Human Neural Tube Research Articles

Small molecule valproic acid enhances ventral patterning of human neural tube organoids by regulating Wnt and Shh signalling.

Valproic acid (VPA), a clinically approved small molecule, has been reported to activate Wnt signalling that is critical for dorsal-ventral (DV) patterning of neural tube. However, little is known about the impact of VPA on DV patterning process. Here, we show that even though VPA has a negative impact on the early formation of human neural tube organoids (hNTOs), it significantly enhances the efficiency of ventrally patterned hNTOs, when VPA is added during the entire differentiation process. RNA sequencing and RT-qPCR analysis demonstrates VPA activates endogenous Wnt signalling in hNTOs. Surprisingly, transcriptome analysis also identifies upregulation of genes for degradation of GLI2 and GLI3 proteins, whose truncated fragment are transcriptional repressors of Shh signalling. The Western-blot analysis confirms the increase of GLI3R proteins after VPA treatment. Thus, VPA might enhance ventral patterning of hNTOs through both activating Wnt, which can antagonise Shh signalling by inducing GLI3 expression, and/or inhibiting Shh signalling by inducing GLI protein degradation. We further obtain results to show that VPA still increases patterning efficiency of hNTOs with a weak influence on their early formation when the initiation time of VPA is delayed and its duration is reduced. Taken together, this study demonstrates that VPA enhances the generation of more reproducible hNTOs with ventral patterning, opening the avenues for the applications of hNTOs in developmental biology and regenerative medicine.

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.

Identification and functional analysis of rare HECTD1 missense variants in human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased extracellular heat shock protein 90 (eHSP90) secretion to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with NTDs in humans.

Mechanical Actuation of Organoids in Synthetic Microenvironments.

Organoids are a powerful model system to explore the role of mechanical forces in sculpting emergent tissue cytoarchitecture. The modulation of the mechanical microenvironment is most readily performed using synthetic extracellular matrices (ECM); however, such materials provide passive, rather than active force modulation. Actuation technologies enable the active tuning of mechanical forces in both time and magnitude. Using such instruments, our group has shown that extrinsically imposed stretching on human neural tube organoids (hNTOs) enhanced patterning of the floor plate domain. Here, we provide a detailed protocol on the implementation of mechanical actuation of organoids embedded in synthetic 3D microenvironments, with additional details on methods to characterize organoid fate and behavior. Our protocol is easy to reproduce and is expected to be broadly applicable to investigate the role of active mechanics with in vitro model systems.

Targeted mechanical stimulation via magnetic nanoparticles guides in vitro tissue development

Tissues take shape through a series of morphogenetic movements guided by local cell-scale mechanical forces. Current in vitro approaches to recapitulate tissue mechanics rely on uncontrolled self-organization or on the imposition of extrinsic and homogenous forces using matrix or instrument-driven stimulation, thereby failing to recapitulate highly localized and spatially varying forces. Here we develop a method for targeted mechanical stimulation of organoids using embedded magnetic nanoparticles. We show that magnetic clusters within organoids can be produced by sequential aggregation of magnetically labeled and non-labeled human pluripotent stem cells. These clusters impose local mechanical forces on the surrounding cells in response to applied magnetic fields. We show that precise, spatially defined actuation provides short-term mechanical tissue perturbations as well as long-term cytoskeleton remodeling in these organoids, which we term “magnetoids”. We demonstrate that targeted magnetic nanoparticle-driven actuation guides asymmetric tissue growth and proliferation, leading to enhanced patterning in human neural magnetoids. This approach, enabled by nanoparticle technology, allows for precise and locally controllable mechanical actuation in human neural tube organoids, and could be widely applicable to interrogate the role of local mechanotransduction in developmental and disease model systems.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

Genetic mutations in ribosomal biogenesis gene TCOF1 identified in human neural tube defects.

