Netrin‐1 is required for efficient neural tube closure

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell–cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin‐1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin‐1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin‐1 results in delayed neural fold apposition and neural tube closure. We further show that netrin‐1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin‐RGMa interactions, neogenin‐netrin‐1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin‐1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure. © 2012 Wiley Periodicals, Inc., 2013     

Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio

We have studied the process of neurulation within the anterior trunk region of the zebrafish by means of serial sectioning of staged embryos and labelling cells by applications of the dye Dil and intracellular injections of fluoresceine dextran amine. The first morphological manifestation of the prospective neural plate is a dorsomedial ectodermal thickening which becomes visible immediately after gastrulation. Within 1–2 h, by the time somatogenesis begins, two bilaterally symmetrical thickenings have appeared more laterally, which eventually fuse with the medial thickening to form the neural keel. The central canal forms next by separation of the cells on either side of the midline of the neural keel, beginning ventrally at the 17-somite stage and progressing towards dorsal levels. By means of fluorescent dye labelling in the late gastrula, we found that both the medial and lateral thickenings contribute to the nerve cord. The medial thickening was found to contain, exclusively, neural progenitor cells from the 90–100% epiboly stage on, whereas the adjacent regions contained a mixture of neural and epidermal progenitor cells, as well as prospective neural crest cells. Between the 90–100% epiboly and 2-somite stages, this heterogeneity of developmental capabilities is resolved into territories, with epidermogenic and neurogenic cells clearly separated from each other. To achieve this segregation into neural and epidermal anlagen, cells from the lateral thickenings have to move over a distance of roughly 400 m within 1–2 h. Epidermal overgrowth of the nerve cord occurs during the morphogenetic movements that accompany nerve cord formation. Correspondence to: J.A. Campos-Ortega     

Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis

Ascidians are a group of invertebrate chordates that exhibit a biphasic life history, with chordate-specific structures developing during embryogenesis (dorsal neural tube and notochord) and metamorphosis (pharyngeal gill slits and endostyle). Here we characterize the expression of a caudal/Cdx gene homologue, Hec-Cdx, from the ascidian Herdmania curvata. Vertebrate Cdx genes are expressed at gastrulation and in the posterior of the developing neural tube and endoderm. Hec-Cdx expression is initiated at the earliest stages of gastrulation, with peaks in RNA abundance occurring first during neurulation and tailbud extension and then in 3- to 5-day-old juveniles. Hec-Cdx is expressed in a pair of cells in the anterior lip of the blastopore in the late gastrula which form the most posterior portion of the neural plate. During tailbud formation expression is maintained in and solely restricted to these cells. During metamorphosis expression is localized to the intestine of the juvenile. These data, along with data for the H. curvata Otx gene, suggest that the evolution of the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a temporal separation of tissue-specific expression. Received: 10 October 1999 / Accepted: 1 November 1999     

Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.     

Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. II. Neural plate morphogenesis and cell lineages during neurulation

We describe the lineage and morphogenesis of neural plate cells in the ascidian, Ciona intestinalis, from reconstructed cell maps of embryos at 12-min intervals during and after neurulation, between 31 and 61% of embryonic development. Neurulation commences in a posterior to anterior wave following in the wake of the ninth cleavage, when all cells, except possibly four, are in their 10th generation. The neural plate then comprises 76 cells, in up to four posterior rows each of eight vegetal-hemisphere cells, and eight anterior rows each of six animal-hemisphere cells. Two cells are lost from the neural plate to the muscle cell line during neurulation and four cells are gained from ectoderm outside the plate. All cells become wedge-shaped. Simple, stereotyped positional changes transform cells from lateral locations in the plate to posterior locations in the tube; bilateral partners shear their midline positions to form the keel, and ectodermal cells zipper up dorsally to form the capstone, of a tube which is four cells in cross section posteriorly, but more complex anteriorly. Neither cell death nor migration occur during neurulation. Divisions become asynchronous and the cell-cycle extends; 170 10th- to 12th-generation cells exist by the time the neural tube becomes completely internalized. Generally, only one further division is required to complete the lineage analysis, two at the most. Neural plate cell divisions were invariant using our observational methods, and their lineage is compared with that from recent studies of H. Nishida (1987, Dev. Biol. 121, 526-541).     

