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Antisera to basal lamina and glial endfeet disturb the normal extension of axons on retina and pigment epithelium basal laminae
Abstract
In order to determine the role of the extracellular matrix in regulating the directed growth of embryonic neurites, antisera to retina (a-RBL I and II), to pigment epithelium (a-PBL) and to glomerular (a-GBL) basal lamina were probed for an effect on the ordered extension of neurites. In the assays, retina explants from chick and quail were cultured on basal lamina from embryonic chick retina and pigment epithelium either in the presence of anti-basal lamina antisera or in the presence of the corresponding preimmune sera. In the presence of all anti-basal lamina antisera, normal extension of axons was greatly inhibited both on retina and on pigment epithelium basal lamina. The antisera affected the growth pattern and the morphology of the individual axons in two ways: in the presence of a-RBL I the short axons were less directed, developed more and longer side branches, and the lamellipodia of the growth cones were reduced in size compared to axons from control cultures. In the presence of a-RBL II and a-GBL, axons grew slowly out from the explants as very thick bundles, strikingly different from axons in control cultures. The antiserum to pigment epithelium basal lamina induced both strong fasciculation and disorganization of the linear fiber extension, being intermediate between the two types of effects observed after antiserum addition. The results suggest that adhesive matrix molecules in basal laminae have important functions in elongation, fasciculation and in the morphology of growing axons.
... /Tween (Tris-buffered saline with 0.5% Tween 20), the blots were incubated with the hybridoma supematants for 1 hr. The blots were rinsed three times in TBYTween, incubated with 1:5000diluted (TBS/Tween) alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson Laboratories) for 1 hr, and finally developed with NBT/ BCIP as described previouslv (Halfter. 1989). Some samoles were nretreated with chondroitinase-ABC: hyaluronidase (Boehringer Mannheim, Indianapolis, IN), or hepantmase (Seikagaku, Rockville, MA) for 2 hr at 37°C under buffer and pH conditions recommended by the manufacturers. Other samples were pretreated with nitric acid to cleave the carbohydrates from the heparan sulfate prote ...
A neuronal heparan sulfate proteoglycan was identified by a panel of four monoclonal antibodies. The antibodies were generated from mice immunized with embryonic chick retina basal lamina (clones 3A12, 3A3, and 9E10) and embryonic chick optic tract (clone 6D2). Cross-reactivity of all four antibodies with the purified proteoglycan confirmed that the antibodies were directed to the same antigen. Antibodies to heparan sulfate proteoglycan from embryonic chick muscle or EHS mouse tumor (perlecan) did not cross-react with the neuronal heparan sulfate proteoglycan, suggesting that the two proteoglycans are not related. In Western blots, the proteoglycan had a molecular weight of 600 kDa that dropped to 250 kDa when the samples were treated with heparitinase or nitric acid. Immunocytochemistry showed that in early stages of chick and quail development, the proteoglycan was exclusively localized in basal laminae and had a distribution similar to that of laminin. During further development, a strong labeling was also found in the extracellular environment of nerve tracts, such as the optic nerve and white matter areas of the brain and spinal cord. The labeling of axonal tracts declined from embryonic day 10 onward, while labeling in basal laminae persisted. Antibodies to muscle heparan sulfate proteoglycan or to perlecan did not label nerve fibers. The data show that embryonic neuronal tissue expresses a new type of heparan sulfate proteoglycan.
... In the wild-type eye, RGC axons grow radially inward toward the optic nerve head to form a spoked pattern of axon bundles (Easter et al., 1984). It has been shown that this ordered pathfinding in the chick eye requires cues present on retinal basal lamina and endfeet of neuroepithelial cells (Halfter, 1989). In our screen, two genes (con, bal) were found that affect pathfinding in the eye, each in a different way. ...
We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum.
82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline.
The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all- or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.
To investigate putative axonal guidance mechanisms used by commissural interneurons in the chick embryo spinal cord, we have examined growth cone morphology, the microenvironment through which the growth cones advance, and interactions Between growth cones and their surroundings. Growth cones of both early and late developing commissural interneurons were examined. The growth cones were visualized by injection of either horseradish peroxidase (HRP) or the fluorescent dye Di-I. Unlabelled growth cones as well as HRP-labelled growth cones were also examined by electron microscopy. The early developing growth cones project circumferentially without fasciculation until they reach the region of the longitudinal pathway in the contralateral ventral funiculus (CVF). In their trajectory towards the floor plate, axons exhibited elaborate growth cones with filopodia and lamellipodia. They projected between processes of neuroepithelial cells within abundant extracellular spaces. Upon arrival at the ipsilateral ventral funiculus, growth cones did not appear to contact preexisting longitudinal axons. Within the floor plate, the growth cones were less complex and lacked long filopodia and exhibited bulbous or varicose shapes with short processes. Electron microscopic observations of the floor plate at this stage revealed that there was only a small amount of extracellular space and that the basal portion of the floor plate cells were directionally oriented (polarized) in the transverse plane. It is of particular interest that contacts between growth cones and the basement membrane in the floor plate were often observed. When the growth cones reached the contralateral ventrolateral region, they again exhibited an elaborate morphology. Close contacts between growth cones and the preexisting contralateral longitudinal axons were observed. Growth cones advancing in the contralateral longitudinal pathway exhibited various shapes and were observed to contact other axons and processes of neuroepithelial cells. Most of the later developing growth cones of commissural cells exhibited lamellipodial shapes irrespective of their location along the circumferential trajectory. Electron microscopic observations revealed that these late developing growth cones always contacted or fasciculated with preexisting axons and that the cellular environment through which they grow is oriented in such a way that the growth cones appear to be guided in specific directions. Growth cones entering the CVF exhibited more elaborated shapes with ramified lamellipodia that made multiple contacts with preexisting longitudinal axons. The present results indicate that differential axonal guidance mechanisms may be employed along the pathway followed by spinal commissural interneurons and that axons and growth cones projecting along this pathway at different developmental stages employ different mechanisms for pathfinding and guidance. These observations suggest that there are developmentally regulated transitions in the molecular cues used by growth cones at different times and in different regions along their pathway.
Optic axons are added to the retinal nerve fibre layer of fish along its vitreal border in a chronotopic manner. Likewise, the optic tract of all vertebrate species acquires axons preferentially along the superficial surface of the pathway. We have examined the developing retina of fetal ferrets ( Mustela putorius furo ) aged between embryonic day 27 (E27) and E34 to see whether a similar segregation of growth cones is apparent within the mammalian retinal nerve fibre layer. The distributions of growth cone, “wrist” (thick trailing portion of the growth cone), axonal, and glial profiles were determined from electron micrographs, and expressed as a percentage of neural profiles for several retinal locations.
The retinal nerve fibre layer of fetal ferrets contains radially elongated bundles of fibres composed of axonal, wrist, and growth cone profiles. Glial processes of varying density divide the adjacent bundles, occasionally subdividing them in the plane of the retina, and give rise to endfeet lining the basal lamina and separating the optic axons from the latter. Growth cones within the developing fibre layer represented about 2.4% of profiles at E28, while at later developmental stages (E34), this value fell to about 0.6%. During this period of axonal outgrowth, growth cones were not preferentially segregated toward the vitreal basal lamina or the glial endfeet within the nerve fibre layer. Rather, they were found scattered throughout the axon bundles of the fibre layer. While there were differences in the proportion of immature profiles found within the vitreal half compared to the scleral half of the fibre layer, such that more growth cones and wrists were found vitreally, there was no clear accumulation of them in association with features of the vitreal margin.
The present results show that young and old optic axons course together throughout the depth of the nerve fibre layer. A chronotopic mode of pathway genesis such as seen in the optic fibre layer of fish or in the optic tract of mammals is not present in the nerve fibre layer of ferrets. Differences in growth cone behaviour in the optic fibre layer and tract indicate that the mechanisms governing pathway formation differ along its course. © 1992 Wiley‐Liss, Inc.
