The extracellular matrix during neural crest formation and migration in rat embryos

Summary Changes in the distribution of extracellular matrix components have been investigated immunohistochemically during neural crest development in the rat. Inside the ectodermal epithelium basal lamina components are formed resulting in a separation of neurectoderm and epidermal ectoderm. Within the presumptive neural crest area fibronectin, hyaluronan and chondroitin sulphate become apparent. Upon subsequent neural crest migration the basal lamina becomes disrupted. As the neural crest cells take part in mesectoderm formation, fragments of the basal lamina remain attached to their surface, as is demonstrated with antibodies against laminin and collagen type IV. The extracellular matrix is therefore active both in the separation of neuroectoderm from epidermal ectoderm and in mesectoderm formation.     

The tight junction scaffolding protein cingulin regulates neural crest cell migration

Neural crest cells give rise to a diverse range of structures during vertebrate development. These cells initially exist in the dorsal neuroepithelium and subsequently acquire the capacity to migrate. Although studies have documented the importance of adherens junctions in regulating neural crest cell migration, little attention has been paid to tight junctions during this process. We now identify the tight junction protein cingulin as a key regulator of neural crest migration. Cingulin knock-down increases the migratory neural crest cell domain, which is correlated with a disruption of the neural tube basal lamina. Overexpression of cingulin also augments neural crest cell migration and is associated with similar basal lamina changes and an expansion of the premigratory neural crest population. Cingulin overexpression causes aberrant ventrolateral neuroepithelial cell delamination, which is linked to laminin loss and a decrease in RhoA. Together, our results highlight a novel function for cingulin in the neural crest.     

Development of the embryonic chick otic placode. II. Electron microscopic analysis

The otic placode takes its origin from surface ectoderm. Prior to the arrival of neural crest cells, surface epithelial cells adjacent to the neural folds are squamous in shape and synthesize primarily interstitial bodies. However, by 26 hours of development, neural crest cells, using the undersurface of the epithelium as a substratum, migrate away from the neural tube. Cells of surface epithelium above the neural crest cells assume a columnar shape, and the amount of intercellular space between adjacent epithelial cells is consequently reduced. Placode cells show extensive interdigitation apically as they pseudostratify and invaginate, while it appears that many of the basal cells contribute components to the underlying extracellular matrix. This extracellular matrix interface between surface epithelium and neural crest cells is distinctly fibrillar and less granular than that found between ordinary head ectoderm and primary mesenchymal cells. Just prior to complete invagination as an otocyst, otic placode cells in a region near the ventrolateral wall of the hindbrain extend cell processes through discontinuities in the basal lamina and leave the otocyst. These are likely to be the cells which contribute to the formation of the acoustico-facialis ganglion. These observations support the hypothesis that the development of the otic placode is the result of a tissue interaction between surface epithelium and neural crest cells.     

Programmed cell death and the morphogenesis of the hindbrain roof plate in the chick embryo

The spatial and temporal distribution of apoptosis in the dorsal midline of the developing chick hindbrain was examined in relation to the development of the neuroepithelium and neural crest using scanning and transmission electron microscopy, immunocytochemistry and in situ hybridization. The pattern of TUNEL labeling and Slug expression in the dorsal midline at stages 10 and 11 differed from that at stages 12-15. At stages 10 and 11, TUNEL labeling and Slug expression were observed in the dorsal part of location II of rhombomere 1/2 (i.e., between the surface ectoderm and the neuroepithelium), but from stage 12 onward, they were observed in both the dorsal and ventral parts of location II. The implication is that whereas apoptosis may be restricted to a subpopulation of the early migrating neural crest at stages 10 and 11, it presumably occurs in subpopulations of both neural crest and neuroepithelial cells from stage 12 onward. Furthermore, as judged by the pattern of TUNEL labeling and Slug expression in r3 and r5, apoptosis in these two rhombomeres likely occurs in subpopulations of both neural crest and neuroepithelial cells. The eminence present in location I of r1/r2 between stages 10 and 12 consisted of both neural crest and neuroepithelial cells. These cells gradually underwent apoptosis until stage 12, when the eminence disappeared in most embryos. The formation of the inner (neuroepithelial) aspect of the hindbrain roof plate involved both cell migration from adjacent neuroepithelium and an alteration in the shapes of the cells, such that cells with flattened surfaces eventually lined the roof plate. During these processes, some of the neuroepithelial cells underwent apoptosis (i.e., in location IV). The results of this study thus demonstrate that subpopulations of both neuroepithelial and neural crest cells may be involved in programmed cell death in the hindbrain. Additionally, apoptosis in the hindbrain contributes significantly to morphogenetic thinning during roof plate formation.     

Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the chick.

Within the hindbrain region, neural crest cell migration is organized into three streams that follow the segmentation of the neuroepithelium into distinct rhombomeric compartments. Although the streaming of neural crest cells is known to involve signals derived from the neuroepithelium, the molecular properties underlying this process are poorly understood. Here, we have mapped the expression of the signaling component of two secreted class III Semaphorins, Semaphorin (Sema) 3A and Sema 3F, at time points that correspond to neural crest cell migration within the hindbrain region of the chick. Both Semaphorins are expressed within rhombomeres at levels adjacent to crest-free mesenchyme and expression of the receptor components essential for Semaphorin activity by neural crest cells suggests a function in restricting neural crest cell migration. By using bead implantation and electroporation in ovo, we define a role for both Semaphorins in the maintenance of neural crest cell streams in proximity to the neural tube. Attenuation of Semaphorin signaling by expression of soluble Neuropilin-Fc resulted in neural crest cells invading adjacent mesenchymal territories that are normally crest-free. The loss or misguidance of specific neural crest cell populations after changes in Semaphorin signaling also affects the integration of the cranial sensory ganglia. Thus, Sema 3A and 3F, expressed and secreted by the hindbrain neuroepithelium contributes to the appropriate positioning of neural crest cells in proximity to the neural tube, a process crucial for the subsequent establishment of neuronal connectivity within the hindbrain region.     

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.     

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker

Summary The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectincoated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.     

The incorporation and dispersion of cells and latex beads on microinjection into the amniotic cavity of the mouse embryo at the early-somite stage

Summary The ability of cells and latex beads to become incorporated into the cranial region of embryos after microinjection into the amniotic cavity was studied. Premigratory neural crest cells isolated from the lateral margins of the neuroepithelium, 3T3 fibroblast cells or H35 hepatoma cells were labelled with WGA-gold conjugates, and were then microinjected into the amniotic cavity of embryos with two to three somites in vitro. Latex beads were similarly microinjected into different groups of embryos. Incorporation of injected cells or latex beads was found in the neural crest of the midbrain and the hindbrain of 5–20% of the recipients 4 h after microinjection. At 6 and 12 h, increasingly more embryos (20–77%) were observed with labelled cells or latex beads in the crest region. While hepatoma cells and latex beads were restricted to the crest region, injected neural crest cells and fibroblasts were also found in the lateral mesenchyme, bounded laterally by the surface ectoderm and medially by the closing neural tube. By 24 h after microinjection, the injected cells or latex beads were found in 50–80% of the recipients. Neural crest cells and fibroblasts, which showed similar patterns of distribution in the embryos, were located on the dorsal aspect of the neural tube, the lateral mesenchyme, the pharyngeal arches and the regions for ganglia. Hepatoma cells and latex beads were limited to the dorsal regions of the neural tube. When microinjection was carried out in embryos with seven to eight somites, incorporation of cells or latex beads was found in 44–75% of embryos, but no dispersion of the incorporated cells or latex beads into the mesenchyme was found 24 h after microinjection. Incorporation and dispersion of cells and latex beads were not observed when embryos with 18–20 somites were used as recipients. The present study showed that neural crest or fibroblast cells when injected into the amniotic cavity could be incorporated into the neural crest, and then undergo migration along the neural crest pathways, whereas hepatoma cells and latex beads could only be incorporated. The incorporation and migration of the exogenous tissues are related to the formation and the accessibility of the neural crest in the recipients.     