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes)

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.     

EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel

During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.     

Stepwise modulation of apical orientational cell adhesions for vertebrate neurulation

Neurulation transforms the neuroectoderm into the neural tube. This transformation relies on reorganising the configurational relationships between the orientations of intrinsic polarities of neighbouring cells. These orientational intercellular relationships are established, maintained, and modulated by orientational cell adhesions (OCAs). Here, using zebrafish (Danio rerio) neurulation as a major model, we propose a new perspective on how OCAs contribute to the parallel, antiparallel, and opposing intercellular relationships that underlie the neural plate–keel–rod–tube transformation, a stepwise process of cell aggregation followed by cord hollowing. We also discuss how OCAs in neurulation may be regulated by various adhesion molecules, including cadherins, Eph/Ephrins, Claudins, Occludins, Crumbs, Na⁺/K⁺-ATPase, and integrins. By comparing neurulation among species, we reveal that antiparallel OCAs represent a conserved mechanism for the fusion of the neural tube. Throughout, we highlight some outstanding questions regarding OCAs in neurulation. Answers to these questions will help us understand better the mechanisms of tubulogenesis of many tissues.     

Stretching cell morphogenesis during late neurulation and mild neural tube defects

Neurulation is defined as a process of neural tube closure. Recent reports suggested that upon completion of this process the major factors of neurulation remain in force at least until the central canal of the neural tube is formed. Hence, an idea has been put forward to define the two periods of neurulation: early neurulation corresponds to the period of neural tube closure and late neurulation corresponds to the period of formation of the central canal. These ideas are discussed in a context of neural tube defects that may affect late neurulation and result in distention of the central canal.     

Neural fold and neural crest movement in the Mexican salamander Ambystoma mexicanum

In studies of amphibian neurulation, the terms "neural ridge," "neural fold," and "neural crest" are sometimes used as synonyms. This has occasionally led to the misconception that grafting of the neural crest is equivalent to grafting of the neural fold. The neural fold, however, is composed of three parts: the neural crest, prospective neural tube tissue, and epidermis. In order to investigate how these neural fold components move during neurulation, time-lapse photography, electron microscopy, and grafting were performed. Ambystoma mexicanum embryos were photographed during neurulation at regular intervals. The photographs were analyzed to find the position of those cells at beginning of neurulation that end up on the line of fusion as the neural folds close. Posteriorly, these cells are already on the emerging neural fold. In the anterior neural folds, however, these cells are located in the lateral epidermis. Electron microscopy of the neural folds confirms the presence of epidermis. To follow the movement of the cells differentiating into melanophores (neural crest), neural fold parts were grafted into albino hosts. The crest cells differentiating into melanophores following ectopic grafting are located in the flank of the neural fold that is in contact with the neural plate. In grafts from the outside (distal) flank, no melanophores developed. Semithin sections show that the third part of the neural fold consists of apically constricted cells known to differentiate into neural tissue. Because the neural folds consist of epidermis, neural tissue, and neural crest, neural fold and neural crest cannot be used as synonyms.     

The polarization of neuroepithelial cells when a portion of the neural plate is inserted into the neural tube cavity in amphibian embryos

Portions of homologous neuroepithelium of various length were introduced into the neural tube cavity in Rana arvalis and Pleurodeles waltlii late neurula. The neural tube in R. arvalis was destroyed independently of the transplant length. In P. waltlii neural primordia were deformed if a large transplant prevented closing of the neural tube. Redundant increase in the host cells length (polarization) was observed during the small transplant-induced retardation of neurulation. Recipient cells elongated 1.5-2 fold as compared to the normal ones. Zones of contact polarization were detected. They arise in slowly relaxing layer under conditions during the transplant-induced retardation of neurulation. The labelled TRITC cells introduced into the neural tube cavity integrated into the wall of the neural tube and retarded polarization of its cells.     

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.     

