Catecholamines and morphogenesis of the chick neural tube and notochord

Catecholamines in the early chick embryo (days 2–3) were studied using the Falck-Hillarp technique. After treating specimens with nor-epinephrine, specific catecholamine fluorescence was localized in the early neural tube of the youngest embryos (stages 7–9). In older embryos (stages 10–12) exposed to exogenous norepinephrine the fluorescence was seen in both the neural tube and notochord and by stage 13 fluorescence was brighter in the notochord than neural tube. Endogenous notochord catecholamine fluorescence was demonstrable in stages 13 through 17 without exposure to exogenous amines although treatment with a monoamine oxidase inhibitor (nialamide) or norepinephrine intensified the fluorescence. Treatment of embryos with nialamide or an inhibitor of catecholamine synthesis (α-Methyltyrosine) caused numerous archencephalic anomalies, ventral flexure of the head without lateral rotation, and spina bifida. The results suggest that catecholamines in the early neural tube and notochord may provide the motive force for morphogenetic movements, including closure of the neural folds and torsion of the embryo.     

Sox10 overexpression induces neural crest-like cells from all dorsoventral levels of the neural tube but inhibits differentiation.

SoxE genes (Sox8, Sox9, and Sox10) are early response genes to neural crest induction. Although the early role of Sox9 has been examined in chick and frog, later roles in neural crest migration and differentiation remain largely unexplored. We first examined which SoxE genes were expressed in trunk neural crest cells and then investigated their function using in ovo electroporation. The results of this analysis reveal that Sox10 is present in migrating neural crest cells, whereas other SoxE genes are only expressed transiently after induction. Ectopic expression of Sox10 in the neural tube at trunk level induced expression of HNK-1 in neuroepithelial cells followed by extensive emigration from all levels of the dorsoventral neuraxis, including the floor plate. Sox10-expressing cells failed to express neuronal, Schwann, or melanocyte markers up to 6 days posttransfection (E8), suggesting these cells were maintained in an undifferentiated state. Overexpression of Sox8 or Sox9 had similar but not identical effects on neuroepithelial cells. These results show that high levels of Sox10, Sox9, or Sox8 expression in the neural tube are capable of inducing a migratory neural crest-like phenotype even in the absence of dorsal signals and can maintain these cells in an undifferentiated state.     

Effect of the notochord on the differentiation of a floor plate area in the neural tube of the chick embryo

Summary The role of a notochord fragment on the origin of an additional floor plate area in the neural tube is investigated by quantitative morphological methods. In 1.5 to 2 day chick embryos a notochordal fragment was implanted in close apposition to the lateral wall of the neural groove in the region between prospective wing and leg bud. At 4 days, adjacent to the implant a distinct area of the neural wall was present, which resembled the natural floor plate with respect to its thickness, the abluminal location of elongated nuclei and the absence of neuroblasts. The mitotic density of this area was reduced. This additional floor plate was distinct when the experiment was performed at 1.5 days but was hardly recognizable when it was carried out at 2 days.From these results it is concluded that a) the notochord induces floor plate like structures and diminishes proliferation, and b) that the period of floor plate induction by the notochord is very restricted.     

Morphogenetic movements during cranial neural tube closure in the chick embryo and the effect of homocysteine

In order to unravel morphogenetic mechanisms involved in neural tube closure, critical cell movements that are fundamental to remodelling of the cranial neural tube in the chick embryo were studied in vitro by quantitative time-lapse video microscopy. Two main directions of movements were observed. The earliest was directed medially; these cells invaginated into a median groove and were the main contributors to the initial neural tube closure. Once the median groove was completed, cells changed direction and moved anteriorly to contribute to the anterior neural plate and head fold. This plate developed into the anterior neuropore, which started to close from the 4-somite stage onwards by convergence of its neural folds. Posteriorly, from the initial closure site onwards, the posterior neuropore started to close almost instantaneously by convergence of its neural folds. Homocysteine is adversely involved in human neural tube closure defects. After application of a single dose of homocysteine to chick embryos, a closure delay at the initial closure site and at the neuropores, flattening of the head fold and neural tube, and a halt of cell movements was seen. A possible interference of Hcy with actin microfilaments is discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube.

