Patterns of neurepithelial cell rearrangement during avian neurulation are determined prior to notochordal inductive interactions

In the epiblast of elongating primitive-streak-stage avian embryos, MHP cells--short wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate--arise from the midline prenodal and nodal area, whereas L cells--tall spindle-shaped neurepithelial cells constituting the lateral neural plate--arise from paired areas flanking the cranial primitive streak. These characteristic differences in neurepithelial cell shape are acquired as a result of inductive interactions with the notochord. Both MHP and L cells undergo extensive rearrangement (intercalation) during shaping and bending of the neural plate, but their pattern of rearrangement differs. MHP cells intercalate with other MHP cells and the population always spans the midline, whereas L cells intercalate with other L cells, remaining in bulk lateral to the midline. The following experiment was performed to establish whether these distinctive rearrangement patterns are determined prior to notochordal inductive interactions. Quail prospective MHP and L cells were transplanted isochronically and heterotopically to chick host blastoderms at stages prior to formation of the notochord (to wit, prospective MHP cells were transplanted into prospective L cell territory and vice versa) and the distribution, fate, and morphological characteristics of grafted cells were determined in chimeras collected 24 hr later. Our results demonstrate that heterotopic MHP and L cells do not adopt the rearrangement pattern characteristic of their new site; rather, they change their position so that grafted MHP cells intermix with MHP cells of the host and grafted L cells intermix with L cells of the host. Thus, patterns of neurepithelial cell rearrangement are determined prior to notochordal inductive interactions. When and how this determination occurs are topics for further studies.     

Induction of an additional floor plate in the neural tube

The role of the notochord in the morphogenesis of the neural tube was investigated by implanting a notochord fragment laterally to the neural wall of a 1.5 day chick embryo. Embryos were sacrificed at 4 days. In the basal part of the neural tube an additional floor plate was induced in the vicinity of the implant. This floor plate was characterized by a low proliferative activity, a thin wall, spindle-like nuclei crowded peripherally and some neuroblast-like cells. It was either blending with the natural floor plate or separated from it, depending on the exact position of the implant. In the latter case neuroblasts were observed in between both floor plates. The additional floor plate was present only when the implanted notochord was less than 25 micron apart from the neural tube; at larger distance an increase of the ventral horn neuroblast area could be seen. It is concluded that the implanted notochord is able to induce a floor plate at 1.5 days of incubation. The specific influence of the notochord on the morphogenesis of the neural tube, its inductive period as well as the presence of the neuroblast-like cells in the additional floor plate are discussed.     

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.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).     

Bone morphogenetic proteins regulate neural tube closure by interacting with the apicobasal polarity pathway

During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.     

Sonic hedgehog and the molecular regulation of mouse neural tube closure

Neural tube closure is a fundamental embryonic event whose molecular regulation is poorly understood. As mouse neurulation progresses along the spinal axis, there is a shift from midline neural plate bending to dorsolateral bending. Here, we show that midline bending is not essential for spinal closure since, in its absence, the neural tube can close by a 'default' mechanism involving dorsolateral bending, even at upper spinal levels. Midline and dorsolateral bending are regulated by mutually antagonistic signals from the notochord and surface ectoderm. Notochordal signaling induces midline bending and simultaneously inhibits dorsolateral bending. Sonic hedgehog is both necessary and sufficient to inhibit dorsolateral bending, but is neither necessary nor sufficient to induce midline bending, which seems likely to be regulated by another notochordal factor. Attachment of surface ectoderm cells to the neural plate is required for dorsolateral bending, which ensures neural tube closure in the absence of sonic hedgehog signaling.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.     

Loss of Occludin and Functional Tight Junctions, but Not ZO-1, during Neural Tube Closure—Remodeling of the Neuroepithelium Prior to Neurogenesis

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.     

Fate mapping the avian neural plate with quail/chick chimeras: origin of prospective median wedge cells

The origin of prospective M cells, which are median neuroepithelial cells that become wedge-shaped during bending of the neural plate and eventually form the midline floor of the neural tube, was determined by constructing quail/chick chimeras and using the quail nucleolar marker to identify quail donor cells in chick host blastoderms. Two possible sites of prospective M-cell origin in the epiblast were examined: a single, midline rudiment located just rostral to Hensen's node and paired rudiments flanking the cranial part of the primitive streak. Our results suggest that M cells arise exclusively from the midline, prenodal rudiment. From this rudiment, M cells extend caudally throughout the entire length of the neuroepithelium. This new information on the origin of prospective M cells will aid in the analysis of their role in neurulation.     

Biomechanical basis of diazepam-induced neural tube defects in early chick embryos: a morphometric study

The biomechanical basis of diazepam (Valium/Roche)-induced neural tube defects in the chick was investigated using a combination of electron microscopy and morphometry. Embryos at stage 8 (four-somite stage) of development were explanted and grown for 6 hr in nutrient medium containing 400 micrograms/ml diazepam. Nearly 80% of these embryos exhibited neural tube defects that were most pronounced in the forming midbrain region and typified by a "relaxation" or "collapse" of neural folds. The hindbrain and spinal cord regions were less affected. Electron microscopy revealed that neuroepithelial cells in diazepam-treated embryos had smoother apical surfaces and broader apical widths than did controls. Morphometric measurements supported this observation and further showed that these effects were focused at sites within the wall of the forming neural tube that typically exhibit the greatest degree of bending and apical constriction (i.e., the floor and midlateral walls). Overall results indicate that neural tube defects associated with exposure to diazepam are due largely to a general inhibition of the contractile activity of apical microfilament bundles in neuroepithelial cells. These findings 1) emphasize the important contribution of microfilament-mediated apical constriction of neuroepithelial cells in providing the driving forces for bending of the neuroepithelium during neural tube formation and 2) suggest that agents or conditions that impair their contractile activity could play a role in the pathogenesis of certain types of neural tube defects.     

