Flat Neural Plate Research Articles

Plate-curving cell culture as a model for assessing curvature-induced cellular responses during neural tube morphogenesis

ABSTRACT Neurulation is an important shape-transforming event during embryonic development where a flat neural plate is converted into a neural tube. Failure in this morphogenetic process accounts for one of the most common birth defects. Mechanical biology has provided key insights into neural tube formation and curvature among many physical properties that are eliciting attention. However, the lack of a proper model to study the effect of curvature has limited the potential to reveal its role in neurulation. In this study, we introduce a novel cell culture method called plate-curving cell culture where a polydimethylsiloxane (PDMS) plate of desired physical properties is curved in either a concave or convex form while the human pluripotent stem cell culture induced to have early neural plate identity is placed on top of its surface. With this method, we observed the elongation of cell colony morphology, as well as the perpendicular alignment of the cell division axis in the concave surface; the oriented cell division does not seem to explain the colony elongation. Transcriptome comparison in search of alternate possibilities suggested selectively altered pathways in the concave surface culture. Our new method is widely available, easy-to-use and culture-friendly, facilitating future mechanobiological studies of neurulation.

Misregulation of cell adhesion molecules in the Ciona neural tube closure mutant bugeye

Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure

During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, −4 and −8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Temperature Sensitivity of Neural Tube Defects in Zoep Mutants.

Neural tube defects (NTD) occur when the flat neural plate epithelium fails to fold into the neural tube, the precursor to the brain and spinal cord. Squint (Sqt/Ndr1), a Nodal ligand, and One-eyed pinhead (Oep), a component of the Nodal receptor, are required for anterior neural tube closure in zebrafish. The NTD in sqt and Zoep mutants are incompletely penetrant. The penetrance of several defects in sqt mutants increases upon heat or cold shock. In this project, undergraduate students tested whether temperature influences the Zoep open neural tube phenotype. Single pairs of adults were spawned at 28.5°C, the normal temperature for zebrafish, and one half of the resulting embryos were moved to 34°C at different developmental time points. Analysis of variance indicated temperature and clutch/genetic background significantly contributed to the penetrance of the open neural tube phenotype. Heat shock affected the embryos only at or before the midblastula stage. Many factors, including temperature changes in the mother, nutrition, and genetic background, contribute to NTD in humans. Thus, sqt and Zoep mutants may serve as valuable models for studying the interactions between genetics and the environment during neurulation.

Morphogenetic movements in the neural plate and neural tube: mouse.

The neural tube (NT), the embryonic precursor of the vertebrate brain and spinal cord, is generated by a complex and highly dynamic morphological process. In mammals, the initially flat neural plate bends and lifts bilaterally to generate the neural folds followed by fusion of the folds at the midline during the process of neural tube closure (NTC). Failures in any step of this process can lead to neural tube defects (NTDs), a common class of birth defects that occur in approximately 1 in 1000 live births. These severe birth abnormalities include spina bifida, a failure of closure at the spinal level; craniorachischisis, a failure of NTC along the entire body axis; and exencephaly, a failure of the cranial neural folds to close which leads to degeneration of the exposed brain tissue termed anencephaly. The mouse embryo presents excellent opportunities to explore the genetic basis of NTC in mammals; however, its in utero development has also presented great challenges in generating a deeper understanding of how gene function regulates the cell and tissue behaviors that drive this highly dynamic process. Recent technological advances are now allowing researchers to address these questions through visualization of NTC dynamics in the mouse embryo in real time, thus offering new insights into the morphogenesis of mammalian NTC.

Apicobasal polarity and neural tube closure

During development, a flat neural plate rolls up and closes to form a neural tube. This process, called neural tube closure, is complex and requires morphogenetic events to occur along multiple axes of the neural plate. Recent studies suggest that cell and tissue polarity play a major role in neural tube morphogenesis. While the planar cell polarity pathway is known to be involved in this process, a role for the apicobasal polarity pathway has only recently begun to be elucidated. These studies show that bone morphogenetic proteins can regulate the apicobasal polarity pathway in the neural plate in a cell cycle dependent manner. This dynamically modulates apical junctions in the neural plate, resulting in cell and tissue shape changes that help bend, shape and close the neural tube.

