Localization of Nogo and its receptor in the optic pathway of mouse embryos

We have investigated the localization of Nogo, an inhibitory protein acting on regenerating axons in the adult central nervous system, in the embryonic mouse retinofugal pathway during the major period of axon growth into the optic chiasm. In the retina, Nogo protein was localized on the neuroepithelial cells at E12 and at later stages (E13-E17) on radial glial cells. Colocalization studies showed expression of Nogo on vimentin-positive glia in the retina and at the optic nerve head but not on most of the TuJ1- and islet-1-immunoreactive neurons. Only a few immature neurons in the ventricular and peripheral regions of the E13 retina were immunoreactive to Nogo. In the ventral diencephalon, Nogo was expressed on radial glia, most strongly on the dense radial glial midline raphe within the chiasm where uncrossed axons turn and in the initial segment of the optic tract. In vitro studies showed that the Nogo receptor (NgR) was expressed on the neurites and growth cones from both the ventral temporal and dorsal nasal quadrant of the retina. In the optic pathway, NgR staining was obvious in the vitreal regions of the retina and on axons in the optic stalk and the optic tract, but not in the chiasm. These expression patterns suggest an interaction of Nogo with its receptor in the mouse retinofugal pathway, which may be involved in guiding axons into the optic pathway and in governing the routing of axons in the optic chiasm.     

Abnormal pigmentation and unusual morphogenesis of the optic stalk may be correlated with retinal axon misguidance in embryonic Siamese cats

Studies of albino rodents have shown that an absence of pigment in the developing optic stalk may alter the position of the first retinal fibers that grow toward the brain, thereby disrupting the gross topographic relationship of fibers in the nerve (Silver and Sapiro: J. Comp. Neurol. 202:521-538, '81). The abnormalities associated with albinism are more extensive in the Siamese cat than-in previously studied species. Therefore, any abnormalities in differentiation of the stalk and axon guidance may be more readily detected. To investigate the guidance and/or misguidance of optic axons, light and electron microscope analyses were made of serial sections through the optic stalk in normally pigmented and Siamese fetal cats. On E20, before axons enter the optic stalk, the only clear morphological distinction between Siamese and normal cats is the distribution of pigment in the stalk. Pigment is found in the dorsal stalk cells of the normal cat for 200 microns from the optic disc. Although the retinal pigment epithelium of the Siamese optic stalk. By E23 axons invade the ventral optic stalk in both strains. Concurrent with the early stages of axonal exit from the retina, there is complete separation of the stalk's dorsal and ventral tiers. As the cleavage occurs, basal lamina invaginates into the zone of separation following along the plane of the old lumen. The ventral stalk fills with axons while the dorsal tier is shed gradually. In contrast, in the Siamese cat, dorsal stalk cells are not sloughed off properly and instead are incorporated ectopically into the nerve. Basal lamina invagination is irregular. Axons do not fill the Siamese stalk symmetrically but enter the region of ectopic cells, which in turn disrupts gross fiber position. Usually, in the mutant, axons originating from the retina temporal to the optic fissure are those that invade the dorsal tier of ectopic cells. The altered position of optic axons in the mutant stalk may provide an explanation for the chiasmatic misrouting of optic axons in this species.     

The early development of the optic nerve and chiasm in embryonic rat

To establish the time course and major features of the development of the optic nerve and chiasm in the embryonic rat, the growth of axons from the retina to the brain has been studied by light and electron microscopy. On embryonic day 14 (E14), the first axons are generated by retinal ganglion cells. Fascicles of axons can be detected in the optic stalk at E14.5 and in the diencephalon by E15.0. In the vitreal retina and optic fissure, large extracellular spaces resemble the oriented channels previously described in the mouse. They form approximately 12 hours before the invasion of optic axons and contain hyaluronic acid. In the optic stalk and diencephalon of the rat, similar spaces are not present, but the timed autolysis of neuroepithelial cells could provide a pathway of minimal resistance for the earliest axons. Degenerating cells are prominent in the ventral stalk and rostral diencephalon prior to the arrival of the first optic axons that preferentially invade these regions. The role of pigment in the development of visual pathways is controversial. In one strain of rat, Manchester Hooded, the retinae are heavily pigmented, but little pigment is seen at any stage in the stalk; in albinos, pigment is absent from both retina and stalk. However, the distribution of axons within the developing optic stalk is very similar in both strains, suggesting that the reduction in size of the ipsilateral pathway observed in the albino rat compared with the Manchester Hooded is not due to a lack of pigment in the optic stalk early in development. Several factors previously reported to contribute to the development of retinotopic order in other species are also present in the rat. These include the sequence in which axons grow into the stalk, and fasciculation. Intermembranous contacts observed between growth cones and adjacent tissues suggest one mechanism by which fasciculation occurs. A small group of fascicles, which may represent the ipsilateral projection, diverges from the crossing fibers on E15.5, without evidence of being deflected by any glial or other structures.     

