Floor Plate Differentiation Research Articles

Forced LMX1A expression induces dorsal neural fates and disrupts patterning of human embryonic stem cells into ventral midbrain dopaminergic neurons

The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson's disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an invitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.

Differences in neural stem cell identity and differentiation capacity drive divergent regenerative outcomes in lizards and salamanders

While lizards and salamanders both exhibit the ability to regenerate amputated tails, the outcomes achieved by each are markedly different. Salamanders, such as Ambystoma mexicanum, regenerate nearly identical copies of original tails. Regenerated lizard tails, however, exhibit important morphological differences compared with originals. Some of these differences concern dorsoventral patterning of regenerated skeletal and spinal cord tissues; regenerated salamander tail tissues exhibit dorsoventral patterning, while regrown lizard tissues do not. Additionally, regenerated lizard tails lack characteristically roof plate-associated structures, such as dorsal root ganglia. We hypothesized that differences in neural stem cells (NSCs) found in the ependyma of regenerated spinal cords account for these divergent regenerative outcomes. Through a combination of immunofluorescent staining, RT-PCR, hedgehog regulation, and transcriptome analysis, we analyzed NSC-dependent tail regeneration. Both salamander and lizard Sox2+ NSCs form neurospheres in culture. While salamander neurospheres exhibit default roof plate identity, lizard neurospheres exhibit default floor plate. Hedgehog signaling regulates dorsalization/ventralization of salamander, but not lizard, NSCs. Examination of NSC differentiation potential in vitro showed that salamander NSCs are capable of neural differentiation into multiple lineages, whereas lizard NSCs are not, which was confirmed by in vivo spinal cord transplantations. Finally, salamander NSCs xenogeneically transplanted into regenerating lizard tail spinal cords were influenced by native lizard NSC hedgehog signals, which favored salamander NSC floor plate differentiation. These findings suggest that NSCs in regenerated lizard and salamander spinal cords are distinct cell populations, and these differences contribute to the vastly different outcomes observed in tail regeneration.

Nrf2 Activation Rescues Dendritic Pathology in hPSC‐Derived And Primary Neuron Based Models of Parkinson's Disease

IntroductionNeuronal loss in Parkinson's disease (PD) is associated with impaired proteostasis and increased oxidative stress in dopaminergic neurons of the substantia nigra pars compacta. In a healthy cell, redox imbalance is controlled by enzymes that detoxify the cell of free radicals. These enzymes are transcriptionally activated as part of the cells antioxidant response. The master regulator of this pathway is a transcription factor called Nrf2 (a.k.a NFE2L2). Aggregated α‐synuclein (α‐syn) is the major constituent of Lewy Bodies (the disease hallmark), however the mechanisms linking α‐syn aggregation with generation of reactive oxygen species (ROS) have been difficult to ascertain. We therefore asked whether a deficit in the antioxidant response might explain the increased susceptibility of PD neurons to ROS generation and dendritic pathology following α‐syn aggregation.MethodsTo study these events, we contrasted human pluripotent stem cells (hPSCs) that harbor the α‐syn‐A53T mutation against isogenic controls. A9‐type dopaminergic neurons (the specific neurons lost in PD) were generated from hPSCs by following a floor plate differentiation paradigm. α‐syn‐A53T neurons show accumulation of insoluble α‐syn consistent with an early disease phenotype. We similarly contrasted rat primary neurons against neurons exposed to α‐syn pre‐formed fibrils (PFF). PFF exposed neurons accumulate large intracellular aggregates of α‐syn consistent with advanced synucleinopathy. We show that aberrant accumulation of α‐syn in both systems promotes increased ROS production coupled to increased dendritic pathology and decreased dendritic length and complexity.ResultsTo evaluate the role of Nrf2 in dendritic pathology we measured Nrf2 protein stability and phospho‐activation in A53T‐mutant and PFF exposed neurons. We found that α‐syn accumulation impaired Nrf2 phospho‐activation leading to the subsequent loss of expression of Nrf2 target genes. To determine whether Nrf2‐mediated activation of Antioxidant Response Elements (AREs) could rescue the observed dendritic pathology, we tested the effects of both Nrf2 overexpression and pharmaceutical modulation of Nrf2 activity on dendritic pathology. We found that Nrf2‐activation increased expression of detoxifying enzymes and reduced ROS levels in both A53T‐mutant and PFF exposed neurons, leading to a significant decrease in dendritic pathology.SignificanceThis investigation established that the Nrf2 transcriptional pathway is impaired by aberrant accumulation of α‐syn in both A53T‐mutant and PFF exposed neurons and determined that restoration of the anti‐oxidant pathway protects PD neurons from dendritic pathology.Support or Funding InformationFunding: This work was supported in part by the Parkinson Society of Canada (2014‐685 to SDR), the Natural Sciences and Engineering Research Council of Canada (RG060805 to SDR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A revised model of Xenopus dorsal midline development: Differential and separable requirements for Notch and Shh signaling