Mouse as a model for multifactorial inheritance of neural tube defects

Neural tube defects (NTDs) such as spina bifida and anencephaly are some of the most common structural birth defects found in humans. These defects occur due to failures of neurulation, a process where the flat neural plate rolls into a tube. In spite of their prevalence, the causes of NTDs are poorly understood. The multifactorial threshold model best describes the pattern of inheritance of NTDs where multiple undefined gene variants interact with environmental factors to cause an NTD. To date, mouse models have implicated a multitude of genes as required for neurulation, providing a mechanistic understanding of the cellular and molecular pathways that control neurulation. However, the majority of these mouse models exhibit NTDs with a Mendelian pattern of inheritance. Still, many examples of multifactorial inheritance have been demonstrated in mouse models of NTDs. These include null and hypomorphic alleles of neurulation genes that interact in a complex fashion with other genetic mutations or environmental factors to cause NTDs. These models have implicated several genes and pathways for testing as candidates for the genetic basis of NTDs in humans, resulting in identification of putative pathogenic mutations in some patients. Mouse models also provide an experimental paradigm to gain a mechanistic understanding of the environmental factors that influence NTD occurrence, such as folic acid and maternal diabetes, and have led to the discovery of additional preventative nutritional supplements such as inositol. This review provides examples of how multifactorial inheritance of NTDs can be modeled in the mouse.

Stepwise Maturation of Apicobasal Polarity of the Neuroepithelium Is Essential for Vertebrate Neurulation

During vertebrate neurulation, extensive cell movements transform the flat neural plate into the neural tube. This dynamic morphogenesis requires the tissue to bear a certain amount of plasticity to accommodate shape and position changes of individual cells as well as intercellular cohesiveness to maintain tissue integrity and architecture. For most of the neural plate-neural tube transition, cells are polarized along the apicobasal axis. The establishment and maintenance of this polarity requires many polarity proteins that mediate cell-cell adhesion either directly or indirectly. Intercellular adhesion reduces tissue plasticity and enhances tissue integrity. However, it remains unclear how apicobasal polarity is regulated to meet the opposing needs for tissue plasticity and tissue integrity during neurulation. Here, we show that N-Cad/ZO-1 complex-initiated apicobasal polarity is stabilized by the late-onsetting Lin7c/Nok complex after the extensive morphogenetic cell movements in neurulation. Loss of either N-Cad or Lin7c disrupts neural tube formation. Furthermore, precocious overexpression of Lin7c induces multiaxial mirror symmetry in zebrafish neurulation. Our data suggest that stepwise maturation of apicobasal polarity plays an essential role in vertebrate neurulation.

Development of the vertebrate central nervous system: formation of the neural tube

The developmental process of neurulation involves a series of coordinated morphological events, which result in conversion of the flat neural plate into the neural tube, the primordium of the entire central nervous system (CNS). Failure of neurulation results in neural tube defects (NTDs), severe abnormalities of the CNS, which are among the commonest of congenital malformations in humans. In order to gain insight into the embryological basis of NTDs, such as spina bifida and anencephaly, it is necessary to understand the morphogenetic processes and molecular mechanisms underlying neural tube closure. The mouse is the most extensively studied mammalian experimental model for studies of neurulation, while considerable insight into underlying developmental mechanisms has also arisen from studies in other model systems, particularly birds and amphibians. We describe the process of neural tube formation, discuss the cellular mechanisms involved and highlight recent findings that provide links between molecular signaling pathways and morphogenetic tissue movements.

