Floor Plate Cells Research Articles

Axon Guidance by Diffusible Chemoattractants: A Gradient of Netrin Protein in the Developing Spinal Cord

Gradients of diffusible long-range attractant and repellent proteins have been proposed to guide growing axons during nervous system development, but such gradients have never been visualized directly. In the embryonic spinal cord, commissural axons pioneer a circumferential trajectory to the floor plate at the ventral midline directed by secreted proteins of the netrin family. In the embryonic chick spinal cord netrin-1 mRNA is expressed by floor plate cells and netrin-2 mRNA by neural epithelial cells. Antibodies to the two netrins reveal a gradient of netrin protein directly in the path of commissural axons. The netrin-1 gradient itself extends many cell diameters dorsal to the floor plate, the site of netrin-1 expression. A similar distribution of netrin-1 protein has been detected in embryonic rat and mouse spinal cord. The detection of a gradient of netrin-1 protein supports the operation of long-range chemotropic mechanisms in the developing nervous system.

Notch signaling regulates midline cell specification and proliferation in zebrafish

Notochord and floor plate cells are sources of molecules that pattern tissues near the midline, including the spinal cord. Hypochord cells are also found at the midline of anamniote embryos and are important for aorta development. Delta–Notch signaling regulates midline patterning in the dorsal organizer by inhibiting notochord formation and promoting hypochord and possibly floor plate development, but the precise mechanisms by which this regulation occurs are unknown. We demonstrate here that floor plate and hypochord cells arise from distinct regions of the zebrafish shield. Blocking Notch signaling during gastrulation entirely prevented hypochord specification but only reduced the number of floor plate cells that developed compared to control embryos. In contrast, elevation of Notch signaling at the beginning of gastrulation caused expansion of hypochord at the expense of notochord, but floor plate was not affected. A cell proliferation assay revealed that Notch signaling maintains dividing floor plate progenitors. Together, our results indicate that Notch signaling regulates allocation of appropriate numbers of different midline cells by different mechanisms.

Early steps in neural development

We studied early neurulation events in vitro by transplanting quail Hensen's node, central prenodal regions (before the nodus as such develops), or upper layer parts of it on the not yet definitively committed upper layer of chicken anti-sickle regions (of unincubated blastoderms), eventually associated with central blastoderm fragments. We could demonstrate by this quail-chicken chimera technique that after the appearance of a pronounced thickening of the chicken upper layer by the early inductive effect of neighboring endophyll, a floor plate forms by insertion of Hensen's node-derived quail cells into the median part of the groove. This favors, at an early stage, the floor plate "allocation" model that postulates a common origin for notochord and median floor plate cells from the vertebrate's secondary major organizer (Hensen's node in this case). A comparison is made with results obtained after transplantation of similar Hensen's nodes in isolated chicken endophyll walls or with previously obtained results after the use of the grafting procedure in the endophyll walls of whole chicken blastoderms.

Homozygous Ft embryos are affected in floor plate maintenance and ventral neural tube patterning

Sonic hedgehog (Shh), produced by the notochord and floor plate cells of the neural tube, plays a critical role in organizing dorsal-ventral patterning in the developing neural tube. We have investigated neural tube development in mouse embryos homozygous for the Fused toes (Ft) mutation, a deletion composed of genes of the Iroquois B (IrxB) cluster and of Fts, Ftm, and Fto. In Ft mutants starting from embryonic day 10.5, the floor plate appeared to degenerate and the notochord failed to undergo ventral displacement from the spinal cord. Consistent with the loss of Shh signalling from the floor plate, V3 neuron generation was reduced in Ft/Ft embryos and the domain of motor neuron generation expanded ventrally at the expense of V2 neurons. These data support the idea that Ft genes play an important role in dorsal-ventral patterning of the neural tube acting to define the extent of motor neuron generation; moreover, the data reveal a previously unanticipated function for Ft genes in the maintenance of the floor plate.

The floor plate: multiple cells, multiple signals

One of the key organizers in the CNS is the floor plate - a group of cells that is responsible for instructing neural cells to acquire distinctive fates, and that has an important role in establishing the elaborate neuronal networks that underlie the function of the brain and spinal cord. In recent years, considerable controversy has arisen over the mechanism by which floor plate cells form. Here, we describe recent evidence that indicates that discrete populations of floor plate cells, with characteristic molecular properties, form in different regions of the neuraxis, and we discuss data that imply that the mode of floor plate induction varies along the anteroposterior axis.

Her9 promotes floor plate development in zebrafish

Notochord, floor plate, and in anamniotes hypochord, are vertebrate embryonic midline structures that are the sources of molecules that pattern the nervous system, somites, and dorsal aorta. Midline precursor cells arise from the dorsal organizer during gastrulation, and Notch signaling is an important regulator of midline cell fate specification. To understand fully how Notch signaling regulates midline development, we investigated the role of potential Notch target genes. We show here that midline precursors express her9, a member of the hairy/Enhancer of split gene family. Although her9 inhibits notochord development and promotes floor plate specification, her9 expression in floor plate cells appears not to require Notch signaling. We show that, instead, her9 is a downstream effector of Nodal signaling for floor plate specification.