The interactions of neurons with extracellular cues are important in directing the formation of precise neuronal networks during the development of the nervous system. This review will focus on recent progress towards the understanding of the molecular machinery involved in the interactions of neurons with the extracellular matrix.
Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin- or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development.
In the developing retina, retinal ganglion cell (RGC) axons elongate toward the optic fissure, even though no obvious directional restrictions exist. Previous studies indicate that axon-matrix interactions are important for retinal ganglion cell axon elongation, but the factors that direct elongation are unknown. Chondroitin sulfate proteoglycan (CS-PG), a component of the extracellular matrix, repels elongating dorsal root ganglion (DRG) axons in vitro and is present in vivo in the roof plate of the spinal cord, a structure that acts as a barrier to DRG axons during development. In this study, we examined whether CS-PG may regulate the pattern of retinal ganglion cell outgrowth in the developing retina.
Immunocytochemical analysis showed that CS-PG was present in the innermost layers of the developing rat retina. The expression of CS-PG moved peripherally with retinal development, always remaining at the outer edge of the front of the developing axons. CS-PG was no longer detectable with immunocytochemical techniques when RGC axon elongation in the retina is complete.
Results of studies in vitro showed that CS-PG, isolated from bovine nasal cartilage and chick limb, was inhibitory to elongating RGC axons and that RGC growth cones were more sensitive to CS-PG than were DRG neurites tested at the same concentrations of CS-PG. The behavior of retinal growth cones as they encounter CS-PG was characterized using time-lapse video microscopy. Filopodia of the RGC growth cones extended to and sampled the CS-PG repeatedly. With time, the growth cones turned to avoid outgrowth on the CS-PG and grew only on laminin.
While numerous studies have shown the presence of positive factors within the retina that may guide developing RGC axons, this is the first demonstration of an inhibitory or repelling molecule in the retina that may regulate axon elongation. Taken together, these data suggest that the direction of RGC outgrowth in the retina may be regulated by the proper ratio of growthpromoting molecules, such as laminin, to growthinhibiting molecules, like CS-PG, present in the correct pattern and concentrations along the retinal ganglion cell pathway.
The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.
Several lines of evidence suggest that glial cells have major effects on neuronal pathfinding. We have examined in vitro whether the outgrowth pattern of Xenopus retinal fibres is influenced by the glial cells encountered as they grow to the optic tectum. Strips of retina were cultured on monolayers of glial cells from the diencephalon and from the rostral and caudal ends of the optic tectum. On glia from the caudal end of the tectum the growth of fibres from the nasal and temporal ends of the strips was different: temporal fibres were shorter and more fasciculated than nasal fibres. This difference was still discernible on glia isolated from the rostral end of the tectum, but to a lesser extent. On glia from the diencephalon there was no difference between nasal and temporal fibres.
Laminin is a large glycoprotein of basement membranes. The best described laminin from a mouse tumor contains three polypeptide chains (A, B1 and B2), but there is recent evidence that some cell types produce laminin isoforms lacking the A chain. We have here studied the occurrence of the isoforms during mouse organogenesis. In all tissues studied, the A chain mRNA and polypeptide were more weakly expressed than those of the B chains. Laminin A chain polypeptides showed a much more restricted tissue distribution than the B chains. Laminin A chain polypeptide was mainly detected in basement membranes of epithelial cells, suggesting that this chain is important for morphogenesis of epithelial sheets. Most endothelial basement membranes and all embryonic mesenchyme matrices studied seemed to lack the A chain even though they contained B chains. Several of the cells producing laminin devoid of A chain seem to produce other polypeptides that become complexed to the B chains. With an anti-laminin antiserum, which in immunoblots reacts only with A and B polypeptide chains, additional polypeptides of 160 and 190 x 10(3) Mr were co-precipitated from all tissues studied. In developing heart, a polypeptide of 300 x 10(3) Mr was co-precipitated in addition. Our data suggest that these laminin-associated polypeptides are not formed by a differential splicing of the known A chain mRNA. Northern blotting of poly (A)+ RNA showed only 10kb A chain transcripts but no truncated forms. We conclude that several cell types in the mouse embryo produce laminin variants that lack the 400 x 10(3) Mr A chain. Since a major cell binding site of laminin contains parts of the A chain, the variants should differ in biological function from laminin containing this A chain.
Axon growth behavior in the optic nerve was examined using a carbocyanine dye, DiI, as a tracer, DiI facilitated clear visualization of the whole growth pattern of the optic nerve, i.e. the initial association of axons, fasciculated growth within the optic fiber layer and flattened growth cones in both living and fixed chick embryo retinae. Retrograde labelling with DiI in fixed retinae revealed that a considerable number of ganglion cells were apparently misdirected, extending their axons toward the periphery of the retina during normal development. The maximum proportion of aberrant ganglion cells reached about 15% of the total upon staining with a single DiI crystal. Misdirection was predominantly observed in retinae prepared from 6- to 8-day-old chick embryos. In embryos more than 9 days old, however, distinction of aberrant ganglion cells from normal ones became difficult, so that any degeneration of misdirected ganglion cells could not be clarified. Almost all of the misdirected ganglion cells were oriented centrifugally to the retinal periphery. These results indicate that misdirection occurs spontaneously during normal development even within the retina.
Helisoma neurons require a factor(s) present in conditioned medium (CM), for successful neurite outgrowth in vitro. A approximately 300 kDa Helisoma extracellular matrix (ECM) protein has been identified in CM and is necessary for neurite initiation. Here we show that purified approximately 300 kDa ECM protein supports outgrowth. Furthermore, anti- approximately 300 kDa Fab fragments cause a rapid, dose-dependent decrease in outgrowth when added to neurons already growing in CM, culminating in growth cone collapse and neurite retraction at 200 micrograms/ml. Collapsing growth cones rapidly lost lamellipodia and filopodia transformed into long filamentous strands. Contortion of microtubules in retracting neurites into serpentine shapes, apparently by compressive forces, suggests that large-scale microtubule depolymerization is not a prerequisite for growth cone retraction. These results imply that substrate-bound approximately 300 kDa CM protein is necessary and sufficient for CM-stimulated growth cone initiation and neurite elongation from Helisoma neurons.
The oblique muscle organizer (Comb- or C-cell) in the embryonic medicinal leech, Hirudo medicinalis, provides an amenable situation to examine growth cone navigation in vivo. Each of the segmentally iterated C-cells extends an array of growth cones through the body wall along oblique trajectories. C-cell growth cones undergo an early, relatively slow period of extension followed by later, protracted and rapid directed outgrowth. During such transitions in extension, guidance might be mediated by a number of factors, including intrinsic constraints on polarity, spatially and temporally regulated cell and matrix interactions, physical constraints imposed by the environment, or guidance along particular cells in advance of the growth cones. Growth cones and their environment were examined by transmission electron microscopy to define those factors that might play a significant role in migration and guidance in this system. The ultrastructural examination has made the possibility very unlikely that simple, physical constraints play a prominent role in guiding C-cell growth cones. No anatomically defined paths or obliquely aligned channels were found in advance of these growth cones, and there were no identifiable physical boundaries, which might constrain young growth cones to a particular location in the body wall before rapid extension. There were diverse associations with many matrices and basement membranes located above, below, and within the layer in which growth cones appear to extend at the light level. Additionally, a preliminary examination of myocyte assembly upon processes proximal to the growth cones further implicates a role for matrix-associated interactions in muscle histogenesis as well as process outgrowth during embryonic development.
Positional identity in the visual system affects the topographic projection of the retina onto its central targets. In this review we discuss gradients and positional information in the retina, when and how they arise, and their functional significance in development. When the axons of retinal ganglion cells leave the eye, they navigate through territory in the central nervous system that is rich in positional information. We review studies that explore the navigational cues that the growth cones of retinal axons use to orient towards their target and organize themselves as they make this journey. Finally, these axons arrive at their central targets and make a precise topographic map of visual space that is crucial for adaptive visual behavior. In the last section of this review, we examine the topographic cues in the tectum, what they are, when, and how they arise, and how retinal axons respond to them. We also touch on the role of neural activity in the refinement of this topography.