Fgfr1 regulates patterning of the pharyngeal region

Development of the pharyngeal region depends on the interaction and integration of different cell populations, including surface ectoderm, foregut endoderm, paraxial mesoderm, and neural crest. Mice homozygous for a hypomorphic allele of Fgfr1 have craniofacial defects, some of which appeared to result from a failure in the early development of the second branchial arch. A stream of neural crest cells was found to originate from the rhombomere 4 region and migrate toward the second branchial arch in the mutants. Neural crest cells mostly failed to enter the second arch, however, but accumulated in a region proximal to it. Both rescue of the hypomorphic Fgfr1 allele and inactivation of a conditional Fgfr1 allele specifically in neural crest cells indicated that Fgfr1 regulates the entry of neural crest cells into the second branchial arch non-cell-autonomously. Gene expression in the pharyngeal ectoderm overlying the developing second branchial arch was affected in the hypomorphic Fgfr1 mutants at a stage prior to neural crest entry. Our results indicate that Fgfr1 patterns the pharyngeal region to create a permissive environment for neural crest cell migration.     

The electrical properties of the ectoderm in the amphibian embryo during induction and early development of the nervous system

1. The electrical properties of ectodermal cells have been studied in embryos of the axolotl Ambystoma mexicanum between gastrulation and the closure of the neural tube.2. At the time of neural induction by the underlying mesoderm the mean membrane potential recorded in ectoderm cells was -30 mV (+/- 1.5 mV S.E. of mean) and in presumptive neural cells -27 mV (+/- 1.6 mV S.E. of mean).3. At late neural fold stages, when specification of the neuroectoderm is complete, the membrane potential in presumptive nerve cells was -44 mV (+/- 1.7 mV S.E. of mean). This is significantly greater than in cells of the surrounding ectoderm at the same developmental stage (-31 mV +/- 1.5 mV S.E. of mean).4. Current injected into an ectoderm cell spread freely throughout the neural and lateral ectoderm both before and after neural specification was complete.5. Voltage-current relations recorded at mid-neural fold stages in the lateral ectoderm and neural plate rectified in opposite directions. In the neural plate the slope conductance rose as the internal potential was made less negative; in the lateral ectoderm the slope conductance fell with depolarization.6. At the time of closure of the neural tube ectoderm and presumptive neural cells lose their low resistance connexions with each other. At the same time low resistance contacts are established across the mid line between ectoderm cells originally separated by the neural plate.7. After the neural tube has closed low resistance connexions remain between presumptive neural cells, although the degree of current spread from one cell to the next is not very great.8. The voltage-current relation recorded in neural tube cells showed a rise in slope conductance as the cell was depolarized.9. Occasionally signs of regenerative activity were seen, but the mechanism for generating a fully fledged action potential does not differentiate until after complete closure of the neural tube.     

Ultrastructure of neural crest formation in the midbrain/rostral hindbrain and preotic hindbrain regions of the mouse embryo

In the mouse embryo, neural crest mesenchyme associated with the first and second pharyngeal arches escapes from the epithelium that forms the tips of the midbrain/rostral hindbrain and preotic hindbrain neural folds. To investigate the ultrastructure of crest formation, embryos with four to eight pairs of somites were processed for transmission electron microscopy. In the earliest event related to crest formation, crest precursors in the midbrain/rostral hindbrain elongated and moved all or most of their contents to the basal region of the epithelium. Elongation was probably mediated by apical bands of micro-filaments and longitudinally oriented microtubules. Elongated cells then relinquished apical associations while nonelongated cells maintained those associations and withdrew from the basal lamina. This resulted in an epithelium stratified into apical and basal (crest precursor) layers. The coalescence of enlarging extra-cellular spaces opened a delaminate gap between the two layers. Additional crest precursors entered this gap from the apical layer. From the time crest precursors began moving basally, some formed microfilament- and/or microtubule-containing processes, which penetrated the basal lamina. Some of these cells moved their contents into the larger, microtubule-containing processes, perhaps thereby escaping from the epithelium. Soon after elongating cells appeared, the basal lamina beneath the epithelium began to degrade in a pattern unrelated to process formation. This ultimately resulted in disruption of the lamina, dispersal of the basal layer of the epithelium, and release of the crest precursors in the delaminate gap. Once crest formation was complete, the apical layer reformed a basal lamina on a patch-by-patch, cell-by-cell basis. In the preotic hind-brain, elongating crest precursors apparently forced their basal faces through the basal lamina and then relinquished apical association to escape. As a result, the lamina was disrupted before the epithelium could stratify, and enlarged extracellular spaces appeared among mesenchymal cells rather than creating a delaminate gap. The failure of elongation to disrupt the basal lamina in the midbrain/rostral hindbrain and its success in the preotic hindbrain might be due to less-vigorous, less-concerted elongation in the midbrain/rostral hindbrain or to earlier, more rapid degradation of the lamina in the preotic hindbrain.     

