Patterning Of Neural Tube Research Articles

Togaram1 is expressed in the neural tube and its absence causes neural tube closure defects

Neural tube closure defect pathomechanisms in human embryonic development are poorly understood. Here we identified spina bifida patients expressing novel variants of TOGARAM gene family. TOGARAM1 has been associated with the ciliopathy Joubert syndrome, but its connection to spina bifida and role in neural development is unknown. We show that Togaram1 is expressed in the neural tube and Togaram1 knockout mice have abnormal cilia, reduced sonic hedgehog (Shh) signalling, abnormal neural tube patterning and display neural tube closure defects. Neural stem cells from Togaram1 knockout embryos showed reduced cilia and defects in Shh signalling. Overexpression in IMCD3 and HEK293 cell of TOGARAM1 carrying the variant found in the spina bifida patient resulted in cilia defect along with reduced pericentriolar material one (PCM1), a critical constituent of centriolar satellites involved in transporting proteins towards the centrosome and primary cilia. Our results demonstrate the role of TOGARAM1 in regulating Shh signalling during early neural development that is critical for neural tube closure and elucidates potential mechanisms whereby the ciliopathy associated gene TOGARAM1 gives rise to spina bifida aperta in humans.

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.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopmentmodels provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

From signalling to form: the coordination of neural tube patterning.

The development of the vertebrate spinal cord involves the formation of the neural tube and the generation of multiple distinct cell types. The process starts during gastrulation, combining axial elongation with specification of neural cells and the formation of the neuroepithelium. Tissue movements produce the neural tube which is then exposed to signals that provide patterning information to neural progenitors. The intracellular response to these signals, via a gene regulatory network, governs the spatial and temporal differentiation of progenitors into specific cell types, facilitating the assembly of functional neuronal circuits. The interplay between the gene regulatory network, cell movement, and tissue mechanics generates the conserved neural tube pattern observed across species. In this review we offer an overview of the molecular and cellular processes governing the formation and patterning of the neural tube, highlighting how the remarkable complexity and precision of vertebrate nervous system arises. We argue that a multidisciplinary and multiscale understanding of the neural tube development, paired with the study of species-specific strategies, will be crucial to tackle the open questions.

Regulation of progenitor cell survival by a novel chromatin remodeling factor during neural tube development.

During development, progenitor cell survival is essential for proper tissue functions, but the underlying mechanisms are not fully understood. Here we show that ERCC6L2, a member of the Snf2 family of helicase-like proteins, plays an essential role in the survival of developing chick neural cells. ERCC6L2 expression is induced by the Sonic Hedgehog (Shh) signaling molecule by a mechanism similar to that of the known Shh target genes Ptch1 and Gli1. ERCC6L2 blocks programmed cell death induced by Shh inhibition and this inhibition is independent of neural tube patterning. ERCC6L2 knockdown by siRNA resulted in the aberrant appearance of apoptotic cells. Furthermore, ERCC6L2 cooperates with the Shh signal and plays an essential role in the induction of the anti-apoptotic factor Bcl-2. Taken together, ERCC6L2 acts as a key factor in ensuring the survival of neural progenitor cells.

Cell mechanics in early vertebrate development: Yap mechanotransduction controls notochord formation and neural tube patterning

A recent study published in Science Advances1 showed the influence of Yap on notochord formation and NT (neural tube) patterning in vertebrate embryonic development, and conducted an in-depth study from the perspective of biomechanical signal mechanotransduction. In addition, this study also explored the possible complex interaction between mechanical signals and gene expression. Together, this study provides new insights into the development mechanism of early vertebrate embryos.

Yap controls notochord formation and neural tube patterning by integrating mechanotransduction with FoxA2 and Shh expression.

Correct notochord and neural tube (NT) formation is crucial to the development of the central nervous system and midline structures. Integrated biochemical and biophysical signaling controls embryonic growth and patterning; however, the underlying mechanisms remain poorly understood. Here, we took the opportunities of marked morphological changes during notochord and NT formation and identified both necessary and sufficient roles of Yap, a key mechanosensor and mechanotransducer, in biochemical signaling activation during formation of notochord and floor plate, the ventral signaling centers that pattern the dorsal-ventral axis of NT and the surrounding tissues. We showed that Yap activation by a gradient of mechanical stress and tissue stiffness in the notochord and ventral NT induces FoxA2 and Shh expression. Hedgehog signaling activation rescued NT patterning defects caused by Yap deficiency, but not notochord formation. Therefore, mechanotransduction via Yap activation acts in feedforward mechanisms to induce FoxA2 expression for notochord formation and activate Shh expression for floor plate induction by synergistically interacting with FoxA2.