Wound closure and repair of embryonic neuroepithelium were studied in organ-cultured embryonic retinae. Eyes from 3 to 4-day-old embryos were cultured after removing pieces of retinal tissue. During the subsequent 24 hours of incubation, the 150 to 200 microns wide holes in the retina closed completely. Histological studies showed that the wound closure was not accomplished by cell migration or cell proliferation, but by an approximation of the wound edges mediated by extracellular matrix fibrils of the vitreous body. The wound contraction facilitated the integration of transplants into the retinal neuroepithelium with a perfect alignment of the implants with the host at the vitreal surface. Within 24 hours, a continuous inner limiting membrane between transplant and host retina was established. The effect of wound healing and tissue transplantation on the navigation of optic axons in the retina was investigated. The wound contraction in the retina caused the optic axons near the lesion site to grow to the wound center, where the axons traversed the retina and formed a neuroma at the ventricular side, resembling the organization of axons at the optic disc. In the transplantation paradigm, axons from the host retina migrated into the transplant and vice versa. However, due to the wound contraction around the transplant, most axons grew into the interface between the transplant and host tissue.
The distribution of retinofugal fibres has been studied by electron microscopy throughout the extent of the developing mouse optic nerve and chiasm at embryonic day (E) 16, in order to determine the course of fibre growth. Growth cones and mature axons, which are randomly distributed in bundles in the extracranial optic nerve, segregate in the juxtachiasmatic optic nerve. Here, growth cones accumulate in subpial regions amongst the endfeet of radial glia, whereas axons lie in the depths of the nerve. Surprisingly, however, growth cones move away from this region toward the ventricular zone in the lateral and midline parts of the chiasm, only to return to subpial regions once more before entering the optic tract, where fibres are again in an age-related order. Superficially, mature axons mingle with growth cones in the chiasm and near the beginning of the optic tract, suggesting that the age-related order begins to be reestablished before growth cones enter the tract. Deep and superficial regions of the pathway were examined in different planes of section. Specialised membrane relationships between retinofugal fibres and radial glial cells were also studied in deep and superficial regions of the lateral part of the chiasm. In addition, the distribution of retinofugal fibre bundles in the adult mouse was looked at by using light microscopy. The changing fibre positions noted in the embryo are maintained in the adult.
We have examined the age-related reordering of optic axons as they pass through the chiasmatic region in fetal ferrets. Proportions of young and old optic axons were determined from electron micrographs taken sequentially through the prechiasmatic nerve, chiasm, and tract. This "chronotopic" reordering of axons was shown to emerge gradually, beginning rostral to the fusion of the two optic nerves, but continuing to develop caudal to the chiasmatic midline. Segregation of young from old optic axons was most pronounced within the optic tract. We then compared the emergence of this fiber reorganization to the distribution of cell adhesion and extracellular matrix molecules and to the glial architecture within the pathway. Using immunohistochemistry, the distributions of the cell adhesion molecules L1, NCAM, and TAG-1 and the extracellular matrix molecules laminin-1 and chondroitin sulfate proteoglycans (CSPGs) were determined. Among these, only the distribution of CSPGs was observed to change in a manner that complemented the segregation of young from old optic axons. CSPGs were densest in the deeper parts of the optic tract, coincident with radial glial fibers that turn to course within the region of the oldest optic axons. Both the glial architecture and the CSPG distribution form as a consequence of the invasion of the first optic axons, shown by the developmental sequence of each, and by the fact that these glial and molecular features fail to form in the absence of optic axons. The data suggest a model in which the gradient of CSPGs across the depth of the tract contributes to the formation of the chronotopic fiber reordering by providing a relatively unfavorable environment for subsequent axonal growth. The CSPGs may do so by interfering with adhesion molecules on optic axons that normally promote elongation.
This study examines morphological changes of the blood-brain barrier (BBB) after spinal cord compression. The lowest thoracic segment (T13) of female guinea pigs was injured and the BBB was tested from 7 days to 5.5 months postinjury using intravenously injected horseradish peroxidase (HRP) as a tracer. Tracer leakage in the injured segment was verified with the light microscope and the fine structure of capillaries was examined. Diffuse tissue staining was observed at T13 up to 2 weeks following injury. A leaky BBB correlated with expected changes in the fine structure of endothelial cell junctions. These were predominantly nonoverlapping cell junctions which, in many instances, were separated by clefts between adjacent cells. At early survival times, numerous capillary profiles with juxtaposed astrocyte foot processes were noted in addition to altered cell associations. Complete sealing of the BBB against interstitial HRP leakage was not observed until 17 days postinjury. After the first week, some of the endothelial cells were contacted by macrophages, processes of perivascular microglia, and processes of swollen and degenerating astrocytes. Perivascular spaces varied in extent and contained amorphous deposits of extracellular materials in addition to supernumerary layers of basal lamina. The early changes were followed by profound tissue restructuring due to loss of both neurons and glia. At longer survival times the BBB to HRP repaired. Endothelial cells formed complex overlapping junctions with zonulae occludentes. Most of the capillaries in the injured segment were no longer in direct contact with astrocyte foot processes, although reactive astrocytes constituted the predominant cell type in the remaining gray matter. Substantial expansion of perivascular spaces was evident. The cytoplasm of endothelial cells had numerous pinocytotic vesicles. Perivascular spaces contained layers of assembled collagen arranged perpendicularly to each other in addition to amorphous matrix materials. The findings suggest that decoupling of astrocyte foot processes from endothelial cell surfaces does not prevent reformation of tight junctions. It remains to be examined what effects the larger perivascular spaces, extracellular matrix deposits, and changes of cell associations may have on transport systems and ionic buffering. The data are relevant for estimating an opportune time for application of barrier-impermeable drugs to the lesion area.
The retino-tectal system has served as a model for the growth and navigation of axons in the developing brain for over 50 years, when the first anatomical studies of embryos revealed details of the ontogeny of this pathway (Herrick 1941). Such observations led to various enthusiastic speculations concerning axon growth, including ideas that growth cones were towed to their targets, or directed there by electric fields. These hypotheses were succeeded by others, slightly less fantastic, which had retinal axons following emerging cracks and channels in the brain that blazed the trail for their long journey (reviewed in Jacobson, 1991). In the 1960s, Sperry made what at first seemed like an equally wild hypothesis, that retinal axons were led to their specific targets in the tectum by cytochemical tags (Sperry 1963). For more than 30 years, Sperry’s hypothesis gained force as attempts to disprove or replace it with less biochemical hypotheses failed. Though success in the search for the molecular basis of retinal axon navigation, and the vindication of Sperry, had to wait until the 1990s and the second coming of age of molecular biology, the attraction of the retino-tectal system for investigating these fundamental issues has never diminished. As a result, this is one of the most carefully studied and well-characterized developing axon pathways known.
Basal laminae, thin sheets of extracellular matrix covering the basal side of all neuroepithelia, are strongly supportive for neurite outgrowth in vitro and may provide a permissive environment for growing neurites in vivo. To gain information about the biological activity and composition of in situ-derived basal laminae the inner limiting membranes from embryonic day (E) 7 to E11 chick and quail retinae were isolated. The basal laminae were solubilized with high-molar guanidine hydrochloride or urea, and the solubilized proteins reconstituted by dialysis. The matrix proteins were spotted or dried onto nitrocellulose or polylysine-coated dishes. When explants from retina or from dorsal root ganglia were incubated on the protein spots, neurite extension was very robust, at a level as high as on authentic basal lamina. Extracts from the pigment epithelial basement membrane did not support neurite extension. Western blot analysis showed that the explant from the retinal inner limiting membrane contained predominantly basal lamina-type proteins, such as laminin, collagen type IV and heparan sulphate proteoglycan, whereas the matrix extract from the pigment epithelium contained predominantly mesenchymal-type proteins, like collagen type I and tenascin. JG22, a beta1 integrin antibody that inhibited neurite extension on EHS tumour laminin substrate, had no effect on neurite outgrowth on retinal basal lamina matrix, indicating that embryonic basal laminae contain other or additional growth promoting substrate molecules.