Morphological and quantitative studies in the otic region of the neural tube in chick embryos suggest a neuroectodermal origin for the otic placode

Careful histological observation of the development of the anlage of the inner ear in chicken embryos led us to question the traditional view of otic placode (OP) formation. First, morphological studies in the cephalic region carried out on stages preceding the appearance of the placodal epithelium revealed that the medial placodal cells are continuous temporally and spatially with cells belonging to the neural fold (NF). Second, both the formation of the basal lamina between the dorsal region of the neural tube (NT) and ectoderm and the pattern of formation of the neural crest present distinctive characteristics between otic levels and regions located anteriorly and posteriorly. Third, numerical comparisons of parameters for the NT and the OP between different levels of the rhombencephalon allowed us to assign a differential behaviour in the growth pattern of the otic region. These results indicated that the medial part of the OP is not derived from already independent ectoderm that increases in thickness under the influence of the NT (as previously accepted) but that it develops directly from the NFs. Although we do not exclude other possibilities, we propose that at least a proportion of the OP cells originate directly from cells committed to be neural crest. After this incorporation, basal laminal formation would delimit the NT from the OP without transition of the otic cells to ectoderm. This hypothesis would imply that part of the otic cells originate directly from neuroepithelial cells having a neuroectodermal (rather than the previously established ectodermal) origin.     

Neural tube and neural crest: A new view with time-lapse high-definition photomicroscopy

The dynamic process of neural tube formation and neural crest migration in live, unstained cultured avian embryos at Hamburger-Hamilton (H.H.) stages 8–11 was investigated by time-lapse cinematography using a high-definition microscope. These studies have demonstrated that neural tube closure in the trunk region differs from that observed in the head. The cephalic neural folds elevate slowly, then make contact rapidly. Following this initial apposition, they gradually “zip-up” in the rostrad and caudad direction. In the trunk region where the neuroepithelium bulges adjacent to the somites, the edges of the folds pulsate and forcefully touch-retract-touch in these bulging regions; the intersomitic epithelia retract, remain open even after more posterior somitic regions have apposed, and then close slowly. Epithelial blebs and N-CAM antibody were observed at the leading edges of the neuroepithelia. Between the open folds only a few bridging cells were seen; they probably represent the sites of initial cell adhesion following epithelial retraction. Focusing into the developing embryo shows that neuroepithelial fusion occurs prior to surface epithelial fusion. A meshwork of synchronously pulsating neural crest cells was identified below the surface epithelium and a preliminary investigation of their initial migration was conducted.     

Neural tube and neural crest: a new view with time-lapse high-definition photomicroscopy.

The dynamic process of neural tube formation and neural crest migration in live, unstained cultured avian embryos at Hamburger-Hamilton (H.H.) stages 8-11 was investigated by time-lapse cinematography using a high-definition microscope. These studies have demonstrated that neural tube closure in the trunk region differs from that observed in the head. The cephalic neural folds elevate slowly, then make contact rapidly. Following this initial apposition, they gradually "zip-up" in the rostrad and caudad direction. In the trunk region where the neuroepithelium bulges adjacent to the somites, the edges of the folds pulsate and forcefully touch-retract-touch in these bulging regions; the intersomitic epithelia retract, remain open even after more posterior somitic regions have apposed, and then close slowly. Epithelial blebs and N-CAM antibody were observed at the leading edges of the neuroepithelia. Between the open folds only a few bridging cells were seen; they probably represent the sites of initial cell adhesion following epithelial retraction. Focusing into the developing embryo shows that neuroepithelial fusion occurs prior to surface epithelial fusion. A meshwork of synchronously pulsating neural crest cells was identified below the surface epithelium and a preliminary investigation of their initial migration was conducted.     