Rare mutations in multiple genes have been associated with human neural tube defects (NTDs), but their causative roles in NTDs disease are poorly understood. Insufficiency of the ribosomal biogenesis gene treacle ribosome biogenesis factor 1(Tcof1) results in cranial NTDs and craniofacial malformations in mice. Here, we aimed to identify genetic association of TCOF1 with human NTDs. High-throughput sequencing targeted on TCOF1was performed on samples from 355humancases affected by NTDs and 225 controls from a Han Chinese population. Four novel missense variants were found in the NTD cohort. Cell-based assays indicated that the p.(A491G) variant carried by an individual, who shows anencephaly and single-nostril abnormality, attenuates production of total proteins, suggesting a loss-of-function mutation in ribosomal biogenesis. Importantly, this variant promotes nucleolar disruption and stabilizes p53 protein, highlighting an unbalancing effect on cell apoptosis. This study explored the functional impact of a missense variant in TCOF1, implicating a set of novel causative biological factors involved in the pathogenicity of human NTDs, particularly whom combined with craniofacial abnormality.

Micronutrient Balance Related to Neural Tube Defects and Prevention

Neural tube defects (NTDs) are a common congenital disorder resulting from failed neural tube formation, the precursor of the brain and spinal cord. The complex etiology of NTDs involve both genetic and environmental factors, thus investigating gene‐environment interactions is critical to understanding how NTDs occur or how NTDs may be prevented. For example, iron deficiency is among the most prevalent micronutrient deficiencies in pregnancy and there is evidence that iron deficiency can increase the risk of NTDs. Folic acid (FA) fortification in grain was implemented in the US in 1998 and now in many countries for the purpose of prevention of neural tube defects (NTDs). In 2017, the US Preventive Services Task Force further recommended the consumption of FA‐containing multivitamin/minerals (MVM) for women of childbearing age. Despite the great benefit of FA on NTD prevention, NTDs remain a serious risk.Our studies focus on mouse models of NTDs and I will discuss two projects. First, the benefits of MVM supplementation are emphasized, but a comparison between MVM supplementation and FA alone on neural tube closure and organ development has not been evaluated, particularly in the context of genetic mutations that lead to NTDs. Using a set of NTD models we have compared MMV to FA supplementation. MVM supplementation shows better improvement than FA alone in the penetrance of spinal and cranial NTDs. We are also using human iPSCs to generate organoids that assume a shape similar to the human spinal neural tube, and display neural tube characteristics, for example apical constriction and interkinetic nuclear migration. Subjecting the organoids to various molecular perturbations, such as Rho‐kinase inhibitor‐induced defects in apical constriction, we have found that both MVM and FA largely prevents this molecular deficit. The goal is to use the mouse NTD model and organoid system to better understand how FA/MVM act to prevent NTDs.Second, we are striving to move beyond the relatively non‐specific recommendations of FA/MVM supplementation to more personalized therapies that take into account the molecular underpinning of the NTD. For example, Iron homeostasis is not only controlled by the concentration of iron in diet, but by proteins responsible for its cellular uptake and metabolism. Sorting nexin 3 (Snx3) is known to regulate recycling of the transferrin receptor (TFRC), a mechanism necessary for cellular uptake of iron, and when Snx3 is knocked out in mice, embryos develop cranial NTDs. We hypothesize that in Snx3 mutants, mistrafficking of TFRC results in disrupted iron homeostasis which contributes to the failure of NT closure. Our current studies are evaluating whether intracellular iron levels are altered in Snx3 mutants and whether maternal diets supplemented with iron can prevent NTDs in the Snx3 background. The hope is that by taking into account the biological process that is disrupted, more tailored therapies can be envisioned to better prevent NTDs.

Convergent extension requires adhesion-dependent biomechanical integration of cell crawling and junction contraction.

SUMMARYConvergent extension (CE) is an evolutionarily conserved collective cell movement that elongates several organ systems during development. Studies have revealed two distinct cellular mechanisms, one based on cell crawling and the other on junction contraction. Whether these two behaviors collaborate is unclear. Here, using live-cell imaging, we show that crawling and contraction act both independently and jointly but that CE is more effective when they are integrated via mechano-reciprocity. We thus developed a computational model considering both crawling and contraction. This model recapitulates the biomechanical efficacy of integrating the two modes and further clarifies how the two modes and their integration are influenced by cell adhesion. Finally, we use these insights to understand the function of an understudied catenin, Arvcf, during CE. These data are significant for providing interesting biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

Rare variants in TULP3 abolish the suppressive effect on sonic hedgehog signaling and contribute to human neural tube defects

Rare variants in TULP3 abolish the suppressive effect on sonic hedgehog signaling and contribute to human neural tube defects

Human neural tube morphogenesis in a dish.