The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube.     

Differential expression of beta3 integrin gene in chick and mouse cranial neural crest cells.

RNA in situ hybridization on early chicken embryos revealed that the beta3 integrin gene started to be expressed after Hamburger and Hamilton (HH) stage 6 in the presumptive epidermis adjacent to the neural plate, before closure of the neural tube. The beta3 integrin gene was also strongly expressed in cephalic neural crest cells at the same stage in which they begin their migration but disappeared progressively in these cells along the route they take to the branchial arches. The gene was weakly expressed in the differentiating cranial neural crest cells. The alphaVbeta3 integrin protein complex was also mainly detected in the migratory cephalic neural crest cells. However, during early mouse embryogenesis and in contrast to the chick, the beta3 integrin gene was expressed in the foregut diverticulum and in the heart and not in the cephalic neural crest cells. Therefore, the difference in the beta3 integrin expression suggests that mouse and chicken cranial neural crest cells may have distinct integrin requirements during their ontogenesis.     

A-current expression is regulated by activity but not by target tissues in developing lumbar motoneurons of the chick embryo

The functional expression of A-type K+ channels (IA) was examined in chick lumbar motoneurons (LMNs) at embryonic days 6 and 11 (E6 and E11). We observed a threefold increase in IA density between E6 and E11 in spinal cord slices and acutely dissociated LMNs. There was no change in current density, kinetics, or voltage dependence of IA in E11 homozygous limbless mutants or in E11 embryos in which hindlimbs were surgically removed at E6. Moreover, chronic in ovo administration of D-tubocurarine, which causes an increase in motoneuron branching on the surface of target muscles, had no effect on IA. Electrical activity played an important role in IA regulation in LMNs in vitro and in ovo. Blocking spontaneous electrical activity of LMNs by chronic in ovo application of mecamylamine or muscimol reduced IA by 80%. LMNs cultured in the presence of TTX also failed to express normal densities of IA, even when the cultures also contained target tissues. The portion of IA that remained after in ovo or in vitro blockade of activity inactivated more quickly than the IA of LMNs that were allowed to discharge spikes. The developmental expression of LMN IA increases significantly during development, and this increase is activity dependent but does not require interactions with target tissues. Ongoing activity also seems to regulate the kinetics of IA inactivation.     

Morphological analysis of the role of the neural tube and notochord in the development of somites

The differentiation of avian somites and skeletal muscles, which themselves are derived from somites, was studied in ovo after the isolation of the unsegmented segmental plate from the notochord and/or neural tube by surgical operations at the level of the segmental plate. In each experiment, the newly formed somites had a normal histological structure, with an outer epithelial somite and core cells in the somitocoeles. Thereafter, the three derivatives of the somites (dermatome, myotome and sclerotome) reacted differently to the different operations. When the somites developed without the notochord, only somitocoele cells showed massive cell death, and muscles developed regardless of the presence or absence of the neural tube. On the contrary, no cell death was observed in any part of the somites that were formed with the neural tube or the notochord present, and muscle cells developed. However, in those embryos that retained only the notochord, striated muscles developed only in the lateral body wall. In each of the experimental operations, the surface ectoderm always covered the somites, and, regardless of the state of sclerotome and/or myotome differentiation, the dermatome always survived. These histological observations indicate that the first step in somite formation is independent of axial structures. The results further suggest that the notochord may produce diffusible factors that are necessary for the survival and further development of sclerotomal cells, and that both the neural tube and notochord can support muscle differentiation. However, it is likely that each structure has a relationship to the development of epaxial muscles and hypaxial muscles respectively. Furthermore, an intimate relationship may also exist between the surface ectoderm and the development of the dermatome.     