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.     

An SEM and TEM study of suppression of eye development in eyeless mutant axolotls

Summary Morphology of the primary optic rudiment of normal eyed and mutant eyeless (e/e) axolotl embryos was studied at light, TEM and SEM levles. The presumptive eyeforming region of eyeless embryos differs from that of normal embryos in several important respects including premature formation of basal lamina, separation from overlying ectoderm by mesenchyme cells and persistence of granules in the interspace surrounding the optic anlage into relatively late developmental stages. These differences suggest that the gene that causes failure of eye formation in the mutant axolotls produces structural differences that interfer with normal physical and/or biochemical inductive interactions between neurectoderm and mesenchyme cells due primarily to the precocious development of basal lamina over the extermal surface of the optic primordia.     

Ganglion formation from the otic placode and the otic crest in the chick embryo: Mitosis, migration, and the basal lamina

Summary We have studied the morphogenesis of the cochleo-vestibular (CVG) and distal cranial ganglia in the early chick embryo (White Leghorn embryos). Light microscopy and immunocytochemical staining for fibronectin and laminin were used to trace the cellular contributions to these ganglia from the otic placode and otocyst. Serial semi-thin plastic sections (3–5 µm) stained with toluidine blue at each Hamburger-Hamilton stage (St.) from 10 to 21 were used. We were able to trace individual groups of cells derived from these epithelial structures into the anlagen of the CVG and the distal parts of cranial n. ganglia VII, IX, and X. For immunostaining, antisera were used to visualize the basal lamina in 15-µm cryostat sections from St. 14 to 21 embryos. Described here for the first time is the otic crest, a ridge of epithelium surrounding the placode. Cells migrate from the otic crest (St. 11 to 14) during the period when the otocyst is forming. These cells become continuous spatially with those derived from the epibranchial placodes and the presumptive ganglia of cranial nerves VII, IX, and X. Furthermore, rostral otic crest cells merge with neural crest cells, which appose the myelencephalon, and they join with the newly formed neuroblasts of the CVG, which migrate from the ventral epithelium of the otocyst at St. 14 to 21. This region of the epithelium forms the bulk of the CVG; it also has many more mitotic figures than the rest of the otocyst. Cells in the rostralmost CVG (vestibular part) are the first to complete their migration and send axons into both the medulla and incipient crista ampullaris. Immuno-staining for fibronectin and laminin shows that these two basal-lamina-associated glycoproteins appear in a continuous layer beneath the otic epithelium just prior to CVG migration. Thus there is no evidence that the migration is launched by a prior decomposition of the basal lamina. The cells migrating from the epithelium bridge the basal lamina with their leading processes while the trailing processes are withdrawing from the epithelium. These trailing processes must withdraw after the neuroblast migrates, since most of the neuroblasts undergo mitosis in subsequent stages. The migrating cells appear to push out of the epithelium by displacing immunostained fragments of the basal lamina ahead of their leading processes. This suggests that the exodus of cells is accompanied by forces within the epithelium itself. Whether this is generated by the migratory neuroblasts themselves or by other sources remains to be determined.Abbreviations BP basilar papilla - CVG cochleo-vestibular ganglion - FN fibronectin - LM laminin     

Artificial neural induction in amphibia. I. Sandwich explants

1. Embryonic tissues (ectoderm, neural plate) of Ambystoma mexicanum and Tritus were killed with hot water and implanted into ectoderm sandwiches. They induced the ectoderm to form neural tissue, lentoids and unspecialized epidermis. Neural tissue always showed retina character. Egg pigment was eliminated and gathered at the outer retinal surface or in the centre of rosettes. Neural crest cells like mesenchyme or melanophores were completely lacking, retinal pigment did not develop. 2. The thus induced living retina tissue was reimplanted into fresh ectoderm after 2 days. It continued histogenetic and morphogenetic differentiation and formed ocular vesicles with numerous eye cups. It induced the enveloping ectoderm to again form retina, lentoids and unspecialized epidermis without neural crest derivatives or RPE. 3. This inductive process can be reproduced several times.