Human neural tube morphogenesis in a dish.

Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides anew route to the study of human organ morphogenesis in health and disease.

Single-cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human-specific features.

ABSTRACTThe spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly distinct neuronal subtypes generated in a characteristic spatiotemporal arrangement from progenitors in the embryonic neural tube. To gain insight into the diversity and complexity of cells in the developing human neural tube, we used single-cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie stages (CS) CS12, CS14, CS17 and CS19 from gestational weeks 4-7. Analysis of progenitor and neuronal populations from the neural tube and dorsal root ganglia identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with mouse revealed overall similarity of mammalian neural tube development while highlighting some human-specific features. These data provide a catalogue of gene expression and cell type identity in the human neural tube that will support future studies of sensory and motor control systems. The data can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

Actuation enhances patterning in human neural tube organoids

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

Organoids as a new model system to study neural tube defects.

The neural tube is the first critically important structure that develops in the embryo. It serves as the primordium of the central nervous system; therefore, the proper formation of the neural tube is essential to the developing organism. Neural tube defects (NTDs) are severe congenital defects caused by failed neural tube closure during early embryogenesis. The pathogenesis of NTDs is complicated and still not fully understood even after decades of research. While it is an ethically impossible proposition to investigate the in vivo formation process of the neural tube in human embryos, a newly developed technology involving the creation of neural tube organoids serves as an excellent model system with which to study human neural tube formation and the occurrence of NTDs. Herein we reviewed the recent literature on the process of neural tube formation, the progress of NTDs investigations, and particularly the exciting potential to use neural tube organoids to model the cellular and molecular mechanisms underlying the etiology of NTDs.

Somatic and de novo Germline Variants of MEDs in Human Neural Tube Defects.

BackgroundNeural tube defects (NTDs) are among the most common and severe congenital defects in humans. Their genetic etiology is complex and remains poorly understood. The Mediator complex (MED) plays a vital role in neural tube development in animal models. However, no studies have yet examined the role of its human homolog in the etiology of NTDs.MethodsIn this study, 48 pairs of neural lesion site and umbilical cord tissues from NTD and 21 case-parent trios were involved in screening for NTD-related somatic and germline de novo variants. A series of functional cell assays were performed. We generated a Med12 p.Arg1784Cys knock-in mouse using CRISPR/Cas9 technology to validate the human findings.ResultsOne somatic variant, MED12 p.Arg1782Cys, was identified in the lesion site tissue from an NTD fetus. This variant was absent in any other normal tissue from different germ layers of the same case. In 21 case-parent trios, one de novo stop-gain variant, MED13L p.Arg1760∗, was identified. Cellular functional studies showed that MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical-WNT signaling pathway. The Med12 p.Arg1784Cys knock-in mouse exhibited exencephaly and spina bifida.ConclusionThese findings provide strong evidence that functional variants of MED genes are associated with the etiology of some NTDs. We demonstrated a potentially important role for somatic variants in the occurrence of NTDs. Our study is the first study in which an NTD-related variant identified in humans was validated in mice using CRISPR/Cas9 technology.

Hypermethylation of PI3K-AKT signalling pathway genes is associated with human neural tube defects

ABSTRACT Neural tube defects (NTDs) are a group of common and severe congenital malformations. The PI3K-AKT signalling pathway plays a crucial role in the neural tube development. There is limited evidence concerning any possible association between aberrant methylation in PI3K-AKT signalling pathway genes and NTDs. Therefore, we aimed to investigate potential associations between aberrant methylation of PI3K-AKT pathway genes and NTDs. Methylation studies of PI3K-AKT pathway genes utilizing microarray genome-methylation data derived from neural tissues of ten NTD cases and eight non-malformed controls were performed. Targeted DNA methylation analysis was subsequently performed in an independent cohort of 73 NTD cases and 32 controls to validate the methylation levels of identified genes. siRNAs were used to pull-down the target genes in human embryonic stem cells (hESCs) to examine the effects of the aberrant expression of target genes on neural cells. As a result, 321 differentially hypermethylated CpG sites in the promoter regions of 30 PI3K-AKT pathway genes were identified in the microarray data. In target methylation analysis, CHRM1, FGF19, and ITGA7 were confirmed to be significantly hypermethylated in NTD cases and were associated with increased risk for NTDs. The down-regulation of FGF19, CHRM1, and ITGA7 impaired the formation of rosette-like cell aggregates. The down-regulation of those three genes affected the expression of PAX6, SOX2 and MAP2, implying their influence on the differentiation of neural cells. This study for the first time reported that hypermethylation of PI3K-AKT pathway genes such as CHRM1, FGF19, and ITGA7 is associated with human NTDs.