The Notch-target gene hairy2a impedes the involution of notochordal cells by promoting floor plate fates in Xenopus embryos

We have previously shown that the early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and that Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Now, we present evidence that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone.

Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphé neurones and cranial motoneurones

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Distinct effects of caudalizing factors on regional specification of embryonic stem cell-derived neural precursors

Recent embryological studies have implicated several “caudalizing factors” in the caudal specification of the central nervous system (CNS). In this study, we have examined the effects of three candidate caudalizing factors on neural precursors induced from embryonic stem (ES) cells by the stromal cell-derived inducing activity (SDIA) method. Among retinoic acid (RA), Wnt and FGF signals, RA causes the strongest level of caudalization: inducing suppression of forebrain differentiation and promotion of caudal CNS specification. Obvious suppression of the telencephalic marker Bf1 and that of the forebrain marker Otx2 occur at 2×10 −8 and 2×10 −7 M, respectively. Activation of the caudal marker genes such as Hoxb9 is observed in a dose-dependent manner over the range of 2×10 −9–2×10 −6 M. Suppression of the forebrain genes has a narrow critical period of RA response during the early culture phase. In contrast, significant induction of the caudal genes is evoked by a 1-day exposure to RA at any time between days 3 and 8. RA treatment not only induces caudal specification but also inhibits differentiation of ventral CNS tissues, particularly of floor plate cells. FGF4 induces partial caudalization while Wnt-3A exhibits weak caudalizing activities only in the presence of RA. These findings provide useful information on the proper selection of combination of signaling molecules, doses and timing for steering ES cell differentiation by caudalizing factors into caudal neural fates.

Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Contacts between the commissural axons and the floor plate cells are mediated by nectins

During development of the central nervous system (CNS), commissural axons grow toward the ventral midline. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. The contacts between the commissural axons and the floor plate cells are involved in this axonal guidance, but their mechanisms or structures have not fully been understood. In this study, we found that nectin-1 and -3, immunoglobulin-like cell–cell adhesion molecules, asymmetrically localized at the contact sites between the commissural axons and the floor plate cells, respectively. In vitro perturbation of the endogenous trans-interaction between nectin-1 and -3 caused abnormal fasciculation of the commissural axons and impairment of the contacts, and resulted in failure in longitudinal turns of the commissural axons at the contralateral sites of the rat hindbrain. These results indicate that the contacts between the commissural axons and the floor plate cells are mediated by the hetero- trans-interaction between nectin-1 and -3 and involved in regulation of the trajectory of the commissural axons.

A dual role for Sonic hedgehog in regulating adhesion and differentiation of neuroepithelial cells

In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, we investigated whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface β1-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promoted differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, our experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through different mechanisms, leading to a coordinated regulation of the various cell interactions essential for neural tube morphogenesis.

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish.

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.

Netrin-1 Is a Chemorepellent for Oligodendrocyte Precursor Cells in the Embryonic Spinal Cord

Netrin-1, secreted by floor plate cells, orients axon extension in relation to the ventral midline of the embryonic spinal cord. Oligodendrocyte precursor (OP) cells are born close to the ventral midline and migrate away from the floor plate. Here we show that OP cells, identified by expression of the platelet-derived growth factor alpha receptor, express the netrin receptors dcc and unc5h1 but do not express netrin-1. Using a microchemotaxis assay, we demonstrate that migrating OPs are repelled by a gradient of netrin-1 in vitro. Furthermore, application of netrin-1 to OPs in vitro triggers retraction of OP processes. In the absence of netrin-1 or Deleted in Colorectal Cancer (DCC) function in vivo, fewer OP cells migrate from the ventral to the dorsal embryonic spinal cord, consistent with netrin-1 acting as a repellent. In addition to their role regulating cell movement, DCC and UNC-5 homologs have been suggested to function as proapoptotic dependence receptors, triggering cell death in the absence of netrin-1. In contrast, we report no evidence of increased OP cell death in vivo or in vitro in the absence of either netrin-1 or DCC. These findings indicate that netrin-1 is a repellent cue for migrating OPs in the embryonic spinal cord.

Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells.

To understand the range of competence of embryonic stem (ES) cell-derived neural precursors, we have examined in vitro differentiation of mouse and primate ES cells into the dorsal- (neural crest) and ventralmost (floor plate) cells of the neural axis. Stromal cell-derived inducing activity (SDIA; accumulated on PA6 stromal cells) induces cocultured ES cells to differentiate into rostral CNS tissues containing both ventral and dorsal cells. Although early exposure of SDIA-treated ES cells to bone morphogenetic protein (BMP)4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the fourth day of coculture causes differentiation of neural crest cells and dorsalmost CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. Notably, high concentrations of Shh efficiently promote differentiation of HNF3beta(+) floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naive precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals.

Cyclops-independent floor plate differentiation in zebrafish embryos.