The development of the vertebrate dorsal midline (floor plate, notochord, and hypochord) has been an area of classical research and debate. Previous studies in vertebrates have led to contrasting models for the roles of Shh and Notch signaling in specification of the floor plate, by late inductive or early allocation mechanisms, respectively. Here, we show that Notch signaling plays an integral role in cell fate decisions in the dorsal midline of Xenopus laevis, similar to that observed in zebrafish and chick. Notch signaling promotes floor plate and hypochord fates over notochord, but has variable effects on Shh expression in the midline. In contrast to previous reports in frog, we find that Shh signaling is not required for floor plate vs. notochord decisions and plays a minor role in floor plate specification, where it acts in parallel to Notch signaling. As in zebrafish, Shh signaling is required for specification of the lateral floor plate in the frog. We also find that the medial floor plate in Xenopus comprises two distinct populations of cells, each dependent upon different signals for its specification. Using expression analysis of several midline markers, and dissection of functional relationships, we propose a revised allocation mechanism of dorsal midline specification in Xenopus. Our model is distinct from those proposed to date, and may serve as a guide for future studies in frog and other vertebrate organisms.

The Cell Surface Membrane Proteins Cdo and Boc Are Components and Targets of the Hedgehog Signaling Pathway and Feedback Network in Mice

Cdo and Boc encode cell surface Ig/fibronectin superfamily members linked to muscle differentiation. Data here indicate they are also targets and signaling components of the Sonic hedgehog (Shh) pathway. Although Cdo and Boc are generally negatively regulated by Hedgehog (HH) signaling, in the neural tube Cdo is expressed within the Shh-dependent floor plate while Boc expression lies within the dorsal limit of Shh signaling. Loss of Cdo results in a Shh dosage-dependent reduction of the floor plate. In contrast, ectopic expression of Boc or Cdo results in a Shh-dependent, cell autonomous promotion of ventral cell fates and a non-cell-autonomous ventral expansion of dorsal cell identities consistent with Shh sequestration. Cdo and Boc bind Shh through a high-affinity interaction with a specific fibronectin repeat that is essential for activity. We propose a model where Cdo and Boc enhance Shh signaling within its target field.

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.

Distinct modes of floor plate induction in the chick embryo.

To begin to reconcile models of floor plate formation in the vertebrate neural tube, we have performed experiments aimed at understanding the development of the early floor plate in the chick embryo. Using real-time analyses of cell behaviour, we provide evidence that the principal contributor to the early neural midline, the future anterior floor plate, exists as a separate population of floor plate precursor cells in the epiblast of the gastrula stage embryo, and does not share a lineage with axial mesoderm. Analysis of the tissue interactions associated with differentiation of these cells to a floor plate fate reveals a role for the nascent prechordal mesoderm, indicating that more than one inductive event is associated with floor plate formation along the length of the neuraxis. We show that Nr1, a chick nodal homologue, is expressed in the nascent prechordal mesoderm and we provide evidence that Nodal signalling can cooperate with Shh to induce the epiblast precursors to a floor-plate fate. These results indicate that a shared lineage with axial mesoderm cells is not a pre-requisite for floor plate differentiation and suggest parallels between the development of the floor plate in amniote and anamniote embryos.