The course of later generated axons in the developing optic nerve of the chick embryo. A morphometric electron microscopic study

The topographic position of growth cones (GCs) shows the course of ingrowing axons within the optic nerve and allows to draw conclusions with respect to the fiber order in this pathway. Therefore, the topographic distribution and frequency of GCs as well as the proximal and distal axon shaft segments were studied within cross-sections of the distal, middle, and prechiasmatic part of the nerve of 3-8-day-old embryos using electron microscopy. The ingrowth of GCs was not confined to a particular region. Initially, GCs were found near the ventral periphery. With increasing age, simultaneous ingrowth occurred within an area that expanded dorsally. In parallel, GCs also occurred in dorsal regions and eventually in the dorsal periphery. GCs intermingled everywhere with more mature axon profiles. However, youngest profiles predominated ventrally, oldest dorsally. Hence, maturity increased from ventral to dorsal. This indicated that the time of arrival of axons and the topographic position in the cross-section correlated significantly. It is concluded that axons are chronotopically organized, but in a probabilistic sense. The predominant ingrowth of axons in the ventral part may be associated largely with the first wave of neurogenesis of retinal ganglion cells. The ingrowth in dorsal regions of the cross section may be related to later generated axons that enter the nerve following older axons of the same retinal sector as well as axons of neighboring ganglion cells which continue to leave the mitotic cycle while the front of neurogenesis has spread into the periphery.     

Axonal pathfinding during the regeneration of the goldfish optic pathway

Retinal ganglion cells in fish and amphibians regenerate their axons after transection of the optic nerve. Fiber tracing studies during the third month of regeneration show that the axons have reestablished a basically normal fiber order in the two brachia of the optic tract; axons originating in the ventral hemiretina are concentrated in the dorsal brachium, axons from the dorsal hemiretina in the ventral brachium. Attardi and Sperry (Exp. Neurol. 7:46-64, 1963) first suggested that the reestablishment of the fiber order reflects path-finding by the regenerating axons. Recently, however, Becker and Cook (Development 101:323-337, 1987) have claimed that the fiber order observed at later stages of regeneration is due to secondary axonal rearrangements and that the initial brachial choice is random. In order to evaluate whether regenerating axons are capable of navigating in the optic tract and brachia and on the tectum, the present study examined the pathway choices and the morphology of regenerating axons en route to their tectal targets in goldfish. Subsets of axons were labeled at various time intervals (2 to 30 days) following an optic nerve crush, by intraretinal application of the lipophilic fluorescent tracer 1,1-dioctadecyl-3-3-3'-3'-tetramethylcarbocyanine (DiI). After a survival time of 18 to 72 hours (to allow for diffusion of DiI along the axons), the experimental animals were perfused with fixative and their right and left optic pathways (nerve, tract, and tectum) were dissected free and separated at the chiasm. Fluorescently labeled axons were traced in whole-mounted pathways. Pathway choices were examined at the brachial bifurcation where axons from ventral and dorsal hemiretinae normally segregate. DiI was found to label axons reliably up to their growth cones, even at the earliest stages of regrowth. The pathway choices of the axons were nonrandom. The majority of the ventral axons reached the appropriate, dorsal hemitectum through the appropriate dorsal brachium of the tract. Dorsal axons reached the ventral hemitectum mainly through the ventral brachium. This suggests the presence of specific guidance cues, accessible to the regenerating axons. Differences in the complexity of the growth cones of the regenerating axons (simple in the nerve and tectal fiber layer, complex in the tract and the synaptic layer of the tectum) provide further evidence for specific interactions between the regenerating axons and their substrates along the pathway. These results argue that regenerating retinal axons in fish are capable of axonal path-finding.     