Prevention of Neural Tube Defects by Periconceptional Use of Folic Acid

Prevention of Neural Tube Defects by Periconceptional Use of Folic Acid

Abnormalities of floor plate, notochord and somite differentiation in the loop-tail ( Lp) mouse: a model of severe neural tube defects

Mouse embryos homozygous for the loop-tail ( Lp) mutation fail to initiate neural tube closure at E8.5, leading to a severe malformation in which the neural tube remains open from midbrain to tail. During initiation of closure, the normal mouse neural plate bends sharply in the midline, at the site of the future floor plate. In contrast, Lp/Lp embryos exhibit a broad region of flat neural plate in the midline, displacing the sites of neuroepithelial bending to more lateral positions. Sonic hedgehog ( Shh) and Netrin1 are expressed in abnormally broad domains in the ventral midline of the E9.5 Lp/Lp neural tube, suggesting over-abundant differentiation of the floor plate. The notochord is also abnormally broad in Lp/Lp embryos with enlarged domains of Shh and Brachyury expression. The paraxial mesoderm shows evidence of ventralisation, with increased expression of the sclerotomal marker Pax1, and diminished expression of the dermomyotomal marker Pax3. While the expression domain of Pax3 does not differ markedly from wild-type, there is a dorsal shift in the domain of Pax6 expression in the neural tube at caudal levels of Lp/Lp embryos. We suggest that the Lp mutation causes excessive differentiation of floor-plate and notochord, with over-production of Shh from these midline structures causing ventralisation of the paraxial mesoderm and, to a lesser extent, the neural tube. Comparison with other mouse mutants suggests that the enlarged floor plate may be responsible for the failure of neural tube closure in Lp/Lp embryos.

Neurulation in the rabbit embryo.

Among a broad range of factors and mechanisms involved in the complex process of neurulation a relationship between the curvature of the craniocaudal body axis and rate of neural tube closure has been proposed, but more examples and models are needed to further substantiate the existence of this relationship. This is particularly true for mammals, where marked differences in embryonic body curvature between species exist. The rabbit embryo has virtually no curvature during the main phase of neurulation and is therefore a suitable model, but neurulation is hardly documented in this species. In the present study, therefore, neural tube closure in the rabbit embryo is presented in detail by morphological and morphometrical parameters, as well as from scanning electron microscopic investigations. At the stages of 6-8 somites, the flat neural plate transforms into a V-shaped neural groove, beginning at the rhombo-cervical level. Between the stages of 8 and 9 somites, multiple closure sites occur simultaneously at three levels: at the incipient pros-mesencephalic transition, at the incipient mes-rhombencephalic transition, and at the level of the first pairs of somites. This results in four transient neuropores. The anterior and rhombencephalic neuropores close between the stages of 9-11 somites. The mesencephalic neuropore is very briefly present. The posterior neuropore is the largest and remains longest. Its tapered (cranial) portion closes fast within somite stages 9-10. Subsequently its wide (caudal) portion closes up to a narrow slit, but further closure slows down till full closure is achieved at the 22-somite stage. In comparing rabbit neurulation with that of chick and mouse, the sequence of multiple site closure resembles that of the mouse embryo, but other important aspects of neurulation resemble those of the chick embryo. In contrast to mouse and chick, no time lag between closure at the three closure sites in the rabbit was seen.

Folic acid and neural tube defects.

The primitive nervous system begins as a flat neural plate 2 weeks after conception, having arisen from ectoderm. This plate becomes indented by a long­ itudinal groove at 20 days with neural folds on the flanks. These folds begin to fuse in the midline forming a cylinder in the middle of the plate and then in a zipper fashion this dorsal closure is promulgated rostrally and caudally resulting in a tubular structure with an open anterior and posterior aperture corresponding to the anterior and the posterior neuropore. At 26 days the anterior neuropore closes and at 29 days the posterior. Factors necessary for the neural tube formation are intrinsic in the neural ectoderm and adjacent mesoderm. A teratological insult in embryogenesis timed to interfere with the closure of the anterior neuropore will result in anencephalus with failed development of the telencephalic vesicle and the normal cerebral hemispheres are replaced by a mass of undifferen­ tiated cells and abnormal vessels. Anencephalus is responsible for about half of all the neural tube defects and is incompatible with life, and the natural history is for spontaneous abortion, still birth or death shortly after delivery. Failure of closure of the posterior neuropore results in an exposed spinal cord. When this is subjected to intrinsic hydrodynamic forces and extrinsic mechanical and chemical forces of the intrauterine environment, then progressive destruction of the spinal cord occurs, which results in the full panoply of permanent neurological damage and deformity recognised at birth as spina bifida (myelomeningoceoele).l Although it is predominantly a neuro-ectodermal abnormality there is often involvement of adjacent mesodermal structures. The resultant spina bifid a deficit includes life-long handicap from motor and sensory paraplegia, deformity, neuropathic bladder and bowel, Chiari II malformation and hydrocepha­ lus etc, with the extent of the deficit depending predominantly on the pattern and level of spinal cord involvement. Encephalocoeles are traditionally considered as closed neural tube defects but they are probably a