Experimental Analyses of the Shaping of the Neural Plate and Tube

Understanding the changing morphology of an embryo presentsspecial challenges. Analyses of neurulation in vertebrate embryosdescribed here required observation from sectioned materialand from time-lapse movies, modeling, computer simulation, andexperiments. All these approaches were essential, and each approachhelped guide the use of the others. Experiments have the specialrole of letting the embryo decide between our alternative hypotheses. In the newt embryo, induction and patterning events establishin the ectoderm boundaries between epidermis and neural plate,and between neural plate and the notoplate at its midline. Thedifferentdomains of cells thus established—epidermis, neural plateand notoplate—develop different adhesive properties suchthat cell motility behavior along the notoplate boundary andalong the spinal cord/epidermis boundary produces forceful intercalationof cells which lengthens the boundaries and distorts (lengthens)the neuroepithelium. Neural plate cells also attempt to crawlbeneath the epidermis along their common boundary, raising neuralfolds and producing a rolling moment directed mediad that islargely responsible for neural tube formation. Both cell motilitythat leads to columnarization of neural plate cells and contractionof organized subapical microfilament bundles reduce the apicalsurface area of the neural plate cells and produce an apicaltension that aids neural tube formation. Cell relocation reducesthe width of the neural plate and increases its length, andthe Poisson buckling forces resulting from this elongation ofthe plate also aid neural tube formation. The newt embryo accomplishes neurulation without growth, butbird and mammal embryos grow during neurulation. Understandingthe organization of the products of growth in the amniote neuralplate is critical in determining whether growth helps or hindersneurulation.     

Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.     

Temporal and spatial requirements for Nodal‐induced anterior mesendoderm and mesoderm in anterior neurulation

Zebrafish with defective Nodal signaling have a phenotype analogous to the fatal human birth defect anencephaly, which is caused by an open anterior neural tube. Previous work in our laboratory found that anterior open neural tube phenotypes in Nodal signaling mutants were caused by lack of mesendodermal/mesodermal tissues. Defects in these mutants are already apparent at neural plate stage, before the neuroepithelium starts to fold into a tube. Consistent with this, we found that the requirement for Nodal signaling maps to mid‐late blastula stages. This timing correlates with the timing of prechordal plate mesendoderm and anterior mesoderm induction, suggesting these tissues act to promote neurulation. To further identify tissues important for neurulation, we took advantage of the variable phenotypes in Nodal signaling‐deficient sqt mutant and Lefty1‐overexpressing embryos. Statistical analysis indicated a strong, positive correlation between a closed neural tube and presence of several mesendoderm/mesoderm‐derived tissues (hatching glands, cephalic paraxial mesoderm, notochord, and head muscles). However, the neural tube was closed in a subset of embryos that lacked any one of these tissues. This suggests that several types of Nodal‐induced mesendodermal/mesodermal precursors are competent to promote neurulation. genesis 54:3–18, 2016. © 2016 Wiley Periodicals, Inc.     

Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo

Background

The neural tube is formed by morphogenetic movements largely dependent on cytoskeletal dynamics. Actin and many of its associated proteins have been proposed as important mediators of neurulation. For instance, mice deficient in MARCKS, an actin cross-linking membrane-associated protein that is regulated by PKC and other kinases, present severe developmental defects, including failure of cranial neural tube closure.

Results

To determine the distribution of MARCKS, and its possible relationships with actin during neurulation, chick embryos were transversely sectioned and double labeled with an anti-MARCKS polyclonal antibody and phalloidin. In the neural plate, MARCKS was found ubiquitously distributed at the periphery of the cells, being conspicuously accumulated in the apical cell region, in close proximity to the apical actin meshwork. This asymmetric distribution was particularly noticeable during the bending process. After the closure of the neural tube, the apically accumulated MARCKS disappeared, and this cell region became analogous to the other peripheral cell zones in its MARCKS content. Actin did not display analogous variations, remaining highly concentrated at the cell subapical territory. The transient apical accumulation of MARCKS was found throughout the neural tube axis. The analysis of another epithelial bending movement, during the formation of the lens vesicle, revealed an identical phenomenon.

Conclusions

MARCKS is transiently accumulated at the apical region of neural plate and lens placode cells during processes of bending. This asymmetric subcellular distribution of MARCKS starts before the onset of neural plate bending. These results suggest possible upstream regulatory actions of MARCKS on some functions of the actin subapical meshwork.