Loss-of-function or gain-of-function variations in VINCULIN (VCL) are risk factors of human neural tube defects.

BackgroundNeural tube defects (NTDs) are severe birth defects resulting from the failure of neural tube closure during embryogenesis. Both genetic and environmental factors contribute to the occurrence of NTDs and the heritability of NTDs is approximately 70%. As a key component of focal adhesions, Vinculin (VCL) plays pivotal roles in cell skeleton remodeling and signal transduction. Vcl deficient mice displayed NTD, but how VCL variants contribute to human NTDs has not been addressed yet.MethodsWe screened VCL variants in a Chinese cohort of 387 NTDs and 244 controls by targeted next‐generation sequencing.ResultsWe identified four case‐specific VCL variations (p.M209L, p.D256fs, p.L555V and p.R586Q). VCL p.D256fs and p.L555V are novel variations that have never been reported. Our analysis revealed that p.D256fs is a loss‐of‐function variant, while p.L555V showed a gain of function in planner cell polarity (PCP) pathway regulation and cell migration, probably due to its enhanced protein stability.ConclusionOur study reports human NTD specific novel variations in VCL and provides the functional evaluation of VCL variants related to the etiology of human NTDs.

Neuroepithelial Organoid Patterning is Mediated by Wnt-Driven Turing Mechanism

Cell patterning in epithelia is critical for the establishment of tissue function during development. The organization of patterns in these tissues is mediated by the interpretation of signals operating across multiple length scales. How epithelial tissues coordinate changes in cell identity across these length scales to orchestrate cellular rearrangements and fate specification remains poorly understood. Here, we use human neural tube organoids as model systems to interrogate epithelial patterning principles that guide domain specification. In silico modeling of the patterning process by cellular automata, validated by in vitro experiments, reveal that the initial positions of floor plate cells, coupled with activator-inhibitor signaling interactions, deterministically dictate the patterning outcome according to a discretized Turing reaction-diffusion mechanism. This model predicts an enhancement of organoid patterning by modulating inhibitor levels. Receptor-ligand interaction analysis of scRNAseq data from multiple organoid domains reveals WNT-pathway ligands as the specific inhibitory agents, thereby allowing for the experimental validation of model predictions. These results demonstrate that neuroepithelia employ reaction-diffusion-based mechanisms during early embryonic human development to organize cellular identities and morphogen sources to achieve patterning. The wider implementation of such in vitro organoid models in combination with in-silico agent-based modeling coupled to receptor-ligand analysis of scRNAseq data opens avenues for a broader understanding of dynamic tissue patterning processes.

Convergent Extension Requires Adhesion-Dependent Biomechanical Integration of Cell Crawling and Junction Contraction

Convergent extension is an evolutionarily conserved collective cell movement that elongates the body axis of all animals and is required for the morphogenesis of several organ systems. Decades of study have revealed two distinct mechanisms of cell movement during CE, one based on cell crawling and the other on junction contraction. How these two behaviors collaborate during CE is not understood. Here, using quantitative live cell imaging we show that these two modes act both independently and in concert during CE, but that cell movement is more effective when the two modes are integrated via mechano-reciprocity. Based on these findings, we developed a novel computational model that for the first time treats crawling and contraction independently. This model not only confirmed the biomechanical efficacy of integrating the two modes, but also revealed for the first time how the two modes -and their integration- are influenced by cell adhesion. Finally, we use these new insights to further understand the complex CE phenotype resulting from loss of the C-cadherin interacting catenin Arvcf. These data are significant for providing new biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.