Spatial and temporal distribution of vinculin and talin in migrating avian neural crest cells and their derivatives.

Neural crest cells express different adhesion modes at each phase of their development starting with their separation from the neural tube, followed by migration along definite pathways throughout the embryo, and finally to settlement and differentiation in elected embryonic regions. In order to determine possible changes in the cytoskeleton organization and function during these processes, we have studied the in situ distribution of two major cytoskeleton-associated elements involved in the membrane anchorage of actin microfilaments, i.e. vinculin and talin, during the ontogeny of the neural crest and its derivatives in the avian embryo. Prior to emigration, neural crest cells exhibited both vinculin and talin at levels similar to the neighbouring neural epithelial cells, and this expression apparently did not change as cells became endowed with migratory properties. However, vinculin became selectively enhanced in neural crest cells as they further migrated towards their final destination. This increase in vinculin amount was particularly striking in vagal and truncal neural crest cells entering cellular environments, such as the sclerotome and the gut mesenchyme. Talin was also expressed by neural crest cells but, in contrast to vinculin, staining was not conspicuous compared to neighbouring mesenchymal cells. High levels of vinculin persisted throughout embryogenesis in almost all neural derivatives of the neural crest, including the autonomous and sensory ganglia and Schwann cells along the peripheral nerves. In contrast, the non-neural derivatives of the neural crest rapidly lost their prominent vinculin staining after migration. The pattern of talin in the progeny of the neural crest was complex and varied with the cell types: for example, some cranial sensory ganglia expressed high amounts of the molecule whereas autonomic ganglia were nearly devoid of it. Our results suggest that (i) vinculin and talin may follow independent regulatory patterns within the same cell population, (ii) the level of expression of vinculin and talin in neural crest cells may be consistent with the rapid, constant modulations of their adhesive properties, and (iii) the expression patterns of the two molecules may also be correlated with the genesis of the peripheral nervous system.

A monoclonal antibody directed against quail tyrosine hydroxylase: description and use in immunocytochemical studies on differentiating neural crest cells.

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

Localization of molecules with restricted patterns of expression in morphogenesis: an immunohistochemical approach.

In a search for molecules with restricted patterns of expression during development, monoclonal antibodies were raised against different transitory structures of the chick embryo. Mice were immunized with cell suspensions from lightly homogenized embryonic tissues explanted from morphogenetically active regions. A convenient immunohistochemical assay was used to screen the hybridoma supernatants on a large scale. It relied on the use of poly(ethylene glycol) as embedding medium. Its water miscibility allowed, in a one-step incubation with antibody-containing supernatants, the dewaxing and rehydration of the tissue sections as well as antibody binding. We report here the usefulness of this approach in selecting monoclonals with unique patterns of immunoreactivity. In this study, cephalic neural crest cells in early or late phase of migration, together with their surrounding tissues, were used as immunogens. The monoclonal antibodies obtained have been classified into regional, cell-lineage, cell-cycle or extracellular material-associated markers. The information provided by the direct visualization of the immunoreactivity of the various monoclonal antibodies on tissue sections, as early as the first round of screening, allows rapid determination of the subsequent strategy to be followed for further characterization of the individual markers.

Neural crest cell differentiation and carcinogenesis: capability of goldfish erythrophoroma cells for multiple differentiation and clonal polymorphism in their melanogenic variants.

Multiple differentiation shown by a single cell line (GEM 81) of goldfish erythrophoroma (tumors of integumental erythrophores) cells after administration of chemical induction in vitro includes 1) melanogenesis, 2) formation of reflecting platelets, 3) synthesis of pteridines heterogeneous to this species, 4) formation of dermal skeletons such as teeth and fin rays, 5) production of neuronal characters, and 6) genesis of lentoid bodies. Melanogenic cells, highest in inducibility, also show remarkable phenotypic diversification in their cell morphology, pigmentation, and physiologic response. In this paper, the following findings are presented; a) multiple differentiation shown by erythrophoroma cells occurs on a clonal basis, making whole component cells of a given induced colony strikingly similar in their cell characters, and b) induced melanogenic clones manifest a remarkable polymorphism in their melanosome ultrastructure and receptor composition associated with motile response. The divergence covers concentric lamellar, multivesicular, fibrillar, and macroglobular types for the former, and a varying combination of receptors for epinephrine, melanin concentrating hormone (MCH), and melatonin for the latter. Because a spectrum of phenotypes expressed by differentiation-induced erythrophoroma cells is restricted to those of neural crest origin (except lentoid bodies) and polymorphism in induced melanized cells is composed mostly of a collection of a variety of known melanogenic characters, it is presumed that erythrophoroma cells are capable of multiple differentiation within the commitment as neural crest cells.

Deposition of laminin and fibronectin in dysraphic mutant mice: an immunofluorescent study.