Effect of the notochord on proliferation and differentiation in the neural tube of the chick embryo

After implantation of a notochord fragment lateral to the neural tube in a 2-day chick embryo, at 4 days the ipsilateral neural tube half was increased in size and axons left the neural tube in a broad dorsoventral area (van Straaten et al. 1985). This enlargement appears to coincide with an increased area of AChE-positive basal plate neuroblasts, as determined with scan-cytophotometry. The effect was ipsilateral and local: clear effects were seen only when the implant was localized less than 80 microns from the neural tube and over 120 microns from the ventral notochord. In order to investigate the expected enhancement of proliferation, the mitotic density and the number of cells at the site of the implant at 3 days was determined and the mitotic index calculated. All three parameters showed an increase. It was concluded that the cell cycle was shorter in the implant area relative to the control area, at least during the third day. At 4 days the number of cells was still increased, predominantly in the basal plate. It appeared that the numerical increase was for the larger part due to neuroblasts. The synergism of two notochords thus resulted in enhancement of proliferation and differentiation in the neural tube. It is suggested that the notochord merely regulates and arranges the surrounding sclerenchymal cells, which are the effective cells in the regulation of neural tube development.     

Electrophysiological evidence for low-resistance intercellular junctions in the early chick embryo

Electrophysiological evidence is presented for the exchange of small ions directly between cells interiors, i.e. "electrical coupling," in the early chick embryo. Experiments with intracellular marking show that coupling is widespread, occurring between cells in the same tissue, e.g. ectoderm, notochord, neural plate, mesoderm, and Hensen's node, and between cells in different tissues, e.g. notochord to neural plate, notochord to neural tube, notochord to mesoderm. The coupling demonstrates the presence of specialized low-resistance intercellular junctions as found in other embryos and numerous adult tissues. The results are discussed in relation to recent electron microscopical studies of intercellular junctions in the early chick embryo. The function of the electrical coupling in embryogenesis remains unknown, but some possibilities are considered.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.     

A critical period for conversion of ectodermal cells to a neural crest fate

Previously, we found that interactions between neural and nonneural ectoderm can generate neural crest cells, with both the ectodermal and the neuroepithelial cells contributing to induced population (M. A. J. Selleck and M. Bronner-Fraser, 1995, Development 121, 525-538). To further characterize the ability of ectodermal cells to form neural crest, we have challenged their normal fate by transplanting them into the neural tube. To ensure that the ectoderm was from nonneural regions, we utilized extraembryonic ectoderm (the proamnion) and transplanted it into the presumptive midbrain of 1. 5-day-old chick embryos. We observed that the grafted ectoderm has the capacity to adopt a neural crest fate, responding within a few hours of surgery by turning on neural crest markers HNK-1 and Slug. However, the competence of the ectoderm to respond to neural crest-inducing signals is time limited, declining rapidly in donors older than the 10-somite stage. Similarly, the inductive capacity of the host midbrain declines in a time-dependent fashion. Our results show that extraembryonic ectoderm has the capacity to form neural crest cells given proper inducing signals, expressing both morphological and molecular markers characteristic of neural crest cells.     

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)     

Early neurogenesis in Amniote vertebrates

Labelling of Hensen's node in a 6-somite stage chick embryo by the quail/chick chimera method has revealed that, while moving caudalwards as the embryo elongates, the node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endoderm. The node itself contains cells endowed with the capacity to yield midline cells (i.e. notochord and floor plate) along the whole length of the neural axis. Caudal node cells function as stem cells. They are responsible for the apical growth of the cord of cells that are at the origin of the midline structures since, if removed, neither the notochord nor the floor plate, are formed caudally to the ablation. The embryo extends however in the absence of midline cells and a neural tube develops posterior to the excision. Only dorsal molecular markers are detectable on this neural tube (e.g. Pax3 and Slug). The posterior region of the embryo in which the structures secreting Shh are missing undergo cell death within the 24 to 48 hours following its formation. Unpublished results indicate that rescue of the posterior region of the embryo can be obtained by implantation of Shh secreting cells. One of the critical roles of floor plate and notochord is therefore to inhibit the cell death programme in the axial and paraxial structures of the embryo at gastrulation and neurulation stages.     

Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord

Individual classes of neural cells differentiate at distinct locations in the developing vertebrate nervous system. We provide evidence that the pattern of cell differentiation along the dorsoventral axis of the chick neural tube is regulated by signals derived from two ventral midline cell groups, the notochord and floor plate. Grafting an additional notochord or floor plate to ectopic positions, or deleting both cell groups, resulted in changes in the fate and position of neural cell types, defined by expression of specific antigens. These results suggest that the differentiation of neural cells is controlled, in part, by their position with respect to the notochord and floor plate.