Neural Induction Process Research Articles

FGF/MAPK/Ets signaling in Xenopus ectoderm contributes to neural induction and patterning in an autonomous and paracrine manner, respectively.

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue.

Linc1548 Promotes the Transition of Epiblast Stem Cells Into Neural Progenitors by Engaging OCT6 and SOX2

The transition of embryonic stem cells from the epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs), called the neural induction process, is crucial for cell fate determination of neural differentiation. However, the mechanism of this transition is unclear. Here, we identified a long non-coding RNA (linc1548) as a critical regulator of neural differentiation of mouse embryonic stem cells (mESCs). Knockout of linc1548 did not affect the conversion of mESCs to EpiSCs, but delayed the transition from EpiSCs to NPCs. Moreover, linc1548 interacts with the transcription factors OCT6 and SOX2 forming an RNA-protein complex to regulate the transition from EpiSCs to NPCs. Finally, we showed that Zfp521 is an important target gene of this RNA-protein complex regulating neural differentiation. Our findings prove how the intrinsic transcription complex is mediated by a lncRNA linc1548 and can better understand the intrinsic mechanism of neural fate determination.

Pcgf1 Regulates Early Neural Tube Development Through Histone Methylation in Zebrafish.

The neural induction constitutes the initial step in the generation of the neural tube. Pcgf1, as one of six Pcgf paralogs, is a maternally expressed gene, but its role and mechanism in early neural induction during neural tube development have not yet been explored. In this study, we found that zebrafish embryos exhibited a small head and reduced or even absence of telencephalon after inhibiting the expression of Pcgf1. Moreover, the neural induction process of zebrafish embryos was abnormally activated, and the subsequent NSC self-renewal was inhibited after injecting the Pcgf1 MO. The results of in vitro also showed that knockdown of Pcgf1 increased the expression levels of the neural markers Pax6, Pou3f1, and Zfp521, but decreased the expression levels of the pluripotent markers Oct4, Hes1, and Nanog, which further confirmed that Pcgf1 was indispensable for maintaining the pluripotency of P19 cells. To gain a better understanding of the role of Pcgf1 in early development, we analyzed mRNA profiles from Pcgf1-deficient P19 cells using RNA-seq. We found that the differentially expressed genes were enriched in many functional categories, which related to the development phenotype, and knockdown of Pcgf1 increased the expression of histone demethylases. Finally, our results showed that Pcgf1 loss-of-function decreased the levels of transcriptional repression mark H3K27me3 at the promoters of Ngn1 and Otx2, and the levels of transcriptional activation mark H3K4me3 at the promoters of Pou5f3 and Nanog. Together, our findings reveal that Pcgf1 might function as both a facilitator for pluripotent maintenance and a repressor for neural induction.

Zinc Promotes Patient-Derived Induced Pluripotent Stem Cell Neural Differentiation via ERK-STAT Signaling.

Nerve regeneration remains a challenge. Patient-derived induced pluripotent stem cell (iPSC)-differentiated neural stem cells (NSCs) provide a promising hope. Zinc is closely involved in central nervous system development and metabolism, but its role on iPSC neural differentiation is elusive and zinc detection methods in live cells are limited. In this study, intracellular zinc was detected in real time by a zinc fluorescent chemosensor and was shown to be increased during the iPSC neural induction process. iPSC neural differentiation was promoted with the addition of zinc chloride (ZnCl2) and inhibited with the addition of zinc chelator N,N,N0,N0-tetrakis(2-pyridylmethyl)-ethylenediamine, indicated by western blot and enzyme-linked immunosorbent assay analysis of NSC marker Nestin expression and measurement of neurite-like structures. Mechanistically, the phosphorylation level of ERK1/2 and STAT3 was changed with the zinc level, suggesting that zinc may affect the neural differentiation of iPSCs through ERK-STAT signaling. In conclusion, our study shows the important role of zinc in iPSC neural differentiation and suggests a new idea for iPSC-derived NSC application in nerve regeneration.

Temporal Modulations of NODAL, BMP, and WNT Signals Guide the Spatial Patterning in Self-Organized Human Ectoderm Tissues

Summary Developmental biology studies using model organisms suggest that the emergence of spatial patterning in the ectoderm is mediated by the morphogen gradients. However, it is still unclear whether the morphogen gradient is necessary and dominates the cell spatial patterning, particularly in human genetic background. Here, we demonstrate that human pluripotent stem cells can self-organize to concentric rings of all major cell types in ectoderm when cultured on micropatterned surfaces in a chemically defined condition. We reveal that modulating the dynamics of NODAL, BMP, and WNT signals is sufficient to control the spatial order of different cell types. Our mathematical model suggests that changes in wavelength and phase of signaling patterns formed via reaction-diffusion may be the mechanism by which temporal information is translated into spatial information. Together, our work demonstrates that in vitro human ectoderm microtissues have great potential in understanding the mechanisms of early-stage human development.

