorostral gradient with maturation. At 8 wk gestation, GFAP was detected in roof plate cells and their processes forming the dorsal median septum. S-100 protein appeared as early as 6 wk and had a more restricted regional distribution than GFAP at all ages. It was strong in the basal plate ependyma of the spinal cord in young fetuses. The temporal and spatial distributions of the immunoreactive proteins studied correlate with evidence that fetal ependymal cells synthesize compounds that attract or repel axonal growth cones to prevent axons from entering the ventricles or deviating from programmed projection pathways. An additional role may be to induce the transformation of radial glial cells in the subventricular zone.