Fate mapping the avian epiblast with focal injections of a fluorescent‐histochemical marker: Ectodermal derivatives

A microinjection technique is described for fate mapping the epiblast of avian embryos. It consists of injecting the epiblast of cultured blastoderms with a fluorescent-histochemical marker, examining rhodamine fluorescence at the time of injection in living blastoderms, and assaying for horseradish peroxidase activity in histological sections obtained from the same embryos collected 24 h postinjection. Our results demonstrate that this procedure routinely marks cells, allowing their fates to be determined and prospective fate maps to be constructed. Two such maps are presented for ectodermal derivatives of the epiblast: one for late stages of Hensen's node progression (stages 3c through 4) and one for early stages of node regression (stages 4 + through 5). These new maps have six significant features. First, they show that regardless of whether the node is progressing or regressing, the flat neural plate extends at least 300 microns cranial to, 300 microns bilateral to and 1 mm caudal to the center of Hensen's node. Second, they confirm our previous fate mapping studies based on quail/chick chimeras. Namely, they show that the prenodal midline region of the epiblast forms the floor of the forebrain and the ventrolateral part of the optic vesicles as well as MHP cells (i.e., mainly wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate); in contrast, paranodal and postnodal regions contribute L cells (i.e., mainly spindle-shaped neurepithelial cells constituting the lateral aspects of the neural plate). Third, they reveal a second source of MHP cells, Hensen's node, verifying previous studies of others based on tritiated thymidine labeling. Fourth, they demonstrate, in contrast to studies of other based on vital staining, carbon marking, and chorioallantoic grafting but in accordance with our previous studies based on quail/chick chimeras, that the cells contributing to the four craniocaudal subdivisions of the neural tube (i.e., forebrain, midbrain, hindbrain, and spinal cord) are not yet spatially segregated from one another at the flat neural plate stage, although more cranial neural plate cells tend to form more cranial subdivision and more caudal cells tend to form more caudal subdivisions. Thus, single injections routinely mark multiple neural tube subdivisions. Probable reasons for the discrepancy between our present results and the previous results of others is discussed. Fifth, they suggest that cells contributing to the surface ectoderm and neural plate are not yet completely spatially segregated from one another at the flat neural plate stage, particularly in caudal postnodal regions. Sixth, they delineate the locations of the otic placodes.(ABSTRACT TRUNCATED AT 400 WORDS)

Roles of neuroepithelial cell rearrangement and division in shaping of the avian neural plate