Embryogenetic expression of glossopharyngeal and vagal excitability in the chick brainstem as revealed by voltage-sensitive dye recording

Using voltage-sensitive dye recording, we traced the ontogenetic expression of neural excitability related to the glossopharyngeal nerve (N. IX) and the vagus nerve (N. X) in the embryonic chick brainstem. At the 3.5-day embryonic stage, by averaging optical signals, we succeeded in recording very small action potential-related optical responses (ΔI/I < 10⁻⁴) induced by N. IX stimulation in the nucleus of the glossopharyngeal nerve (motor nucleus). This suggests that glossopharyngeal excitability in the motor nucleus is first generated no later than this developmental stage. On the other hand, action potential-related optical responses induced by N. X stimulation were first detected at the 4-day embryonic stage. Comparison with morphogenesis indicated that glossopharyngeal and vagal motoneurons acquire their excitability and send their axons to the periphery soon after they leave the proliferative pool.     

Changes in peanut lectin binding sites on the neuroectoderm during neural tube formation in the bantam chick embryo

Summary Cell surface carbohydrates in the neurulating ectoderm of bantam chick embryos of stage 6–11 were examined using the fluorescein isothiocyanate-labeled and ferritin-labeled peanut lectin,Arachis hypogaea agglutinin (PNA), which is Gal₁₃GalNAc specific. Weak fluorescence showing PNAbinding sites was seen on the apical surfaces of neural plate cells. On the surfaces of neural tube cells the fluorescence was more intense and appeared as a band. When using ferritin particles as a quantitative EM marker, only a few PNA binding sites covering the apical surfaces of the basal plate cells during the neural plate stage were seen (274.3±18.67 ferritin particles/m²). As neural tube formation advanced, the number of the ferritin labeled PNA binding sites increased as was to be expected from the fluorescent label experiment. At the neural ridge contact stage there were 2.5 times more binding sites than at the neural plate stage. After this period, the lectin binding sites showed no significant changes. These results were the inverse of those for RCA_I or WGA lectins previously reported by us. These observations suggest that sugar residues or the sugar-chain skeletons on the neuroectoderm are altered during neurulation.     

Differentiation of the neural plate and neural tube in the young chick embryo. A study by scanning and transmission electron microscopy.

The differentiation of the presumptive neural plate, the neural plate and the neural tube have been investigated in the chick embryo by SEM, TEM and histochemical techniques. The relationship of these tissues to neighbouring structures, including extracellular materials, has also been studied. When SEM micrographs of primitive streak stage embryos were examined in stereo, it was found that cells which had been invaginating at the time of fixation were similar in shape to fibroblasts migrating in vitro. It was concluded that SEM stereo pairs could provide evidence about the mode and direction of cell migration. Many more mid-bodies have been found associated with the developing neural tissue than with the lateral ectoderm. It was found possible to recognise mid-bodies not only by TEM but also by SEM. It is therefore proposed that SEM montages may be used for assessing which regions of a tissue have recently undergone extensive mitosis. The beads on the specialised threads seen in the early stages of development are now considered to be formed from mid-bodies. Similar, but unbeaded threads have been described which span the gap between the neural folds just prior to the dorsal closure of the neural tube and it seems probably that these threads help to close the neural tube. It is suggested that the beaded threads arise by incomplete separation of two daughter cells at mitosis, whereas the unbeaded threads form by outgrowth of cell processes.     

Universal absence of merlin, but not other ERM family members, in schwannomas.

NF2 (neurofibromatosis 2, encoding the merlin protein) gene mutations and chromosome 22q loss have been demonstrated in the majority of sporadic and NF2-associated schwannomas, but many schwannomas fail to demonstrate genetic evidence of biallelic NF2 gene inactivation. In addition, the role of the merlin-related ERM family members (ezrin, radixin, and moesin) remains unclear in these tumors. We therefore studied expression of NF2-encoded merlin as well as ezrin, radixin, and moesin in 22 vestibular and peripheral schwannomas that had been evaluated for NF2 mutations and chromosome 22q loss. Western blotting and immunohistochemistry with antibodies directed against the amino and carboxy termini of merlin demonstrated loss of merlin expression in all studied schwannomas, including 12 tumors lacking genetic evidence of biallelic NF2 gene inactivation. Western blotting with antibodies directed against ezrin, radixin, and moesin, however, showed expression of these proteins in all schwannomas. In addition, immunohistochemistry with an antibody to moesin revealed widespread expression in tumor and endothelial cells. These data indicate that the specific loss of merlin is universal to schwannomas and is not linked to loss of ezrin, radixin, or moesin expression.     