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.

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.

A Floor Plate Enhancer of the Zebrafish netrin1 Gene Requires Cyclops (Nodal) Signalling and the Winged Helix Transcription Factor FoxA2

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3β, axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2—given its importance for midline development also in the mouse—may be one such component.

Severe neural tube defects in the loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor plate specification.

Neural tube defects (NTD) are clinically important congenital malformations whose molecular mechanisms are poorly understood. The loop-tail (Lp) mutant mouse provides a model for the most severe NTD, craniorachischisis, in which the brain and spinal cord remain open. During a positional cloning approach, we have identified a mutation in a novel gene, Lpp1, in the Lp mouse, providing a strong candidate for the genetic causation of craniorachischisis in LP: Lpp1 encodes a protein of 521 amino acids, with four transmembrane domains related to the Drosophila protein strabismus/van gogh (vang). The human orthologue, LPP1, shares 89% identity with the mouse gene at the nucleotide level and 99% identity at the amino acid level. Lpp1 is expressed in the ventral part of the developing neural tube, but is excluded from the floor plate where Sonic hedgehog (Shh) is expressed. Embryos lacking Shh express Lpp1 throughout the ventral neural tube, suggesting negative regulation of Lpp1 by SHH: Our findings suggest that the mutual interaction between Lpp1 and Shh may define the lateral boundary of floor plate differentiation. Loss of Lpp1 function disrupts neurulation by permitting more extensive floor plate induction by Shh, thereby inhibiting midline bending of the neural plate during initiation of neurulation.

Cloning and characterization of zebrafish smad2, smad3 and smad4.

smad genes encode transcription factors involved in the signal transduction of members of the TGFbeta superfamily. We report here the cloning, characterization and genomic mapping of smad2, smad3 and smad4 from the zebrafish, Danio rerio. In Xenopus, smad2 overexpression has been shown to interfere with gastrulation and dorsal cell fate specification. However, full-length zebrafish smad2, although functionally active in Xenopus explants, has no effect when overexpressed in zebrafish embryos. In contrast, an N-terminally truncated, constitutively active version of Smad2 protein causes severe dorsalization or partial secondary axis formation, pointing to a role of Smad2 during mesoderm and axis formation. The temporal and spatial expression patterns of zebrafish smad2, 3 and 4 were investigated by developmental RT-PCR and whole mount in-situ hybridization. All three genes show strong and ubiquitous maternal expression. Zygotic expression is weak and ubiquitous in the case of smad2, and strong and ubiquitious in the case of smad4, while smad3 shows a spatially restricted zygotic expression pattern. It is expressed in migrating neural crest cells of the trunk and a subset of cells in the diencephalon in close proximity to the expression domain of the Nodal-related protein Cyclops/Ndr2/Znr1, a potential signal upstream of Smad2/3 required for eye-field separation and floor plate specification. Overexpression of truncated smad2 in cyclops mutant embryos leads to a rescue of the eye and floorplate defects. These data suggest that Smad2 acts as a mediator of Nodal signals during zebrafish midline signaling, while Smad3 might be involved in later steps of eye field separation.

Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice.

Floor plate cells at the midline of the neural tube are specified by high-level activity of Sonic hedgehog (Shh) secreted by notochord, whereas motor neurons are thought to be specified by a lower level activity of Shh secreted in turn by floor plate cells. In Drosophila, the Gli zinc finger protein Cubitus interruptus functions as a transcription factor activating Hedgehog-responsive genes. We report that the expression of known Shh-responsive genes such as Ptc and Gli1 is downregulated in mutant mice lacking Gli2 function. Gli2 mutants fail to develop a floor plate yet still develop motor neurons, which occupy the ventral midline of the neural tube. Our results imply that Gli2 is required to mediate high level but not low level Shh activity and show that the development of motor neurons can occur in the absence of floor plate induction.