Factors guiding optic fibers in developing Xenopus retina

We have characterized, by electron microscopy, the growth of pioneering axons from the retina into the visual pathway during early development of Xenopus laevis. The subsequent development of following fibers from the growing retinal margin as they accumulated in the ganglion cell fiber layer (GCFL) of the retina was also studied. Extracellular channels bordered by neuroepithelial cells appear in the developing retina in a dorsal to ventral gradient before any pioneering axons are seen. Pioneering axons are subsequently observed in these channels, usually surrounded by neuroepithelial cell processes. Ruthenium red treatment of embryonic retinas reveals extracellular matrix (ECM) within these retinal channels, while extracellular spaces in the proximal optic stalk, just beyond the optic disc, lack this material. ECM is also seen in optic tectum wherever ingrowing retinal and nonretinal axons are found. The channels and the ECM contained within them may provide guidance cues for pioneering retinal axons. The early association of pioneering retinal axons with neuroepithelial cell processes (putative glia) appears to be important in further development of the GCFL. The so-called following fibers of ganglion cells, arising later in development, fasciculate with pioneer axons in extracellular spaces and form fiber bundles of the GCFL on top of the layer of glial cell endfeet. It is not clear whether pioneering axons, glial cell surfaces, or both serve as guidance cues for following fiber migration.     

Growth cone distribution patterns in the optic nerve of fetal monkeys: implications for mechanisms of axon guidance

The distribution of growth cones was studied in the optic nerve of monkeys during the first half of prenatal development using quantitative electron microscopic methods. Our aim was to test the hypothesis that ganglion cell growth cones extend predominantly along the surfaces of the nerve, just beneath the pia mater. A complete census of growth cones in cross sections of the nerve during the early phase of axon ingrowth, from embryonic day 39 (E39) to E41, demonstrates that growth cones are scattered within the majority of fascicles, even those located far from the surface of the nerve. By E45, growth cones are concentrated around the nasal, dorsal, and ventral edge of the optic nerve. They are less concentrated in the core and around the temporal edge. However, even as late as E49, virtually all fascicles in the nerve, whether deep or superficial, contain growth cones. Growth cones are dispersed within single fascicles and are often located far from glia. Thus, the newest fibers penetrate deep parts of the pathway and push through centers of densely packed bundles of older axons. This finding is consistent with the vagrant paths of growing axons reported in previous work on embryonic monkey optic nerve (Williams and Rakic, 1985). Our data challenge the hypotheses that growth cones extend selectively along the basal lamina, the pia mater, or glial end feet. Gradients found at later stages of development in the nerve are not due to a particular affinity of growth cones for non-neuronal substrata. The pattern we observed is much more likely to result from central-to-peripheral gradients in ganglion cell generation and possible associations between growth cones originating from the same regions of the retina.     

Changing glial organization relates to changing fiber order in the developing optic nerve of ferrets

The structures of the developing eye-stalk and the relationships of early retinofugal fibers as they pass through the stalk, chiasm, and tract have been studied by light and electron microscopical methods in fetal ferrets aged 23–27 days. The early eye-stalk can be divided into two parts: a narrow extracranial part has a narrow lumen and is lined by few cells, whereas a thicker intracranial part has a wider lumen and is lined by several rows of cells. At the earliest stages no axon bundles are recognizable in the stalk, but fibers of the supraoptic commissure are already beginning to cross the midline in the diencephalon. Subsequently, as retinofugal axons invade the stalk, the glia of the extracranial part of the stalk have an interfascicular distribution and axon bundles are separately encircled by glial cytoplasm. In the intracranial part, as in the chiasm and tract, the glial cells occupy a periventricular position and send slender radial cytoplasmic processes to the subpial surface; these pass between groups of axons that here lie immediately deep to the subpial glia. Whereas axonal growth cones have no evident preferred distribution in the extracranial stalk, they tend to accumulate near the pial surface intracranially. The boundary between the two types of organization shifts as development proceeds so that the interfascicular glial structure of the early extracranial stalk first encroaches upon the intracranial parts and later appears in the chiasm. The characteristic adult arrangement of fibers in an age-related order in the optic chiasm and tract, but not in the optic nerve, can be understood if axonal growth cones are guided toward the pial surface by radial glia but not by interfascicular glia. From the distribution of the growth cones, this is what appears to happen.     