In the developing or regenerating nervous system, migrating growth cones are exposed to regulatory molecules that positively and/or negatively affect guidance. Chondroitin sulfate proteoglycans (CSPGs) are complex macromolecules that are typically negative regulators of growth cone migration in vivo and in vitro. However, in certain cases, neurites sometimes traverse regions expressing relatively high levels of CSPGs, seemingly a paradox. In our continuing efforts to characterize CSPG inhibition in vitro, we manipulated the ratio of CSPGs to growth-promoting laminin-1 to produce a substratum that supports outgrowth of a subpopulation of dorsal root ganglia (DRG) neurites, while still being inhibitory to other populations of DRG neurons [Exp. Neurol. 109 (1990), 111; J. Neurobiol. 51 (2002), 285]. This model comprises a useful tool in the analysis of mechanisms of growth cone guidance and is particularly useful to analyze how CSPGs can be inhibitory under some conditions, and growth permissive under others. We grew embryonic (E9-10) chicken DRG neurons on nervous system-isolated, substratum-bound CSPGs at a concentration that supports an intermittent pattern of outgrowth, alternating with regions adsorbed with growth-promoting laminin-1 alone, and analyzed outgrowth behaviors qualitatively and quantitatively. A novel finding of the study was that DRG neurites that elongated onto CSPGs were predominantly fasciculated, but immediately returned to a defasciculated state upon contact with laminin-1. Further, cursory inspection suggests that outgrowth onto CSPGs may be initially accomplished by pioneer axons, along which subsequent axons migrate. The outgrowth patterns characterized in vitro may accurately reflect outgrowth in vivo in locations where inhibitory CSPGs and growth-promoting molecules are coexpressed, e.g., in the developing retina where fasciculated outgrowth may be instrumental in the guidance of retinal ganglion cells from the periphery to the optic fissure.
The vitreous surface of the embryonic avian retinal neuroepithelium was isolated by mechanical disruption of the retina mounted between 2 adhesive substrata. The 200-micron-thick sheath covered an area of up to 1 cm2 and consisted of the vitreal basal lamina with a lamina densa, 2 laminae rarae, and a carpet of ventricular cell endfeet on top of the lamina. The vitreal endfeet were removed by detergent treatment and an extracellular basal lamina was obtained. The laminae were further characterized by immunohistochemistry and immunoblotting. A 190 kDa laminin protein was detected in laminae with and without vitreal endfeet, whereas the membrane-bound neural cell adhesion molecule (N- CAM) was detectable only on the endfeet of the ventricular cells and was absent in the detergent-treated basal laminae. Neither immunoblotting nor immunostaining revealed fibronectin in these preparations. Explants of retina, sensory ganglia, and cerebellum from chick, quail, and mouse were cultured on the basal lamina as a substratum. In all cases axonal outgrowth was excellent, with a growth rate similar to that in situ. Outgrowing axons from sensory ganglia and cerebellar explants were accompanied by migratory cells, which, in the case of sensory ganglia, were flat cells and, in the case of cerebellar explants, resembled granular neurons. Optic axons grew on the laminae in an asymmetric, explant-inherent pattern specific for the position of origin of the explant. On detergent-treated basal laminae, as well as on laminin, the retinal axons grew in a clockwise orientation. This axonal growth pattern was specific for retinal tissue and was not observed with axons from other neural explants. In spite of the excellent substrate properties provided by the substratum, cues for growing axons (toward or away from the optic disk) were not detectable in the basal lamina preparations.
Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
The adhesion of chick embryo sensory neurons to glass coverslips was examined with interference reflection optics. On untreated glass, adhesive contacts are common only beneath growth cones and are small. On polylysine-treated glass growth cones are highly spread, microspikes reach treat lengths and extensive adhesive contacts underlie growth cones, microspikes and nerve fibers. Veils, expanded from the growth cone, are adherent to the substratum either centrally or laterally, while the extending edge of the cell margin is non-adherent. Linear adhesions are frequent beneath microspikes and pass centrally beneath the growth cone margin. The distribution of linear adhesions resembles that of microfilament bundles seen within whole mounts of growth cones. Adhesive contacts stabilize extensions of the growth cone margin and may influence the organization of the microfilamentous network within the growth cone. Regulation of microfilament organization by adhesion may influence microfilament functions in growth cone mobility and the assembly of neurite structures.
The role of cell-to-substratum adhesion in the initiation, elongation, and branching of axons from embryonic sensory neurons was investigated. Cells from sensory ganglia of 4–8-day-old chicken embryos were cultured on several substrata: including collagen; polyornithine-, polylysine-, and polyglutamate-coated surfaces, and tissue culture dishes. The air-blaster method was used to measure growth cone-substratum adhesion.Growth cones adhere much more strongly to polyornithine- or polylysine-coated surfaces and to the upper surfaces of glial cells than to tissue culture plastic. Axons, too, adhere tightly to these substrata, and are crooked, whereas on tissue culture plastic, axons are not adherent and are straight. The fraction of neurons that form axons and the rates of axonal elongation and branching are markedly increased when cells are cultured on polyornithine-coated dishes as compared to tissue culture dishes.This correlation of strong adhesion and enhanced neuronal morphogenesis suggests that adhesive interactions between the growth cone and the microenvironment in an embryo are crucial parts of the initiation and elongation of neuronal processes. Regulation of neuronal morphogenesis may be expressed through the physicochemical properties of the interacting cell surfaces and extracellular environment.
A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.
The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.
The optic nerve of the developing rat was examined for the presence of laminin, an adhesive glycoprotein, to assess whether it might serve as a substrate for retinal axon growth in vivo. The optic stalk and nerve of developing rats were screened immunohistochemically for the presence of laminin before, during, and after the period of retinal axon growth. On embryonic day 14 (E14), laminin immunoreactivity was present in the ventral portion of the optic stalk, the same region in which the first retinal axons grow. Between E16 and postnatal day 10 (P10), cells positive for laminin were distributed throughout the cross-sectional area of the nerve. There was a progressive appearance of glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for astrocytes, from the optic nerve head towards the chiasm beginning on E20. At the advancing front of GFAP immunoreactivity, cells were positive for both laminin and GFAP. Behind the front, laminin immunoreactivity disappeared from the cells. By P12, the only laminin immunoreactivity that remained within the optic nerve surrounded the vasculature. This is a time after the last retinal axons grow through the optic nerve. Monolayer cell cultures were prepared from perinatal rat optic nerves and processed for immunohistochemistry to determine which astrocyte type was laminin-positive. Type 1 astrocytes, which primarily compose the immature nerve, are GFAP-positive, A2B5-negative, and laminin-positive. Type 2 astrocytes, a major component of the mature optic nerve, were GFAP-positive, A2B5-positive and laminin-negative. An extract of developing optic nerve was analyzed by immunoblot along with laminin purified from Engelbreth-Holm-Swarm (EHS) sarcoma. Purified laminin ran with SDS-PAGE under reducing conditions as 2 bands with Mrs of 200,000 and 4000,000. Both bands reacted with antibodies to laminin. A low-salt extraction of whole optic nerve from E18 rats resulted in 2 bands with the same Mr as seen with laminin from EHS sarcoma. When only the inside of the optic nerve (which lacked the basal lamina and meninges that surround the outside) was processed, there was a dark 200,000 D band, but the 400,000 D band was virtually absent. These results are consistent with the hypothesis that laminin, or a variant form of laminin, serves as a substrate for retinal axon growth in the developing rat optic nerve.