Small molecules re-establish neural cell fate of human fibroblasts via autophagy activation.

The generation of neural cells is of great interest in medical research because of its promising in neurodegenerative diseases. Small chemical molecules have been used for inducing specific cell types across lineage boundaries. Therefore, to direct neural cell fate, small molecule is a feasible approach for generating clinically relevant cell types without genetic alterations. Human fibroblasts have been directly induced into neural cells with different combinations of small molecules; however, the mechanism underlying neural induction is still not fully understood. In this study, human fibroblasts were induced into neural cells by using only 4 small molecules in a short time period, 5d. Small molecules used in this study included WNT activator, DNMT inhibitor, Notch inhibitor, and retinoic acid. Neural-specific genes, including NESTIN, TUJ1, and SOX2, were upregulated upon the induction for 5d. Noteworthy, this neural induction process by small molecules coincided with the activation of autophagy. Autophagy-related genes, such as LC3, ATG12, and LAMP1, were enhanced upon neural induction, and the number of induced-neural cells decreased when autophagy was suppressed by chloroquine. The activation of autophagy was found to reduce ROS generation within the induced-neural cells, and the inhibition of autophagy by chloroquine suppressed the expression of antioxidant genes, CATALASE, SOD, and GPX. This implied that autophagy maintained the optimal level of ROS for neural induction of human fibroblasts. Altogether, this study presented the effective and convenient condition to induce neural cells from human fibroblasts and revealed the positive roles of autophagy in controlling neural cell induction.

Stem Cells Engineered During Different Stages of Reprogramming Reveal Varying Therapeutic Efficacies.

Stem cells are emerging as promising treatment strategies for several brain disorders and pathologies. In this study, we explored the potential of creating induced pluripotent stem cell-derived neural stem cells (ipNSC) by using either unmodified or gene-modified somatic cells and tested their fate and therapeutic efficacies in vitro and in vivo. We show that cells engineered in somatic state lose transgene-expression during the neural induction process, which is partially restored by histone deacetylase inhibitor treatment whereas cells engineered at the ipNSC state have sustained expression of transgenes. In vivo, bimodal mouse and human ipNSCs engineered to express tumor specific death-receptor ligand and suicide-inducing therapeutic proteins have profound anti-tumor efficacy when encapsulated in synthetic extracellular matrix and transplanted in mouse models of resected-glioblastoma. This study provides insights into using somatic cells for treating CNS disorders and presents a receptor-targeted cancer therapeutic approach for brain tumors. Stem Cells 2018;36:932-942.

Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems.

CXCR4 activation promotes differentiation of human embryonic stem cells to neural stem cells

G protein-coupled receptors (GPCRs) are involved in many fundamental cellular responses such as growth, death, movement, transcription and excitation. Their roles in human stem cell neural specialization are not well understood. In this study, we aimed to identify GPCRs that may play a role in the differentiation of human embryonic stem cells (hESCs) to neural stem cells (NSCs). Using a feeder-free hESC neural differentiation protocol, we found that the expression of several chemokine receptors changed dramatically during the hESC/NSC transition. Especially, the expression of CXCR4 increased approximately 50 folds in NSCs compared to the original hESCs. CXCR4 agonist SDF-1 promoted, whereas the antagonist AMD3100 delayed the neural induction process. In consistence with antagonizing CXCR4, knockdown of CXCR4 in hESCs also blocked the neural induction and cells with reduced CXCR4 were rarely positive for Nestin and Sox1-staining. Taken together, our results suggest that CXCR4 is involved in the neural induction process of hESC and it might be considered as a target to facilitate NSC production from hESCs in regenerative medicine.