Shaping of the neural plate, one of the most striking events of neurulation, involves rapid craniocaudal extension. In this study, we evaluated the roles of two processes in neural plate extension: neuroepithelial cell rearrangement and cell division. Quail epiblast plugs of constant size were grafted either just rostral to Hensen's node or paranodally and the resulting chimeras were examined at selected times postgrafting. By comparing the size of the original plug, the number of cells it contained and the distribution of cells within it to those same features of the grafts in chimeras, we were able to ascertain that, during transformation of the flat neural plate into the closed neural tube (a period requiring 24 h), the graft undergoes a maximum of 3 rounds of craniocaudal extension (each round of craniocaudal extension was defined as a doubling of graft length, so 3 rounds equaled an 8-fold increase in length). Such extension is accompanied by 2 rounds of cell rearrangement and 2-3 rounds of cell division (cell rearrangement occurred mediolaterally, so each round was defined as a halving of the number of cells in the width of the graft and a doubling of the number of cells in its length; each round of cell division was defined as a doubling of graft cell number). Modeling studies demonstrate that these amounts of cell rearrangement and division are sufficient to approximate the shaping of the neural plate that normally ensues during neurulation, provided that some of the cell division occurs within the longitudinal plane of the neural plate and some within its transverse plane: longitudinal cell division results in craniocaudal extension of the neural plate, whereas transverse cell division results in lateral expansion of the neural plate such as that occurring at its cranial end; cell rearrangement results in craniocaudal extension of the neural plate as well as in its narrowing. In conclusion, our results provide evidence that shaping of the neural plate involves mediolateral cell rearrangement and cell division, with the latter occurring within both the longitudinal and transverse planes of the neural plate.

Cell cycle and neuroepithelial cell shape during bending of the chick neural plate.

Neuroepithelial cells change shape from spindle-like to wedge-like within three restricted areas (hinge points) of the bending neural plate. The mechanisms underlying these localized cell shape changes and the specific role that these changes play in bending are unclear. This study was designed to determine whether changes in neuroepithelial cell shape involve basal cellular expansion owing to alteration of the cell cycle. Neurulating chick embryos were treated with colchicine to arrest and accumulate cells in metaphase, and colchicine indices and cell generation times were calculated for the neural plate. During bending of the neural plate, cell generation time in the median hinge point, which contains predominantly wedge-shaped cells, was significantly longer than that in adjacent lateral areas of the neural plate, which contain predominantly spindle-shaped cells. In addition, cell generation time in the flat neural plate, which contains predominantly spindle-shaped cells and has not yet differentiated into the median hinge point and lateral subdivisions, was identical to that in lateral areas of the bending neural plate but was significantly shorter than that in the median hinge point. These results support the hypothesis that changes in neuroepithelial cell shape from spindle-like to wedge-like involve basal cellular expansion owing to alteration of the cell cycle. Additional tests of this hypothesis and studies on the role of localized cell shape changes in neurulation are in progress.

Quantitative analyses of changes in cell shapes during bending of the avian neural plate

It is widely believed that changes in cell shapes play important roles in the bending or folding of epithelial sheets, but few studies have actually examined cell shapes in such systems. We have determined the percentages of four types of neuroepithelial cells (i.e., spindle, flask, inverted flask, and globular) present during bending of the avian neural plate. Serial transverse plastic sections through seven craniocaudal levels of the neuroepithelium were examined. Four distinct periods of bending were chosen based on the morphology of the neuroepithelium: period I, flat neural plate; period II, midline furrow without elevation of the neural folds; period III, midline furrow with elevation; and period IV, bilateral furrows with convergence of the neural folds. We compared statistically the percentages of different cell types in bending (furrowed) and nonbending regions of the neuroepithelium, as well as changes in cell shapes with time. Our results demonstrate that dramatic changes in cell shapes occur in the midline and bilateral furrows during bending of the neural plate, such that as many as 70% of the neuroepithelial cells in the midline and 55% in the bilateral furrows are wedge shaped by the end of bending. In contrast, less than 35% of the neuroepithelial cells are wedge shaped outside of the three morphological loci of bending. These results support the hypothesis that localized changes in cell morphologies have roles in bending and shaping of the neural plate, but exactly how cells change shapes and what precise roles such changes play in bending remain to be determined.

A reinvestigation of some of the tissue movements involved in the formation of the neural tube and the eye/lens system of Triturus alpestris and Xenopus laevis

ABSTRACT A scheme of eye/lens development suggested by Chanturishvili is described and compared with the ‘classical’ scheme of Spemann. A cine-micrographic examination of the ectodermal movements involved in neural tube formation in Triturus alpestris and an experimental embryological investigation of the process in Xenopus laevis provided data consistent with the classical scheme and irreconcilable with Chanturishvili’s suggestion that the whole eye/lens system originates as a single continuous anlage within the flat neural plate of Amphibia.