Both the polarity of the axonal growth and the formation of the optic fiber pattern early in retinal morphogenesis were studied in silver stained whole mounts of embryonic chick, quail, and pigeon retinae. The surface area of the retina and of the optic fiber layer increases in size exponentially, the optic fiber layer expanding faster than the retina. The optic fiber layer covers the retinal surface at E5 in quail and at E6 in chick and pigeon. In all species studied, the retinal fiber layer does not expand homogeneously with the optic nerve head as the center. Instead, the retinal fiber layer enlarges with polarities in the dorsal to ventral and nasal to temporal direction. The very first axon bearing ganglion cells appear at stage 16 in the dorsal and central portion of the retina and grow ventrally to merge at the optic disk. From stage 23 on, the optic fiber layer expands faster in the temporal than in the nasal side. Measurements on the initial polarization of young axonal processes show that the axonal growth is directed toward the optic fissure and the optic nerve head. This growth polarization is found at the onset of growth cone formation and in axons far from the nearest ganglion cells or ganglion cell axons. Therefore axon-axon interaction cannot be involved in the initial axon orientation early in retinal morphogenesis.
A monoclonal antibody was obtained that binds to cell membrane molecules distributed in a topographic gradient in avian retina. Thirty-five-fold more antigen was detected in dorsoposterior retina that in ventroanterior retina. Most of the antigen was associated with the synaptic layers of the retina. Less antigen was detected in cerebrum, thalamus, cerebellum, and optic tectum, but little or none was found in non-neural tissues tested. The antigen was found on most or all cell types in retina, and the concentration of antigen found is a function of the square of the circumferential distance from the ventroanterior pole of the gradient toward the dorsoposterior pole. Thus, the antigen can be used as a marker of cell position along the ventroanterior-dorsoposterior axis of the retina.
The retinal fiber layer and the juxtaretinal portion of the optic nerve of goldfish have been studied with light and electron microscopy in order to determine whether the age-related order of fibers in the nerve originates in the retina. In the retina, no patent spaces (channels) were noted. The fibers ran in fascicles and consisted of two classes: nonmyelinated fibers, which ran superficially (close to the vitreal surface), and "myelinated" fibers, which ran more deeply and were loosely wrapped by processes presumed to be glial. The myelinated fibers were larger and presumably older. The nonmyelinated fibers are believed to be the young ones, from the peripheral, more recently generated, ganglion cells, for the following reasons. (1) Their size and cytoskeletal elements were typical of young axons. (2) They were the only axons in peripheral retina. (3) They were continuous with the nonmyelinated fibers in the nerve, previously shown to be the young ones. (4) When retinal axons were cut peripherally, the degenerating axons were in the superficial part of the fiber layer. (5) Growth cones, presumably from the newest ganglion cells, were always observed at the most superficial position in the fiber layer, in direct contact with the basal lamina of the inner limiting membrane superficially and nonmyelinated fibers deeply. The nonmyelinated fibers always clustered together in the retinal fiber layer and occupied the most central portion in the cross-section of the optic nerve head. Thus, the age-related organization of fibers in the nerve is established in the retina. These results are discussed in the context of growth, with the aim of evaluating the relative importance of four factors that might influence the intraretinal course of the growth cone. Its interactions with other fibers and with the basal lamina of the inner limiting membrane seem to be more important than interactions with the glial end feet or guidance into open, preformed channels.
The cell adhesion molecule (CAM) is involved in adhesion among embryonic retinal and brain cells and has been detected in a variety of neural tissues. This paper describes the use of spinal ganglion cultures and specific anti-CAM antibodies to determine the distribution of CAM on plasma membranes of nerve processes, and to assess the results of perturbation of its function during the growth of neurites from ganglia. The results indicate that CAM is distributed over the entire surface of nerve processes, and that specific anti-CAM Fab' fragments alter the morphology of neurite outgrowth. In particular, it was observed that anti-CAM inhibits formation of nerve bundles, so that the ganglion becomes surrounded by a tangled net of fine processes. Growth cone functions, such as neurite elongation, motility, and attachment to the substratum, did not appear to be affected by the antibody. These studies suggest that one of the major functions of CAM is to mediate side-to-side adhesion between neurites to form fascicles, and raise the possibility that this molecule serves a key role in embryogenesis of nerve tissues.
Lewis rats were injected intravenously with rabbit anti-rat glomerular basement membrane (GBM) antisera in doses that were sufficient to cause glomerular fixation of rabbit gamma globulin (RGG) detectable by immunofluorescence, but which failed to induce histologically detectable lesions. 24 h later, groups of rats received lymph node cells or serum from syngeneic donors that had been immunized with either RGG or ovalbumin; they were injected with [3H]thymidine three times during the next 2 days, and sacrificed 48 or 96 h after transfer. Only the rats given anti-GBM antiserum plus lymph node cells from donors sensitized to RGG showed histological glomerular lesions, in the form of segmental hypercellularly and necrosis. Autoradiographs revealed the greatest number of labeled cells in glomeruli in the same group. In analogous experiments, it was shown that T-cell-enriched populations could induce hypercellular glomerular reactions. On the basis of electronmicroscopic and autoradiographic observations, it appears that the glomerular hypercellularity resulted from both infiltration of mononuclear cells and proliferation of endothelial cells. The findings indicate that interaction of specifically sensitized lymphocytes with glomerular-bound antigen can induce a cell-mediated (delayed-type) reaction in glomeruli.
Retinal ganglion neurons extend axons that grow along astroglial cell surfaces in the developing optic pathway. To identify the molecules that may mediate axon extension in vivo, antibodies to neuronal cell surface proteins were tested for their effects on neurite outgrowth by embryonic chick retinal neurons cultured on astrocyte monolayers. Neurite outgrowth by retinal neurons from embryonic day 7 (E7) and E11 chick embryos depended on the function of a calcium-dependent cell adhesion molecule (N-cadherin) and beta 1-class integrin extracellular matrix receptors. The inhibitory effects of either antibody on process extension could not be accounted for by a reduction in the attachment of neurons to astrocytes. The role of a third cell adhesion molecule, NCAM, changed during development. Anti-NCAM had no detectable inhibitory effects on neurite outgrowth by E7 retinal neurons. In contrast, E11 retinal neurite outgrowth was strongly dependent on NCAM function. Thus, N-cadherin, integrins, and NCAM are likely to regulate axon extension in the optic pathway, and their relative importance varies with developmental age.
Several mechanisms seem to contribute to the formation of the retinotectal projection, including interactions of retinal axons with each other and with tectal tissue. Spatially graded distributions of positional markers, in particular, may be involved in axonal guidance. Recently, new assays have been developed for measuring directional effects on growing fibres. They have been applied to investigations of pathways taken by misrouted fibres in vivo and by fibres which encounter a situation in vitro where they have to choose between two artificial pathways. The results demonstrate the existence of directional cues and spatially graded markers on the tectum which are capable of guiding invading axons. Guidance by gradients may sometimes be due to differential adhesion. Chemotactic guidance may also be involved particularly if slight directional cues are amplified within the growth cone. Suitable interactions of graded components, as revealed by mathematical analysis, would be capable of generating reliable projections.
Optic fibers were experimentally deflected from their normal courses in the ganglion cell fiber layer (GCFL) of the embryonic chick retina. Fibers deflected sclerad to the GCFL travelled randomly with no tendency to re-enter the GCFL. Fibers growing in the more sclerad portion of the GCFL travelled bidirectionally, i.e. either toward or away from the optic nerve, sometimes exiting through the retinal periphery. Fibers in the most vitread portion of the GCFL tended to grow unidirectionally, toward the optic nerve. The results indicate that the factors that steer optic fibers toward the optic nerve do not operate through the full thickness of the retina but are narrowly confined to the GCFL, possibly to its most vitread portion. The GCFL acts like a glue that entraps but does not attract optic axons.
Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin-N-hydroxysuccinimidester, biotin-epsilon-aminocaproic acid-N-hydroxysuccinimidester, and desthiobiotin-N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5-E5.5) to 89% (stage 29 to 35; E6-E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9-E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.