THE INFLUENCE OF NICOTINAMIDE ON THE DEVELOPMENT OF NEURONS

A major challenge in translating the promise of stem cell therapies to treat a myriad of neurodegenerative disorders is to rapidly and efficiently direct pluripotent stem cells to generate differentiated neurons. The application of active vitamin metabolites known to function in embryonic development and maintenance in the adult brain such as retinoic acid (vitamin A), ascorbic acid (vitamin C) and calcitriol (vitamin D3) have proven effective in current in-vitro differentiation protocols. Therefore, in this study we investigated whether the biologically active vitamin B3 metabolite, nicotinamide could enhance the differentiation of mouse embryonic stem cells, cultured as monolayers, into mature neurons at either early or late stages of development. Interestingly, nicotinamide elicited a dose-responsive increase in the percentage of neurons when added at an early developmental stage to the cells undergoing differentiation (days 0–7). Nicotinamide (10 mM) increased the proportion of β-III tubulin positive neurons by two fold and concomitantly decreased the total number of cells in culture, measured by quantification of 4′, 6-diamidino-2-phenylindole positive cells. This effect could result from induction of cell-cycle exit and/or selective cell death in non-neural populations. Higher levels of nicotinamide (20 mM) induced cytoxicity and cell death. This study supports previous evidence that vitamins and their metabolites can efficiently direct stem cells into neurons. Current work is focusing on the effect of nicotinamide on the process of neural induction and whether nicotinamide influences the generation of particular neuronal subtypes implicated in neurodegenerative diseases, specifically focusing on midbrain dopamine neurons; towards a therapy for Parkinson9s disease.

Selective Roles of Normal and Mutant Huntingtin in Neural Induction and Early Neurogenesis

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt) and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt) causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an in vitro mouse embryonic stem cell (ESC) clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs) severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively) during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs), mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2)-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF)-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of Notch, Hes1 and Hes5. These cumulative observations indicate that Htt is required for multiple stages of neural induction, whereas mHtt enhances this process and promotes precocious neurogenesis and oligodendrocyte progenitor cell elaboration.

Molecular genetics of neurulation.

The formation of the neural tube begins during gastrulation when ectoderm, an epithelial sheet on the outside of the embryo, is induced to form the neural plate. During the process of neural induction, the epithelium of the neural plate is regionalized along both the dorsoventral and anteroposterior axes of the embryo; this regionalization is likely to contribute to the cellular processes underlying neurulation. Genes whose expression marks the formation and regionalization of the neural plate and which encode cell adhesion molecules or putative transcription factors have been recently identified. The differential expression of these genes apparently subdivides the epithelium of the neural plate into small regions. Evidence from transgenic embryo experiments supports the idea that the differential expression of these genes in the neural plate plays a role in neural tube formation.

Tangential Neuronal Migration Controls Axon Guidance: A Role for Neuregulin-1 in Thalamocortical Axon Navigation

Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain.

Neural induction, neural fate stabilization, and neural stem cells.

The promise of stem cell therapy is expected to greatly benefit the treatment of neurodegenerative diseases. An underlying biological reason for the progressive functional losses associated with these diseases is the extremely low natural rate of self-repair in the nervous system. Although the mature CNS harbors a limited number of self-renewing stem cells, these make a significant contribution to only a few areas of brain. Therefore, it is particularly important to understand how to manipulate embryonic stem cells and adult neural stem cells so their descendants can repopulate and functionally repair damaged brain regions. A large knowledge base has been gathered about the normal processes of neural development. The time has come for this information to be applied to the problems of obtaining sufficient, neurally committed stem cells for clinical use. In this article we review the process of neural induction, by which the embryonic ectodermal cells are directed to form the neural plate, and the process of neural�fate stabilization, by which neural plate cells expand in number and consolidate their neural fate. We will present the current knowledge of the transcription factors and signaling molecules that are known to be involved in these processes. We will discuss how these factors may be relevant to manipulating embryonic stem cells to express a neural fate and to produce large numbers of neurally committed, yet undifferentiated, stem cells for transplantation therapies.

The neural induction process; its morphogenetic aspects.

This posthumous review of early embryonic inductions concludes: 1) the amphibian egg has only two distinct components, animal and vegetal. Interactions at their mutual boundary forms meso-endoderm. This is "meso-endoderm induction", not just "mesoderm induction". 2) The dorso-ventral polarity of the yolk mass implies a dorsally situated inducing centre. 3) Accumulation of cells into one, two, three or many cell masses [problastopores] along the circumference of the meso-endoderm results in as many axes, implying a self-organizing capacity of meso-endoderm. 4) Induction of the meso-endoderm is slow, spreading cell to cell through the animal moiety from the boundary of the vegetal yolk mass towards the animal pole. 5) Interaction between mesoderm and ectoderm is a separate step leading to cranio-caudal differentiation of the archenteron roof. 6) The initial invaginating endoderm and mesoderm, representing the future pharynx endoderm and prechordal plate mesoderm, first contacts the most posterior presumptive neurectoderm after having passed the still uninvaginated trunk mesoderm. At that moment an antero-posterior level neural induction actually starts. 7) The ectoderm contraction wave coincides spatially and temporally with the induced neural plate. 8) Two successive homoiogenetic waves of inductive activity pass through the presumptive neurectoderm in the anterior direction, the first one, "activation", giving rise to neural differentiation and ultimately forebrain, the second one, "transformation", to more caudal CNS structures. These are separate, successive steps in CNS regional induction. 9) The midbrain represents a secondary formation in the neural plate. 10) The observed changes in morphogenesis may depend upon separate, successive binary decisions via [cell and] nuclear state splitters [involving differentiation waves].