Abnormal loop-tail (Lp/Lp) mutant mouse embryos exhibiting severe exencephaly and myeloschisis were analyzed and compared with their normal (+/+; Lp/+) littermates by means of immunofluorescence histochemistry to determine regional differences in the distribution of laminin (L) and fibronectin (FN). In the neural basement membrane and adjacent mesenchymal cell matrix of the abnormal embryos, regional differences in the deposition of L and FN were similar to those in normal littermates. Moreover, most of the putative neural crest (NC) cells appeared to emigrate normally in terms of their site of detachment and migration pathways, despite the severe topographic distortions and loss of neuroepithelial integrity. However, some putative NC cells projected incorrectly from the 'luminal' surface of the neuroepithelium, suggesting that some of the NC may be abnormal or sequestered and prevented from appropriate detachment and emigration from the neural tube.

Heart conduction system: a neural crest derivative?

Using the anti-neurofilament monoclonal antibody iC8 we report here that muscle fibers of the conduction system of the adult and developing rabbit heart express a cytoskeletal protein antigenically and electrophoretically similar to the middle subunit of neurofilaments (NF-M). In the 11-day embryo a number of cardiac muscle cells also express a neural crest surface marker recognized by the monoclonal antibody HNK-1. Both markers are found in many cells of the 3rd and 4th branchial arches, which are populated by cells of neural crest origin. In the 11-day embryo cells of the 4th branchial arch are in close proximity to and intermingled with the atrial myocardium: cells co-expressing sarcomeric myosin heavy chain with iC8 and HNK-1 immunoreactivity are seen at these sites. The findings suggest that conduction tissue cells of the rabbit heart originate from a population of neural crest-derived cells migrating from the branchial arches into the developing heart.

Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest.

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.

In vitro differentiation of quail neural crest cells into sensory-like neuroblasts.

This study shows that quail neural crest cells can differentiate in vitro into sensory-like neuroblasts. The putative sensory neuroblasts were large and spherical, possessing large diameter, bipolar or pseudo-unipolar, long processes that lacked multiple varicosities characteristic of autonomic neurons. They bound HNK-1, a monoclonal antibody against a cell surface epitope expressed by early neural crest cells but not by young neural tube-derived cells. Many of the sensory-like neuroblasts had substance P (SP)-like immunoreactivity. Some exhibited histochemical carbonic anhydrase activity; carbonic anhydrase is shown in this study to stain a subpopulation of spinal sensory neurons in adult quail and embryos 9 days and older, whereas ventral root axons and neurons in sympathetic ganglia are non-reactive at all ages. Double staining indicated that unlike the multipolar neuroblasts developing in the same cultures, SP-like immunoreactive neuroblasts do not contain detectable levels of tyrosine hydroxylase or dopamine-beta-hydroxylase. Finally, the neuronal nature of the cultured sensory-like neuroblasts was further documented by double labeling for antibodies against the 68 kDa neurofilament polypeptide and substance P.

The relationship between emerging neural crest cells and basement membranes in the trunk of the mouse embryo: a TEM and immunocytochemical study.

The earliest stage of neural crest cell (NCC) migration is characterized by an epitheliomesenchymal transformation, as the cells leave the neural tube. There is evidence that in a number of cell systems this transformation is accompanied by alteration or depletion of associated basement membranes. This study examines the ultrastructural relationship between mouse NCCs and adjacent basement membranes during the earliest stages of migration from the neural tube. Basement membranes were identified by transmission electron microscopy (TEM) and immunofluorescence using antibodies to type-IV collagen. The ultrastructural features of NCCs and their relationship with surrounding tissues were also examined using TEM. In the dorsal region of the neural tube, from which NCCs originate, the basement membrane was depleted or absent, and with the immunofluorescence technique it was shown that this pattern was reflected in a deficit of type-IV collagen. TEM observations indicated that ultrastructurally NCCs differ from their neuroepithelial neighbours only in overall cell shape and their relationship to other cells and the extracellular matrix.

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo.

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.

The migration of neural crest cells and the growth of motor axons through the rostral half of the chick somite.

We have studied the pathway of migration of neural crest cells through the somites of the developing chick embryo, using the monoclonal antibodies NC-1 and HNK-1 to stain them. Crest cells, as they migrate ventrally from the dorsal aspect of the neural tube, pass through the lateral part of the sclerotome, but only through that part of the sclerotome which lies in the rostral half of each somite. This migration pathway is almost identical to the path which presumptive motor axons take when they grow out from the neural tube shortly after the onset of neural crest migration. In order to see whether the ventral root axons are guided along this pathway by neural crest cells, we surgically excised the neural crest from a series of embryos, and examined the pattern of axon outgrowth approximately 24 h later. In somites which contained no neural crest cells, ventral root axons were still found only in the rostral half of the somite, although axonal growth was slightly delayed. These axons were surrounded by sheath cells, which had presumably migrated out of the neural tube, to a point about 50 micron proximal to the growth cones. With appropriate antibodies we found that the extracellular matrix components fibronectin and laminin are evenly distributed between the rostral and caudal halves of the somite. Neither of these molecules therefore plays a critical role in determining the specific pathway of neural crest cells or motor axons through the rostral half of the somite.