Combinatorial Gli gene function in floor plate and neuronal inductions by Sonic hedgehog.

Within the developing vertebrate nervous system, it is not known how progenitor cells interpret the positional information provided by inducing signals or how the domains in which distinct groups of neural cells differentiate are defined. Gli proteins may be involved in these processes. In the frog neural plate, we have previously shown that the zinc finger transcription factor Gli1 is expressed in midline cells and mediates the effects of Shh inducing floor plate differentiation. In contrast, Gli2 and Gli3 are expressed throughout the neural plate except for the midline. Here, it is shown that Gli3 and Shh repress each other whereas Gli2, like Gli1, is a target of Shh signaling. However, only Gli1 can induce the differentiation of floor plate cells. In addition, Gli2 and Gli3 repress the ectopic induction of floor plate cells by Gli1 in co-injection assays and inhibit endogenous floor plate differentiation. The definition of the floor plate domain, therefore, appears to be defined by the antagonizing activities of Gli2 and Gli3 on Gli1 function. Because both Gli1 and Gli2 are induced by Shh, these results establish a regulatory feedback loop triggered by Shh that restricts floor plate cells to the midline. We have also previously shown that the Gli genes induce neuronal differentiation and here it is shown that there is specificity to the types of neurons the Gli proteins induce. Only Gli1 induces Nkx2.1/TTF-1(+) ventral forebrain neurons. Moreover, Gli2 and Gli3 inhibit their differentiation. In contrast, the differentiation of spinal motor neurons can be induced by the two ventrally expressed Gli genes, Gli1 and Gli2, suggesting that Gli2 directly mediates induction of motor neurons by Shh. In addition, Gli3 inhibits motor neuron differentiation by Gli2. Thus, combinatorial Gli function may pattern the neural tube, integrating positional information and cell type differentiation.

Two Critical Periods of Sonic Hedgehog Signaling Required for the Specification of Motor Neuron Identity

Antibodies that block Sonic Hedgehog (SHH) signaling have been used to show that SHH activity is required for the induction of floor plate differentiation by the notochord and independently for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: an early period during which naive neural plate cells are converted into ventralized progenitors and a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. The ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.

Inhibition of floor plate differentiation by Pax3: evidence from ectopic expression in transgenic mice.

The Pax genes containing a complete paired-type homeobox are expressed in restricted dorsoventral domains of the undifferentiated neuroepithelium. Their expression respond to signals that pattern the neural tube and which emanate from the notochord, floor plate and overlying ectoderm. In order to determine whether the dorsally restricted Pax3 gene can influence the fate of cells within the neural tube, we produced transgenic mice overexpressing Pax3 in the entire neural tube under the Hoxb-4 promoter/region A enhancer. In two distinct transgenic mouse lines, we observed embryos with abnormal limb, eye, brain and neural tube development. Expression of Pax3 was not sufficient to dorsalize cells from the ventral intermediate zone, which still expressed ventral markers. However, in these embryos, expression of Pax3 at the ventral midline was associated with the absence of floor plate differentiation. Under these conditions, motor neurons still differentiated in the ventral spinal cord, although in fewer numbers.

Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation.