In zebrafish, development of the ventral neural tube depends on the Nodal-related signal Cyclops (Cyc). One-day-old cyc mutant embryos lack the medial floor plate (MFP). We show here that cells expressing MFP marker genes differentiate gradually in cyc mutant embryos in a delayed manner during the second day of development. This late differentiation is restricted to the hindbrain and spinal cord and depends on an intact Hedgehog (Hh) signalling pathway. Cells expressing MFP marker genes in cyc mutant embryos appear to be derived from lateral floor plate (LFP) cells as they coexpress LFP and MFP marker genes. This finding suggests that the correct temporal development of the MFP is required for the distinction of LFP and MFP cells in wild-type embryos.

Bovine floor plate explants secrete SCO-spondin

SCO-spondin is a large-molecular mass glycoprotein, secreted by the subcommissural organ (SCO), which has been implicated in neuronal development during ontogeny of the central nervous system. The expression of SCO-spondin is not restricted to the SCO but it also occurs in the floor plate, a key structure participating in neuronal differentiation and patterning of the neural tube. It has been postulated that SCO-spondin detected in the floor plate is released into the lumen of the neural tube, but this new route of secretion of floor plate cells needs to be further substantiated. For this purpose, we standardized long-term organ culture of bovine floor plate and performed morphological, immunological, biochemical, and gene expression analyses. The study of floor plate explants and their conditioned media allowed us to demonstrate that: (1) organ-cultured floor plate cells are actively secretory for up to 25 days; (2) SCO-spondin gene is actively transcribed and translated by the cultured floor plate cells; (3) SCO-spondin is released into the culture medium via the apical cell pole; and (4) upon release, SCO-spondin does not aggregate in the conditioned medium but remains soluble. Furthermore, in the cultured floor plate cells, SCO-spondin may be secreted through a route bypassing the Golgi apparatus.

Floor plate, glia and other support cells in the anterior nerve cord of amphioxus larvae

AbstractSerial electron micrograph reconstructions and interval series were used to examine the support cells, including glia, of the anterior nerve cord in 6‐, 8‐ and 12.5‐day larvae of Branchiostoma floridae and one newly metamorphosed juvenile. The floor plate begins immediately behind the infundibular cells. It consists for the most part of a single file of midline cells, but adjacent lateral floor plate cells occur in some places. The floor plate is interrupted at one point, in the posterior part of the cerebral vesicle above the tegmental neuropile. A class of early developing axons crosses the midline at this point, which suggests that the floor plate may have a developmental role in axon guidance. The structural integrity of the cord is maintained by ependymal and ependymoglial cells that attach to its sides. Two other glial cell types were found in larvae. Both appear to originate adjacent to the floor plate and hence are referred to here as midline glia. Those in somites 1 and 2 remain connected to the central canal; they appear to be a mixed population that may include precursors of midline support cells which are present later in the juvenile. Those caudal to somite 3 detach early from the central canal and develop an extensive network of axial processes; they are referred to here as axial glia and treated as a subcategory of midline glia. Based on their site of origin and the absence of glial filaments, their closest counterpart among vertebrate glia may well be the oligodendrocyte. To our knowledge, this is the first report of a possible amphioxus homologue of this important vertebrate cell type.

Opponent Activities of Shh and BMP Signaling during Floor Plate Induction In Vivo

We performed in vivo experiments in chick embryos that examined whether application of an exogenous source of Shh protein mimics the ability of the notochord to induce ectopic floor plate cells in the neural tube. Shh cannot act alone to induce a floor plate. However, coapplication of Shh and chordin, a BMP antagonist normally coexpressed with Shh in the notochord, results in a marked switch from dorsal to ventral cell fate, including a dramatic and widespread induction of floor plate cells. These data provide in vivo evidence that notochord-derived BMP antagonists may normally generate a permissive environment for the Shh-mediated induction of floor plate. Further experiments performed to address the source of BMPs that are inhibited by the action of chordin suggest that they derive specifically from the surface ectoderm and dorsal-most neuroepithelium. These data indicate that, at neural groove stages, dorsally derived BMPs affect ventral-most regions of the neural plate, suggesting a novel long-range action of BMPs. Together, these studies suggest that the balance of dorsally derived signals and notochord-derived signals determines the extent of floor plate cell induction.

Dual function of polysialic acid during zebrafish central nervous system development.

Polysialic acid (PSA), a carbohydrate epitope attached to the neural cell adhesion molecule, serves as a modulator of axonal interactions during vertebrate nervous system development. We have used PSA-specific antibodies and whole-mount immunocytochemistry to describe the spatiotemporal expression pattern of PSA during zebrafish central nervous system development. PSA is transiently expressed on all cell bodies and, except for the posterior commissure, it is not found on axons. Floorplate cells in the spinal cord and hindbrain strongly express PSA throughout development. Enzymatic removal of PSA leads to a defasciculated growth pattern of the posterior commissure and also affects distinct subsets of commissural axons in the hindbrain, which fail to cross the midline. Whereas the disordered growth pattern of hindbrain commissures produced by PSA-removal could be mimicked by injections of soluble PSA, the growth of axons in the posterior commissure was unaffected by such treatment. These results suggest that there are distinct mechanisms for PSA action during axon growth and pathfinding in the developing zebrafish CNS.