Short historical survey of pattern formation in the endo-mesoderm and the neural anlage in the vertebrates: the role of vertical and planar inductive actions.

After some introductory remarks about vertical versus horizontal inductive interactions and about planar versus homoiogenetic induction, the author discusses: a) the historical development of the more recently studied endo-mesoderm induction in the Urodeles and in the anuran Xenopus laevis, b) the possible causal relationship between endo-mesoderm induction and the initiation of the gastrulation process, and c) the older history of the regional neural induction as initially studied in the Urodeles and only recently analysed in the anuran Xenopus laevis. The essential vertical interaction in the neural induction process both in urodelian and in anuran amphibians is emphasized.

Vertebrate neural induction.

During early vertebrate development, the cells of the ectoderm choose between two possible fates: neural and epidermal. The process of neural induction was discovered nearly 70 years ago in vertebrates, and molecular analyses in recent years using Xenopus laevis embryos have identified several secreted factors with direct neural-inducing ability. There is considerable evidence that the mechanism of neuralization by these inducing factors is under inhibitory control and involves derepression. This review focuses on factors involved in the specification of neural fate within the frame of the default model of neural induction.

Developmental neurobiology of the anterior areas in amphibians: urodele perspectives.

Because of its evolutionary grade and its relative simplicity, the Urodele brain provides an excellent archetype for the study of forebrain development. Early experiments on Primary Induction took advantage of the Urodele's manipulatability and ease of use, but due to the fact that its ectoderm was very readily neuralized Anurans (especially Xenopus) became the vertebrate of choice for early developmental neurobiology. Recent advances in the molecular biology of neuralization in Xenopus may rejuvenate Urodele use in solving the complicated sequence of events during this process of neural induction and to ascertain if separate or a combination of events (de fault and inductive) are involved. In the future, the combined use of Urodeles and Anurans will provide much information with regard to the evolutionary conservation of the mechanisms of regional specification, gene expression events, neurulation, neuroblast migration, and axonogenesis during the development of the nervous system. The present review provides some recent examples of this approach of using Urodeles and Anurans in a combinatorial fashion to decipher specific aspects of developmental neurobiology.

Hepatocyte growth factor/scatter factor (HGF/SF) in early development: evidence for a role in neural induction

Since the pioneering work of Spemann and Mangold, who found that transplantation of the dorsal lip of the frog blastopore produces a secondary neural axis, embryologists have been intrigued by the process of neural induction. However, the molecular nature of the inducer(s) has remained obscure, largely due to the complication that neural induction often occurs as a secondary consequence of mesodermal induction. In the past few years, candidate molecules have been identified that are able to induce neural tissue in the absence of mesodenn in the frog. These include noggin and follistatin (reviewed in Ref. 1), which are thought to function by inhibiting activin. In the chick, recent experiments have implicated hepatocyte growth factor/scatter factor (HGF/SF) as a neural inducer based on it5 expression in the primitive streak and Hensen's node of the early chick embryo, and its ability to induce neural tissue in naive epiblast. This review describes the evidence supporting the possible function of HGF/SF as an early embryonic inducer.

Expression of the guanine nucleotide-binding protein Go correlates with the state of neural competence in the amphibian embryo

The nucleotide-binding protein Go is a transducing molecule closely associated with neural structures in vertebrates. Because of the potential importance of molecules of this type during the first step of neurogenesis, we have investigated the kinetics of expression of Go in the amphibian ( Pleurodeles waltl) embryo, focusing our attention on the stages corresponding to the acquisition of neural competence by presumptive ectoderm and to the process of neural induction. Using affinity-purified IgGs directed against the α subunit of Go, Go-like immunoreaction (GoLI) is first detected at the midblastula stage in some animal cap (future ectodermal) cells just before they have attained competence to be neuralized. At the early gastrula stage, GoLI is almost exclusively expressed by neural-competent tissue as a whole, with no obvious difference between the dorsal (prospective neural) and the ventral (prospective epidermal) ectoderm. The expression of GoLI is therefore related to the state of competence of the tissue rather than to its fate. At the early neurula stage, immediately following neural induction, the expression of GoLI persists essentially in that part of ectoderm that has been diverted from epidermal differentiation towards the neural pathway; in the ventral ectoderm, as neural competence is lost GoLI disappears. Furthermore, in the neurectoderm, only approximately 70% of the cells conserve GoLI, demonstrating that immediately following neural induction the population of neurectodermal cells is not homogeneous.