Cytoskeletal architecture of the matrix cell and neuroblast in the neural tube of the chick embryo.

Cytoskeletal architecture of the matrix cell and neuroblast in the wall of midbrain of 4-6 day-old chick embryos was examined by electron microscopy and immunohistochemistry. The matrix cell, the undifferentiated stem cell later producing neurons and glial cells in the central nervous system, is characterized ultrastructurally by abundant free ribosomes and a poorly developed cytomembrane system. A few microtubules running in random directions are observed in the matrix cell body. In the cell processes, microtubules are oriented longitudinally, and linked with each other by cross-bridges, presumably composed of microtubule-associated proteins (MAPs). The cell processes contain abundant cytoplasmic filaments including a large amount of actin filaments which adhere to the plasma membrane of junctional complexes located immediately below the inner surface of the neural tube. In the neuroblast which has been differentiated from the matrix cell, the cytomembranous organelles, especially rough endoplasmic reticulum are markedly better developed than in the matrix cell; microtubules are more numerous in the cell body. The cell process contains many microtubules with cross-bridges and a few intermediate filaments, which are relatively characteristic of the cytoskeleton of the neuroblast. Phalloidin-staining and immunohistochemistry showed that the neuroblast was richer in F-actin, beta-tubulin, MAP1, MAP2, tau, calspectin, and synapsin I than the matrix cell. As the matrix cell differentiates into the neuroblast, both the cytoskeletal and cytomembranous systems proved to develop features, characteristic of a neuron.     

Molecular differentiation of the otic vesicle and neural tube in the chick embryo demonstrated by monoclonal antibodies

Monoclonal antibodies (MAbs) were produced against membrane fractions of the chick neural tube and somite. These MAbs selectively stained the neural tube and neural crest cells; the antigens for some of these MAbs were identified as cell adhesion molecules or glycolipids. Histochemistry of the otic vesicle and its progeny in the chick embryo was performed with these MAbs and other MAbs obtained previously in our laboratory. It was demonstrated that differentiation of the otic placode and vesicle from the ectoderm, and development of the acoustic ganglion from the otic vesicle were accompanied by the appearance and disappearance of various molecules.     

Disruption of proteoglycans in neural tube fluid by β-D-xyloside alters brain enlargement in chick embryos

Following neurulation, the anterior end of the neural tube undergoes a dramatic increase in size due mainly to the enlarging of the brain cavity. This cavity is filled with so-called neural tube fluid (NTF), whose positive pressure has been shown to play a key role in brain morphogenesis. This fluid contains a watersoluble matrix, rich in chondroitin sulfate (CS), which has been proposed as an osmotic regulator of NTF pressure genesis. The purpose of the present study is to observe the influence of CS on NTF osmolality and its relation to NTF hydrostatic pressure and brain expansion. NTF was obtained by means of microaspiration from the mesencephalic cavity of chick embryos. The osmolality of NTF between H.H. stages 20 and 29 was measured on the basis of its cryoscopic point. CS synthesis was disrupted by using β-D-xyloside and the induced variations in brain volume were measured by means of morphometry. We also measured the variations in NTF osmolality, hydrostatic pressure, and the concentration of CS and sodium induced by means of β-D-xyloside. Our data reveal that, at the earliest stages of development analyzed, variations in NTF osmolality show a characteristic pattern that coincides with the developmental changes in the previously described fluid pressure. Chick embryos treated with β-D-xyloside, a chemical that disrupts CS synthesis, displayed a notable increase in brain volume but no other apparent developmental alterations. Morphometric analysis revealed that this increase was due to hyperenlargement of the brain cavity. β-D-xyloside brings about specific changes in the biochemical composition of NTF, which entails a large increase in CS concentration, mainly in the form of free chains, and in that of sodium. As a result, the fluid's osmolality and brain intraluminal pressure increased, which could account for the increase in size of the brain anlage. These data support the hypothesis that the intraluminal pressure involved in embryonic brain enlargement is directly dependent on NTF osmolality, and that the concentrations of CS and its associated microions could play a key role in the regulation of this process. Anat. Rec. 252:499–508, 1998. © 1998 Wiley-Liss, Inc.