Appearance of nerve growth factor receptors on cultured neural crest cells.

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.

Cell adhesion mechanisms in gangliogenesis studied in avian embryo and in a model system.

Neural crest cells undergo rapid changes in their cell-to-cell and cell-to-extracellular matrix adhesion during the ontogeny of the peripheral nervous system. The mechanisms of adhesion have been analyzed to assess the respective roles played by the cell adhesion molecules (CAMs) and the differentiated junctions. Crest cells which lose their terminal bar junctions after emigration from the neural tube contain only very few gap junctions during gangliogenesis. The calcium-dependent cell adhesion molecules, L-CAM, disappear from the neural crest and never reappear in crest cell derivatives. In contrast, the number of calcium-independent cell adhesion molecules, N-CAM, diminishes transiently during the migratory phase. In vitro, N-CAM is expressed de novo either just before or at the onset of aggregation into autonomic ganglion rudiments, whereas it is delayed in the dorsal root ganglion cells. In vitro, N-CAM mediates the calcium-independent aggregation mechanism; the rate of aggregation is, however, similar whether crest cells are derived from well-spread cultures or from two- and three-dimensional clusters. Crest cells also exhibit a calcium-dependent mechanism of adhesion controlled by molecules differing from N-CAM but which may codistribute on many different cell types during embryogenesis. These two classes of cell adhesion molecules are present on the surface of neural precursors prior to their differentiation into neurons and glial cells.

Brain "identifier sequence" is not restricted to brain: similar abundance in nuclear RNA of other organs.

A repeated 82 base pair sequence in genomic DNA of the rat was previously proposed as being a control element governing brain (neuron) specific genetic expression. This intronic sequence, termed the brain "identifier" (ID), is complementary to small RNA species localized in brain cytoplasm, and it was thought to be represented specifically in RNA produced by brain nuclei in vitro. The RNA blot analyses of total nuclear and polyadenylated heterogeneous nuclear RNA described in the present report show that this ID sequence is also present in the liver and kidney in abundances similar to those in the brain. This repeated sequence is not, therefore, restricted to transcripts produced in the brain as suggested from previous transcriptional "runoff" experiments. Measurements on rat and mouse nuclear RNA indicate that the abundance of ID sequence transcript is roughly proportional to the number of copies of this repeat in the respective genomes. This suggests a rather random genomic location and transcription of this sequence. From these results it seems improbable that the ID sequence functions as a transcriptional-level control element in genes expressed specifically in the brain.

In vivo and in vitro development of somatostatin-like-immunoreactivity in the peripheral nervous system of quail embryos.

The appearance and development of somatostatin-like immunoreactivity (SLI) in the peripheral nervous system of quail embryos were studied using radioimmunoanalysis and immunocytochemistry. In vivo, no SLI is observed in neural crest cells before or during migration. SLI appears between days 3 and 4 of incubation in sympathetic ganglia, immediately following ganglion formation, and between days 4 and 5 of incubation in the adrenal gland, soon after the adrenal gland primordium first appears. The development of SLI in the adrenal gland differs from that in the sympathetic ganglia. While in the former the amount of SLI and the number of SLI-containing cells increase as the embryo ages, in the sympathetic ganglia the amount of SLI and the percentage of SLI-containing cells decrease. When migrating neural crest cells are obtained from the sclerotomal part of 3-day embryos and grown in culture, they first display SLI after 48 hr, and the amount of SLI increases thereafter. When the sympathoadrenal precursors are removed at 4 days of incubation and grown in vitro, SLI appears after 24 hr in culture and increases during the next few days. Our results demonstrate that SLI is present very early in the quail embryo and that its appearance parallels the differentiation of neural crest cells into autonomic sympathetic ganglionic cells. We also show that the differentiation of neural crest into SLI-containing cells can be reproduced in culture, thus permitting the study of peptide production and expression in vitro.

Studies on the mechanisms of neurulation in the chick: possible involvement of myosin in elevation of neural folds.

The possible involvement of myosin in elevation of neural folds in the chick was studied. Indirect immunofluorescence revealed the presence of myosin in the neuroepithelium as early as the neural-plate stage and was concentrated in the apical regions of neuroepithelial cells where microfilaments are known to be organized into discrete bundles. This fluorescent pattern persisted until closure of the neural tube. Actin-specific fluorescence followed a similar distribution pattern as myosin. Diazepam (Valium/Roche), at 400 micrograms/ml, was found to preferentially inhibit elevation of neural folds in explanted stage 8 embryos within 6 hr of incubation. Affected neuroepithelial cells were often less elongated, contained thinner and less conspicuous microfilament bundles, and had apical surfaces which were smoother and broader than the controls. These morphological changes were accompanied by a considerable reduction in the intensity of myosin-specific fluorescence, particularly in the cell apices. Results suggest that (1) diazepam inhibits elevation of neural folds through its disruptive effects on the organization and contractility of apical microfilament bundles in developing neuroepithelial cells and (2) myosin may be directly involved in elevation of neural folds.