Primate gastrulation and early organogenesis at single-cell resolution

Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8–11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species—for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation—and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.

Loss-of-Function of p21-Activated Kinase 2 Links BMP Signaling to Neural Tube Patterning Defects.

Closure of the neural tube represents a highly complex and coordinated process, the failure of which constitutes common birth defects. The serine/threonine kinase p21-activated kinase 2 (PAK2) is a critical regulator of cytoskeleton dynamics; however, its role in the neurulation and pathogenesis of neural tube defects (NTDs) remains unclear. Here, the results show that Pak2-/- mouse embryos fail to develop dorsolateral hinge points (DLHPs) and exhibit craniorachischisis, a severe phenotype of NTDs. Pak2 knockout activates BMP signaling that involves in vertebrate bone formation. Single-cell transcriptomes reveal abnormal differentiation trajectories and transcriptional events in Pak2-/- mouse embryos during neural tube development. Two nonsynonymous and one recurrent splice-site mutations in the PAK2 gene are identified in five human NTD fetuses, which exhibit attenuated PAK2 expression and upregulated BMP signaling in the brain. Mechanistically, PAK2 regulates Smad9 phosphorylation to inhibit BMP signaling and ultimately induce DLHP formation. Depletion of pak2a in zebrafish induces defects in the neural tube, which are partially rescued by the overexpression of wild-type, but not mutant PAK2. The findings demonstrate the conserved role of PAK2 in neurulation in multiple vertebrate species, highlighting the molecular pathogenesis of PAK2 mutations in NTDs.

Cell fate decisions, transcription factors and signaling during early retinal development.

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases.

Transcriptomic architecture of nuclei in the marmoset CNS

To understand the cellular composition and region-specific specialization of white matter — a disease-relevant, glia-rich tissue highly expanded in primates relative to rodents — we profiled transcriptomes of ~500,000 nuclei from 19 tissue types of the central nervous system of healthy common marmoset and mapped 87 subclusters spatially onto a 3D MRI atlas. We performed cross-species comparison, explored regulatory pathways, modeled regional intercellular communication, and surveyed cellular determinants of neurological disorders. Here, we analyze this resource and find strong spatial segregation of microglia, oligodendrocyte progenitor cells, and astrocytes. White matter glia are diverse, enriched with genes involved in stimulus-response and biomolecule modification, and predicted to interact with other resident cells more extensively than their gray matter counterparts. Conversely, gray matter glia preserve the expression of neural tube patterning genes into adulthood and share six transcription factors that restrict transcriptome complexity. A companion Callithrix jacchus Primate Cell Atlas (CjPCA) is available through https://cjpca.ninds.nih.gov.

Pax3/7 regulates neural tube closure and patterning in a non-vertebrate chordate.

Pax3/7 factors play numerous roles in the development of the dorsal nervous system of vertebrates. From specifying neural crest at the neural plate borders, to regulating neural tube closure and patterning of the resulting neural tube. However, it is unclear which of these roles are conserved in non-vertebrate chordates. Here we investigate the expression and function of Pax3/7 in the model tunicate Ciona. Pax3/7 is expressed in neural plate border cells during neurulation, and in central nervous system progenitors shortly after neural tube closure. We find that separate cis-regulatory elements control the expression in these two distinct lineages. Using CRISPR/Cas9-mediated mutagenesis, we knocked out Pax3/7 in F0 embryos specifically in these two separate territories. Pax3/7 knockout in the neural plate borders resulted in neural tube closure defects, suggesting an ancient role for Pax3/7 in this chordate-specific process. Furthermore, knocking out Pax3/7 in the neural impaired Motor Ganglion neuron specification, confirming a conserved role for this gene in patterning the neural tube as well. Taken together, these results suggests that key functions of Pax3/7 in neural tube development are evolutionarily ancient, dating back at least to the last common ancestor of vertebrates and tunicates.