In wholemounted retinae of cat, rat and monkey, in which ganglion cells were retrogradely labelled with horseradish peroxidase, a quantitative analysis of the direction of the axon initial segment with respect to the optic disc and of the relationship between the axon initial segment and the direction and distribution of primary dendrites was performed on the class of largest ganglion cells. The results show the following. (1) In all 3 species, the majority of primary dendrites of ganglion cells are directed away from the axon initial segment. (2) Primary dendrites arise with a greater frequency from the region of the cell body opposite to the axon initial segment than close to it. (3) In cat the direction of the axon initial segments show less variance in their initial direction with respect to the optic disc than in rat or monkey. In adult cats the nucleus of alpha-ganglion cells occupies a central position. In the kitten the position of the nucleus is eccentric and lies in a part of the cell body opposite to the axon initial segment. The nucleus moves to a central position over the next 3 weeks. The position of the axon initial segment is discussed as a possible determinant of ganglion cell dendritic geometry.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
A striking example of topographic specificity in synapse formation is the preferential reinnervation of original synaptic sites on denervated muscle fibres by regenerating motor axons. This specificity is mediated by the basal lamina of the synaptic cleft. A glycoprotein, s-laminin, has now been identified that is selectively associated with synaptic basal lamina and is recognized by motoneurons. Molecular cloning reveals that s-laminin is a novel homologue of laminin, a potent promoter of neurite outgrowth.
Chick embryo retinal ganglion cell (RGC) axons grow to the optic tectum along a stereotyped route, as if responding to cues distributed along the pathway. We showed previously that, in culture, RGCs from embryonic Day 6 retina are responsive to the neurite-promoting effects of the extracellular matrix glycoprotein laminin and that this response is lost by RGCs at a later stage of development. Here we report that, before axon outgrowth is initiated in vivo, laminin, is expressed along the optic pathway at nonbasal lamina sites that are accessible to the growth cones of RGC axons. The distribution of laminin within the pathway is consistent with its localization at the end-feet of neuroepithelial cells that line the route, and it continues to be expressed at these marginal sites during the first week of embryonic development. At later stages, concomitant with the loss of response by RGCs in culture, laminin becomes restricted to basal laminae at the retinal inner limiting membrane and pial surface of the optic pathway. Neurofilament-positive RGC axons bind a monoclonal antibody, JG22, which recognizes the laminin/fibronectin receptor complex, and continue to do so throughout embryonic development. We show that, in vitro, the JG22 antigen expressed by RGCs appears to function as a laminin receptor, by demonstrating that JG22 antibody blocks neurite outgrowth on a substrate of laminin. These findings are consistent with the possibility that laminin defines a transient performed pathway specifically recognized by early RGC growth cones as they navigate toward their central target.
In order to study cell translocation in vitro on a physiological substrate a novel cell migration assay was developed using the inner limiting membrane of the avian embryonic retina. The matrix sheet consists of a laminin-rich basal lamina covered by a dense layer of neuroepithelial endfeet. The retina basal lamina does not contain fibronectin. Cells translocating on this substrate displace the neuroepithelial endfeet, leaving behind tracks in the endfeet monolayer. Motility of cells and the relative forward to lateral migration can be quantitated by measuring lengths, widths, and areas of the tracks. Using this assay system, the conditions and patterns of cell migration for a variety of cells have been examined. In the absence of serum all cell types show only minor migratory activity and addition of serum to the culture medium always enhances the rate of cell migration in a saturable, dose-response manner. The serum cannot be replaced by fibronectin or vitronectin (serum spreading factor). For maximum cell migration, serum has to be constantly present in the medium; however, 58% cell migration is obtained in serum-free medium when the matrix is preincubated with serum. According to the area and linearity of the tracks, the migratory behavior of the different cells can be classified into three groups: (i) fibroblasts and the nonpigmented Bowes melanoma cells form straight and long tracks; (ii) glioma, sarcoma, and carcinoma cells from straight but short tracks, and (iii) neuronal tumor cells, epithelial cells, and pigmented B16 melanoma cells form wide and short tracks. Comparative studies with low and high metastatic clones of tumorgenic cell lines show that migratory activity and metastatic potential of cells do not necessarily correlate. Finally, we show that fibroblasts deposit fibronectin fibrils on their paths as they migrate on the basal lamina. Fibronectin trails are also seen when fibroblasts are cultured on plain basal laminae that are pretreated with detergent to remove the endfeet monolayer. Likewise, when fibroblasts are cultured in the presence of antifibronectin antibodies, the fibronectin secreted by cells is detectable. Due to antibody treatment the cellular fibronectin is precipitated and its normal fibril formation is inhibited; however, the translocation of fibroblasts is not impaired.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
In Drosophila melanogaster certain mutations alter the polarity of trichomes and bristles, cuticular structures secreted by the epithelial cells of the adult fly. Since sensory neurons arise from epithelial cell precursors, and sensory axons grow along the inner faces of epithelial cells, we have studied the developing wings of these mutants to see whether the change in epithelial cell polarity has an influence on the direction of axon outgrowth. The nerve patterns formed in the mutants prickled, inturned, and frizzled, however, were largely normal, indicating that in these cases the polarity of the cuticular structures produced by the epithelial cells is altered without any effect on the polarity of the associated axons.
During axonogenesis, contacts made by the growth cone with its substratum are important in guiding the direction of neurone outgrowth. This study examines the contacts made by the growth cones of pioneer neurones in the embryonic grasshopper limb. Individual pioneer neurones at different stages of development were injected with horseradish peroxidase and the contacts made by the filopodia at the tip of their growth cones were examined by electron microscopy.
Filopodia made few contacts with mesodermal cells, some contacts with ectodermal cells and very frequent contacts with basal lamina underlying the ectoderm. Components of the basal lamina may therefore play a role in guiding pioneer axon outgrowth.
The function of the neurite growth-promoting antigen INO has been tested in an in vivo neurite regeneration system, the rat iris. The sympathetic innervation of the irides was removed by a single systemic injection of 6-hydroxydopamine. The subsequent regeneration of sympathetic axons into the iris of one eye bathed by the INO antibody, which inhibits neurite growth in vitro, was compared with the regrowth of sympathetic axons into the iris of the animal's other eye, which contained control antibody. Antibodies were released within the eye by implanted hybridoma cells. Neurite regeneration was measured by assaying [3H]norepinephrine uptake into freshly explained irides. The blockage of the function of the INO antigen by the antibody resulted in a decreased rate of axonal regeneration, thus suggesting the involvement of the INO antigen in the process of neurite regeneration in vivo.
Examination of a large number of retinal pigment epithelia revealed that, in a small proportion, optic axons in chick and quail eyes aberrantly entered the pigment cell layer between embryonic day (E) 7 to E14. The aberrant retinal axons originated from the main stream of retinal fibers in the optic nerve and invaded the pigment layer from various positions of the optic nerve head or fissure by growing along the basal side of the pigment epithelium. The axon bundles grew several millimeters into the epithelial sheet and arborized at the margin of the eye. As shown by electron microscopy the nerve fibers occurred as bundles of three to several hundred axons. They always were located at the basal side of the epithelium, and were enveloped by processes of epithelial cells. Very large bundles of axons, however, displaced the epithelial cells from the basal matrix. These retinal axons contacted the pigment epithelial basal lamina. The basal extracellular matrix from the retinal pigment epithelium was isolated and used as substratum for in vitro cultures of various types of neural explants. The matrix preparations consisted of a sheet of a 50 nm thick basal lamina with a central lamina densa, two laminae rarae, and a 15 micron thick stroma. Axons from avian retina explants, as well as sensory ganglia, grew on the basal lamina side of the pigment cell matrix with the same growth rate and with the same fiber density as on similarly prepared basal laminae from the neural retina. These experiments show that the matrix from the pigment epithelium of the avian eye does not have negative effects on axonal growth and indicate that a basal lamina from a normally non-innervated tissue can provide a favorable matrix for axonal growth.