The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. The secreted protein encoded by the Sonic hedgehog (Shh) gene is expressed by axial midline cells and can induce floor plate cells in vivo and in vitro. Motor neurons can also be induced in vitro by cells that synthesize Sonic hedgehog protein (Shh). It remains unclear, however, if the motor-neuron-inducing activity of Shh depends on the synthesis of a distinct signaling molecule by floor plate cells. To resolve this issue, we have developed an in vitro assay which uncouples the notochord-mediated induction of motor neurons from floor plate differentiation, and have used this assay to examine whether Shh induces motor neurons in the absence of floor plate differentiation. Floor plate cells and motor neurons were induced in neural plate explants grown in contact with the notochord, but only motor neurons were induced when explants were separated from the notochord. COS cells transfected with Shh induced both floor plate cells and motor neurons when grown in contact with neural plate explants, whereas only motor neurons were induced when the explants were grown at a distance from Shh-transfected COS cells. Direct transfection of neural plate cells with an Shh-expression construct induced both floor plate cells and motor neurons, with motor neuron differentiation occurring prior to, or coincidentally with, floor plate differentiation. The induction of motor neurons appears, therefore, not to depend on floor plate differentiation. The induction of motor neurons by Shh does not depend on distinct floor-plate-derived signaling molecules. Shh can, therefore, initiate the differentiation of two cell types that are generated in the ventral region of the neural tube. These results show that the early development of motor neurons involves the inductive action of Shh, whereas the survival of motor neurons at later stages of embryonic development requires neurotrophic factors.

Restrictions to Floor Plate Induction by hedgehog and Winged-Helix Genes in the Neural Tube of Frog Embryos

Intercellular signaling molecules of the vertebrate hedgehog family and transcription factors of the winged-helix family have been implicated in floor plate development. We have examined the consequences of misexpressing the vertebrate hedgehog gene vhh-1 (sonic hedgehog, shh) and the winged-helix gene HNF-3β in the neural plate and neural tube of frog embryos. Misexpression of either of these genes induces floor plate differentiation at ectopic locations. However, ectopic floor plate induction in response to both vhh-1 and HNF-3β was temporally and spatially restricted. At neural plate stages, ectopic floor plate differentiation was not detected. After neural tube closure, ectopic floor plate differentiation was detected but was restricted predominantly to the dorsal region of the neural tube. The ability of winged-helix and vertebrate hedgehog genes to induce floor plate differentiation in vivo may, therefore, be constrained by additional signals that specify the time and position of floor plate differentiation.

Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord

The differentiation of distinct cell types in the ventral neural tube depends on local inductive signals from the notochord. We have isolated a vertebrate homolog of the Drosophila segment polarity gene hedgehog ( hh) from zebrafish and rat, termed vhh-1. vhh-1 is expressed in the node, notochord, floor plate, and posterior limb bud mesenchyme. Each of these cell groups has floor plate inducing activity, suggesting that the vhh-1 gene may encode a floor plate-inducing molecule. Widespread expression of rat vhh-1 in frog embryos leads to ectopic floor plate differentiation in the neural tube. In vitro tests for the signaling functions of vhh-1 demonstrate that COS cells expressing the rat vhh-1 gene induce floor plate and motor neuron differentiation in neural plate explants. vhh-1 may, therefore, contribute to the floor plate and motor neuron inducing activities of the notochord.

Induction of floor plate differentiation by contact-dependent, homeogenetic signals.

The floor plate is located at the ventral midline of the neural tube and has been implicated in neural cell patterning and axon guidance. To address the cellular mechanisms involved in floor plate differentiation, we have used an assay that monitors the expression of floor-plate-specific antigens in neural plate explants cultured in the presence of inducing tissues. Contact-mediated signals from both the notochord and the floor plate act directly on neural plate cells to induce floor plate differentiation. Floor plate induction is initiated medially by a signal from the notochord, but appears to be propagated to more lateral cells by homeogenetic signals that derive from medial floor plate cells. The response of neural plate cells to inductive signals declines with embryonic age, suggesting that the mediolateral extent of the floor plate is limited by a loss of competence of neural cells. The rostral boundary of the floor plate at the midbrain-forebrain junction appears to result from the lack of inducing activity in prechordal mesoderm and the inability of rostral neural plate cells to respond to inductive signals.