Cilia and their role in neural tube development and defects

Abstract Cilia are microtubule-based filamentous organelles that play a vital role in embryogenesis. Multiple signal transduction pathways are orchestrated by cilia, such as Hedgehog and planar cell polarity signals. Various studies, spanning over last 2 decades, have emphasized the role of cilia-mediated signaling cascades in regulating neural tube patterning and development. Moreover, the deficiency of certain ciliary genes have been reported to cause neural tube defects (NTDs), which are a set of disorders that occur due to perturbation of normal neural tube closure. However, the mechanisms underlying cilia dysfunction resulting NTDs remain unclear. Recent studies have highlighted the association of phosphoinositide signaling with cilia, thereby conferring novel insights into the function of cilia during neural tube development. Here, we have reviewed recent studies on cilia, focusing on the molecular mechanism underlying the involvement of cilia in neural tube development and the role of ciliary disruption in the development of NTDs.

Rnf220 is Implicated in the Dorsoventral Patterning of the Hindbrain Neural Tube in Mice.

Rnf220 is reported to regulate the patterning of the ventral spinal neural tube in mice. The brainstem has divergent connections with peripheral and central targets and contains unique internal neuronal groups, but the role of Rnf220 in the early development of the hindbrain has not been explored. In this study, Nestin-Cre-mediated conditional knockout (Rnf220 Nestin CKO) mice were used to examine if Rnf220 is involved in the early morphogenesis of the hindbrain. Rnf220 showed restricted expression in the ventral half of ventricular zone (VZ) of the hindbrain at embryonic day (E) 10.5, and as development progressed, Rnf220-expressing cells were also present in the mantle zone outside the VZ at E12.5. In Rnf220 Nestin CKO embryos, alterations of progenitor domains in the ventral VZ were observed at E10.5. There were significant reductions of the p1 and p2 domains shown by expression of Dbx1, Olig2, and Nkx6.1, accompanied by a ventral expansion of the Dbx1+ p0 domain and a dorsal expansion of the Nkx2.2+ p3 domain. Different from the case in the spinal cord, the Olig2+ pMN (progenitors of somatic motor neuron) domain shifted and expanded dorsally. Notably, the total range of the ventral VZ and the extent of the dorsal tube were unchanged. In addition, the post-mitotic cells derived from their corresponding progenitor domain, including oligodendrocyte precursor cells (OPCs) and serotonergic neurons (5-HTNs), were also changed in the same trend as the progenitor domains do in the CKO embryos at E12.5. In summary, our data suggest similar functions of Rnf220 in the hindbrain dorsoventral (DV) patterning as in the spinal cord with different effects on the pMN domain. Our work also reveals novel roles of Rnf220 in the development of 5-HTNs and OPCs.

Δ9-Tetrahydrocannabinol inhibits Hedgehog-dependent patterning during development

Many developmental disorders are thought to arise from an interaction between genetic and environmental risk factors. The Hedgehog (HH) signaling pathway regulates myriad developmental processes, and pathway inhibition is associated with birth defects, including holoprosencephaly (HPE). Cannabinoids are HH pathway inhibitors, but little is known of their effects on HH-dependent processes in mammalian embryos, and their mechanism of action is unclear. We report that the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) induces two hallmark HH loss-of-function phenotypes (HPE and ventral neural tube patterning defects) in Cdon mutant mice, which have a subthreshold deficit in HH signaling. THC therefore acts as a 'conditional teratogen', dependent on a complementary but insufficient genetic insult. In vitro findings indicate that THC is a direct inhibitor of the essential HH signal transducer smoothened. The canonical THC receptor, cannabinoid receptor-type 1, is not required for THC to inhibit HH signaling. Cannabis consumption during pregnancy may contribute to a combination of risk factors underlying specific developmental disorders. These findings therefore have significant public health relevance.