The effective regeneration of severed neuronal axons in the peripheral nerves of adult mammals may be explained by the presence of molecules in situ that promote the effective elongation of neurites. The absence of such molecules in the central nervous system of these animals may underlie the relative inability of axons to regenerate in this tissue after injury. In an effort to identify neurite growth-promoting molecules in tissues that support effective axonal regeneration, we have developed an in vitro bioassay that is sensitive to substrate-bound factors of peripheral nerve that influence the growth of neurites. In this assay, neonatal rat superior cervical ganglion explants are placed on longitudinal cryostat sections of fresh-frozen sciatic nerve, and the regrowing axons are visualized by catecholamine histofluorescence. Axons are found to regenerate effectively over sciatic nerve tissue sections. When ganglia are similarly explanted onto cryostat sections of adult rat central nervous system tissue, however, axonal regeneration is virtually absent. We have begun to identify the molecules in peripheral nerve that promote effective axonal regeneration by examining the effect of antibodies that interfere with the activity of previously described neurite growth-promoting factors. Axonal elongation over sciatic nerve tissue was found to be sensitive to the inhibitory effects of INO (for inhibitor of neurite outgrowth), a monoclonal antibody that recognizes and inhibits a neurite growth-promoting activity from PC-12 cell-conditioned medium. The INO antigen appears to be a molecular complex of laminin and heparan sulfate proteoglycan. In contrast, a rabbit antiserum that recognizes laminin purified from mouse Engelbreth-Holm-Swarm (EHS) sarcoma, stains the Schwann cell basal lamina of peripheral nerve, and inhibits neurite growth over purified laminin substrata has no detectable effect on the rate of axonal regeneration in our assay.
Antibodies against laminin (LN), fibronectin (FN), collagen type IV (Col IV), neural cell adhesion molecule (N-CAM), T-61 antigen, actin, tubulin and neurofilament protein were injected into the eyes of quail embryos (Coturnix coturnix japonica) of different ages. Twenty h after injection, the heads of the embryos were fixed and the antibodies visualized in sections with the use of fluorescein-isothiocyanate (FITC) or peroxidase-labeled second antibodies by light- and electron microscopy. Antibodies against cell surface molecules, such as N-CAM, LN, Col IV and T 61, labeled matrix and membrane components of the retinal cells in different antigen-specific patterns. Antibodies against intracellular antigens, such as actin, tubulin and neurofilament protein labeled nonspecifically the vitreous body and the inner basal lamina of the retina, but resulted in only a very weak and diffuse labeling of retinal cells. N-CAM was detected in high concentration in the optic fiber layer on the surface of axons and on the membranes of all retinal cells. Col IV, LN and T 61 antigen were found predominantly in the optic fiber layer. LN and Col IV were located on the surface of axons and the endfeet of ventricular (neuroepithelial) cells in a patchy distribution. The T-61 antigen was found in early stages in the cell-free space of the optic fiber layer, on the surface of ventricular cells and axons, and at later stages also in high-density patches between nerve fibers. The distribution of LN and T-61 antigen together with data from in vitro experiments suggests a crucial role of these proteins in axon extension in the avian retina during early development of the optic fiber layer.
We have examined the neural tube in Xenopus laevis tadpoles to investigate the anatomical guidance elements which may be present in the presumptive marginal zone. With appropriate fixation protocols the neuroepithelial cells appeared in contact; electron microscopic observations failed to show any specialized intercellular spaces preceding the growing axons. The first fibres were found in the intercellular clefts between the neuroepithelial cells near the surface of the neural tube. Reconstructions of the neural tube from examination of serial 1 micron sections showed that the intercellular clefts are non-aligned at this stage and branching. Scanning electron microscopy of the surface of the neural tube confirmed that the intercellular spaces are non-aligned and often branch caudal to the growing front of descending axons. Thus to grow in a consistent direction the developing axons may have to make consistent and selective (specific) selections of pathway at numerous branch points if their growth is restricted to these intercellular clefts. As more axons grow along the neural tube, the intercellular clefts become wider, and the neuroepithelial cells bounding the clefts become indented. At later stages many fibres were observed with both scanning and transmission electron microscopy to grow along the surface of the neural tube. These changes in neuroepithelial cell morphology and fibre pathway allow axons to form bundles which take a fairly straight course in contrast to the winding path which must be taken by the first axons to grow through the intercellular clefts.
Brain topography may have its earliest expression as spatial gradients of molecules controlling the deposition of neurones and neuronal processes. In the vertebrate visual system there is evidence that the stereotyped alignment of central retinal projections relies on an initial spatially organized distribution of molecules in both the retina and its central target nuclei. We used an immunological approach to look for molecules that are so organized and produced a monoclonal antibody (JONES) which shows a pronounced dorsal to ventral gradient of binding in the rat retina throughout the period when retinal ganglion cell axons are forming topographically organized projections within the central nervous system (CNS). Binding is present throughout the radial thickness of the retinal epithelium in regions where postmitotic neurones are generated but is not associated with any consistent histological characteristic of the tissue. The antibody was shown to bind on the cell surface of freshly dissociated retinal cells, and dorsal retinal quadrants were found in vitro to have nearly twice as much antigen as ventral retinal quadrants. Initial biochemical characterization of the target epitope reveals that it is a lipid present in chloroform/methanol extracts from perinatal retina and is sensitive to neuraminidase digestion.
How is the adult pattern of connections between motoneurones and the muscles that they innervate established during vertebrate development? Populations of motoneurones are thought to follow one of two patterns of development: (1) motor axons initially follow stereotyped pathways and project to appropriate regions of the developing muscle or (2) motor axons initially project to some regions that are incorrect, the inappropriate projections being eliminated subsequently. Here we observed individually identified motoneurones in live zebra fish embryos as they formed growth cones and as their growth cones navigated towards their targets. We report that from axogenesis, each motor axon followed a stereotyped pathway and projected only to the specific region of the muscle appropriate for its adult function. In addition, the peripheral arbor established by each motoneurone was restricted to a stereotyped region of its own segment and did not overlap with the peripheral arbor of the other motoneurones in that segment. We conclude that the highly stereotyped pattern of innervation seen in the adult is due to initial selection of the appropriate pathway, rather than elimination of incorrect projections.
In embryonic nervous systems, growing axons must often travel long distances through diverse extracellular terrains to reach their postsynaptic partners. In most embryos, axons grow to their appropriate targets along particular tracts or nerves, as though they were following guidance cues confined to specific pathways. For example, in all vertebrates, axons from the retina invariably grow to the tectum along the well-defined optic tract. Yet, transplant experiments demonstrate that retinal axons make tectal projections even though they enter the brain at locations which are distinctly off the optic tract. Only recently has it become possible to label discreet growing projections in the embryonic vertebrate brain. Thus, it is not yet known whether displaced retinal axons grow directly towards the tectum or find it accidently, through random extension. To resolve this question, pioneering axons from normal and transplanted eyes in embryonic Xenopus were labelled using a short-survival horseradish peroxidase (HRP) method, and their orientation during growth was quantitatively assessed. The finding that the ectopic fibres head towards their distant targets implies that guidance cues are not restricted to specific pathways but are distributed throughout the embryonic brain. The significance of this result is discussed with respect to the ontogeny and evolution of the visual pathway.
Outgrowing neurites in Xenopus embryos were labeled with horseradish peroxidase which had been injected into a single blastomere at the 32-cell stage and had been inherited by all the descendants, including neurons. Neurite outgrowth was traced from labeled trigeminal ganglion cells and most or all types of neurons present in the spinal cord at embryonic stages 20-30: primary motoneurons, commissural, dorsal longitudinal, ventral longitudinal, and Rohon-Beard neurons. All types of nerve fibers grew by the most direct pathway, apparently without errors of initial outgrowth, pathway selection, or target selection. An initial transient phase of outgrowth of filopodial processes from neuronal cell bodies and shafts of short neurites was observed which disappeared after further elongation of the neurites. The first pioneer fibers grew out from all types in a 2-hr period, from stage 20 to 22, and these fibers arrived at the targets within 3.5 hr after initial outgrowth. Additional fibers grew later in contact with the pioneers to form fascicles. Nerve fibers elongated without branching until they neared or contacted their targets. The rate of elongation at 20 degrees C was 30-75 micron/hr. The rapid, unbranched, error-free initial outgrowth and elongation of neurites to their targets is discussed in relation to theories of development of nerve pathways.