Neural tube patterning: From a minimal model for rostrocaudal patterning toward an integrated 3D model

SummaryRostrocaudal patterning of the neural tube is a defining event in vertebrate brain development. This process is driven by morphogen gradients which specify the fate of neural progenitor cells, leading to the partitioning of the tube. Although this is extensively studied experimentally, an integrated view of the genetic circuitry is lacking. Here, we present a minimal gene regulatory model for rostrocaudal patterning, whose tristable topology was determined in a data-driven way. Using this model, we identified the repression of hindbrain fate as promising strategy for the improvement of current protocols for the generation of dopaminergic neurons. Furthermore, we combined our model with an established minimal model for dorsoventral patterning on a realistic 3D neural tube and found that key features of neural tube patterning could be recapitulated. Doing so, we demonstrate how data and models from different sources can be combined to simulate complex in vivo processes.

Exploring Sonic Hedgehog Cell Signaling in Neurogenesis: Its Potential Role in Depressive Behavior.

Depression is the most prevalent form of neuropsychiatric disorder affecting all age groups globally. As per the estimation of the World Health Organization (WHO), depression will develop into the foremost reason for disability globally by the year 2030. The primary neurobiological mechanism implicated in depression remains ambiguous; however, dysregulation of molecular and signaling transductions results in depressive disorders. Several theories have been developed to explain the pathogenesis of depression, however, none of them completely explained all aspects of depressive-pathogenesis. In the current review, we aimed to explore the role of the sonic hedgehog (Shh) signaling pathway in the development of the depressive disorder and its potential as the therapeutic target. Shh signaling has a crucial function in neurogenesis and neural tube patterning during the development of the central nervous system (CNS). Shh signaling performs a basic function in embryogenesis and hippocampal neurogenesis. Moreover, antidepressants are also known to enhance neurogenesis in the hippocampus, which further suggests the potential of Shh signaling. Furthermore, there is decreased expression of a glioma-associated oncogene (Gli1) and Smoothened (Smo) in depression. Moreover, antidepressants also regulate brain-derived neurotrophic factor (BDNF) and wingless protein (Wnt) signaling, therefore, Shh may be implicated in the pathogenesis of the depressive disorder. Deregulation of Shh signaling in CNS results in neurological disorders such as depression.

Neural Tube Patterning: From a Minimal Model for Rostrocaudal Patterning Towards an Integrated 3D Model

Rostrocaudal patterning of the neural tube is a key event in vertebrate brain development. Underlying this process are morphogen gradients which specify the fate of neural progenitor cells dose-dependently, leading to the partitioning of the tube. Although this is extensively studied experimentally, an integrated view of the genetic circuitry is currently lacking. Here, we present a minimal gene regulatory model for rostrocaudal patterning, whose configuration was determined in a data-driven way, resulting in a tristable network motif. Further analysis identified the repression of hindbrain fate as promising strategy for the improvement of current protocols for the generation of dopaminergic neurons. To test our model further, we integrated it with an established model for dorsoventral patterning to simulate the steady state pattern of a physiological 3D neural tube. Simulations of elevated morphogen secretion illustrate the sensitivity of neural tube patterning to morphogen levels, implying that these are tightly regulated during development.

An ontology for developmental processes and toxicities of neural tube closure

In recent years, the development and implementation of animal-free approaches to chemical and pharmaceutical hazard and risk assessment has taken off. Alternative approaches are being developed starting from the perspective of human biology and physiology.Neural tube closure is a vital step that occurs early in human development. Correct closure of the neural tube depends on a complex interplay between proteins along a number of protein concentration gradients. The sensitivity of neural tube closure to chemical disturbance of signalling pathways such as the retinoid pathway, is well known. To map the pathways underlying neural tube closure, literature data on the molecular regulation of neural tube closure were collected. As the process of neural tube closure is highly conserved in vertebrates, the extensive literature available for the mouse was used whilst considering its relevance for humans. Thus, important cell compartments, regulatory pathways, and protein interactions essential for neural tube closure under physiological circumstances were identified and mapped. An understanding of aberrant processes leading to neural tube defects (NTDs) requires detailed maps of neural tube embryology, including the complex genetic signals and responses underlying critical cellular dynamical and biomechanical processes. The retinoid signaling pathway serves as a case study for this ontology because of well-defined crosstalk with the genetic control of neural tube patterning and morphogenesis. It is a known target for mechanistically-diverse chemical structures that disrupt neural tube closureThe data presented in this manuscript will set the stage for constructing mathematical models and computer simulation of neural tube closure for human-relevant AOPs and predictive toxicology.