The presence of serum in the culture medium affects the morphology of spinal ganglion neurons. With serum present, neuronal cell bodies are rounded and axons are predominantly straight. In serum-free medium axons curve all along their lengths, while both cell bodies and growth cones are spread on the substratum. Such “curved” axons straighten if serum is added to the culture dish.Serum-free medium may increase the adhesion of neurons to the substratum. This can account for the curved morphology of axons in serum-free medium, since such axons may represent a “history” of the movement of the growth cone during axon elongation.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Intraocular HRP injections in E16-21 embryos show that during the normal development of the central optic projections in hooded and albino rats many optic axons grow through the chiasm into the contralateral eye. This retino-retinal projection disappears shortly after birth. This suggests that an initial, imprecise guidance of growing axons is followed by a selective elimination of axons taking aberrant pathways and failing to make appropriate synapses.
Whole retinae of 4- to 10-day-old chick and quail embryos were spread on membrane filters and kept in culture for up to 4 days. Axon growth during culture was demonstrated by silver staining, anterograde labeling of fibers with RITC, time-lapse recording, and SEM. Fiber growth was observed in specimens from chick embryos up to 7 days old, with a growth maximum at E6 and from quail embryos up to E6 with the maximum at E5. Newly growing axons followed the optic fiber pattern already existing and, like axons in vivo, grew predominantly toward the optic fissure. Directional and orientational adaptation of newly growing axons to the preexisting fibers increased with the donor age. Retinae from donors up to E5 in chick and up to E4 in quail showed a high proportion of axons which crossed the optic fissure during the culture period and invaded the opposite retinal fiber layer. These fibers showed a correct radial orientation while growing in the opposite direction to normal. Likewise, in cultures from these young donors some fibers grew out initially in the diametrically opposite direction to normal toward the tissue periphery. Since all of the wrongly directed axons grew at the same rate as normal and adapted correctly to the already formed axon pattern, this suggests independent signals for the direction and orientation of growing fibers. Treatment of mounted retinae with collagenase or trypsin removed the vitreal retinal surface, leaving the existing axon pattern intact. Subsequently, new axons grew profusely in culture, but lost both their orientational and directional characteristics.
Antibodies against the neural cell adhesion molecule (NCAM) were used in vivo both to localize NCAM antigenic determinants in developing tissues of the chicken visual system and to perturb cell-cell adhesion during growth of optic fibers to the tectum. The immunohistochemical studies revealed a staining pattern on neuroepithelial cells which coincided with certain regions of the presumptive route for optic axons, not only with respect to the overall pathway from the eye to the tectum, but also in the preferential distribution of the antigen on the marginal endfeet which are contacted by optic axon growth cones. The antibody-perturbation studies, which involved intraocular injection of anti-NCAM Fab at embryonic Day 3.5, demonstrated that inhibition of NCAM-mediated adhesion results in a dramatic distortion of growth cone-neuroepithelial cell relationships and consequently of the optic pathway. Together, these studies suggest that guidance of optic axons along the margin of the brain is at least in part influenced by a preformed adhesive pathway on neuroepithelial cells associated with NCAM antigens.
Excerpt
Nerve cells often establish precise synaptic connections with target cells at distant locations, both within the CNS and in the periphery. During embryonic development, axonal growth cones have to navigate over long distances from the site of the cell body to the site of the target cell to establish these connections. We are interested in the developmental mechanisms underlying growth cone navigation.
Grasshopper embryogenesis offers an unusual opportunity for studying the formation of long-distance neural pathways between the CNS and the periphery. As embryonic limb buds begin to evaginate from the body wall, they are initially devoid of neurons. The first nerve cells in the limb are afferent (sensory) neurons (termed “pioneers”), which arise within the limb epithelium and project axons toward the CNS (Bate 1976; Keshishian 1980a). The pioneer growth cones are the first to traverse the limb, reaching the CNS just as the first efferent growth cones are...
The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged varied with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explanation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.
The scanning electron microscope was used to examine the growth cones of sensory neurites on the basal lamina of the trunk skin and on the myotomes in dissected embryos of the amphibian, Xenopus laevis. On the myotomes growth cones are large and flat with extensive lamellipodia and many filopodia. On the skin growth cones are smaller and have simpler processes particularly in more ventral positions. Where growth cones contact each other or other neurites they are very intimately apposed and show many indications of strong mutual adhesion. Fasciculation and separation of growing neurites is described and the conditions leading to fasciculation are considered. Measurements of growth cones on the myotomes and different dorsoventral regions of the skin are interpreted in terms of possible differences in the adhesiveness of these substrates. We conclude that many of our observations can be explained by differences in substrate adhesion to the growth cones but that the skin may have some special, unknown attraction for them.
We have reported previously that during optic nerve regeneration in Rana pipiens, axons are misrouted into the opposite nerve and retina. In the present investigation we have examined the time course of formation of these "misrouted" axons and their cells of origin. The right eye of 31 frogs was injected with 3H-proline at various times after right optic nerve crush. In every frog examined 2 weeks and later after nerve crush, the distribution of autoradiographic label indicated that axons from the right eye had grown into the left optic nerve at the chiasm. The amount of label increased from 2 weeks to reach a maximum at 6 weeks where the entire left nerve was filled with silver grains. At 5 to 6 weeks after crush, labeled axons were found within the ganglion cell fiber layer (GCFL) of the retina of the opposite eye for a maximum distance of 2.3 mm from the optic disc. In frogs examined at intervals later than 6 weeks after crush, the amount of label within the left eye and nerve progressively decreased, indicating a gradual disappearance of the misrouted axons. Studies using anterograde transport of horseradish peroxidase (HRP) after nerve injection confirmed these autoradiographic findings. The position of ganglion cells in the right eye whose axons were misrouted to the left eye was determined by retrograde transport of HRP. Five or 6 weeks after crushing the right optic nerve, the left eye was injected with HRP and labeled ganglion cells were found throughout the right eye retina. The largest percentage of labeled cells was found within the ventral half of the retina, particularly within the temporal quadrant, and nearly all of the labeled cells were found in more peripheral portions of the retina. Since few retino-retinal axons are found during normal development, the present results show that the factors guiding regenerating axons in the adult frog differ substantially from those present during development.
The formation of the sensory neurite plexus on the basal lamina of trunk skin in Xenopus embryos has been examined using the scanning electron microscope. It is formed by Rohon-Beard and extramedullary neurons which provide the first sensory innervation of the skin. By observing the distribution of growth cones on the inside surface of the skin of embryos at different ages, the development of the plexus has been followed and related to the development of sensitivity to sensory stimulation. The general features of the plexus are illustrated using a photomontage taken at x 1100. Measurements on neurites from this, and of growth cone orientations demonstrate a general ventral growth pattern with some small regional variations. Interactions of neurites within the plexus are examined. Neurites meeting at shallow angles tend to fasciculate, while those meeting at close to 90 degrees tend to cross each other. Angles of incidence and separation of neurites show few angles less than 30 degrees, which suggests that active adjustments occur after a growth cone meets or leaves another neurite. The observations allow comparison of behaviour of growing neurites in vivo and in vitro. Our evidence suggests that adhesion between growth cones and neurites is stronger than that between growth cones and the basal lamina of the skin.
An important question in developmental neurobiology is how a neuron finds its way over long distances to its correct target
during embryogenesis. Peripheral pioneer neurons in insect embryos have been used for study because of the relative simplicity
of the early embryonic appendages, and the accessibility of the identified neurons whose growth cones traverse this terrain.
The data presented suggest an adhesive hierarchy of both epithelial and neuronal surfaces that guides the first growth cones
from the appendages of the grasshopper embryo.