Axonal guidance during development of the optic nerve: The role of pigmented epithelia and other extrinsic factors

It is well established that a congenital lack of ocular melanin (albinism) can lead to developmental abnormalities of the central visual pathways. However, it is yet unknown how the pigmentation per se acts to influence formation of the optic projection. In order to study the possible interaction between eye pigment and optic axons during development, we have examined, with the use of serial section techniques, a series of timed embryos at stages when the ocular pigment and outgrowing axons first become apparent. Our results have demonstrated that, in mice and rats, the upper wall of the distal half of the primitive eye stalk (a region which lies along the potential route to be taken by the earliest developing nerve fibers) is transiently pigmented prior to and during the migration of the pioneer optic axons. All outgrowing neurites avoid this stretch of melanotic tissue and instead grow preferentially through a system of extracellular tunnels in the ventral, pigment-free zones of the distal eye stalk. The stalk remains unpigmented from about its midpoint and continuing toward the brain. At the pigment/pigment-free interface many of the axons shift upward from their ventral positions, forming a marginal annulus. In the chick, on the contrary, pigmentation of the stalk does not occur and as the optic axons exit the globe they grow immediately in an annulus configuration. In Xenopus, the entire stalk becomes pigmented and the optic fibers congregate in one discrete bundle of fascicles along the length of the stalk's most ventral margin. These observations suggest that melanin-producing stalk cells may play a role in controlling the topographic patterning of optic fibers within the developing nerve by inhibiting the lateral spread of axonal growth cones into or within their territory. To test this hypothesis we have charted the distribution of optic fibers in the developing optic stalks of timed albino rat embryos. Indeed, as fibers leave the mutant eye, it was found that a small but consistent number of pioneering axons (day E15) become ectopic and immediataely invade nonpigmented regions (those normally pigmented and axon-free) in the distal optic stalk. Thus, the usual topographic arrangement of the collection of pioneer optic fibers is altered in the albino.     

Observations on the early development of the optic nerve and tract of the mouse.

The early development of the retinofugal pathway of mice has been studied by light and electron microscopic methods in order to define the spatial distribution and the structure of the growth cones as they advance from the eye to the brain. We have studied the relationships of the growth cones to each other, to the glia and, in the older individuals, to the nerve fibers that are already terminating in the brain. We have looked at the rate of advance of the growth cones and have paid particular attention to the changing relationships of the growth cones as they approach the optic chiasm. We have also looked to see whether, at early stages, it is possible to recognise any characteristic features distinguishing the fibers destined to be the thickest in the adult, which come from ganglion cells that are generated among the earliest ganglion cells. In transverse sections through the optic stalk about 50-100 microns behind the eye, the first bundles of fibers are seen on embryonic day 12.5 (E12.5) as a mixture of thin (less than 0.5 micron) axons, thicker growth cones, and fine filopodial and foliopodial extensions. During the next two days, as these bundles in the intraorbital nerve increase in size and number, growth cones can be seen in all of the bundles and in all parts of the bundles. They show only a slight preference for one part of the nerve relative to another, and our material provides no evidence for the view that axons are particularly inclined to follow pre-existing bundles. The structure of the pathway changes significantly as it is traced towards the chiasm, and no section or small stretch of sections can be regarded as representative of the nerve as a whole. As the fibers approach the optic chiasm the growth cones come to lie predominantly close to the pial surface, with the deeper regions occupied almost entirely by fine axons. The change occurs in a region where the glial environment also changes, and where a characteristic neural tube-like organization first becomes recognizable. Here the glial cells lie in a periventricular position and send slender radial processes out towards the subpial surface. The newly invading axons in the early optic nerve taper from a broad growth cone back to an extremely slender axon, less than 0.5 micron in diameter. The tapered region is of the order of 100-300 microns in length and advances through the nerve at approximately 60 microns per hour.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth cones, dying axons, and developmental fluctuations in the fiber population of the cat's optic nerve

We have studied the rise and fall in the number of axons in the optic nerve of fetal and neonatal cats in relation to changes in the ultrastructure of fibers, and in particular, to the characteristics and spatiotemporal distribution of growth cones and necrotic axons. Axons of retinal ganglion cells start to grow through the optic nerve on the 19th day of embryonic development (E-19). As early as E-23 there are 8,000 fibers in the nerve close to the eye. Fibers are added to the nerve at a rate of approximately 50,000 per day from E-28 until E-39--the age at which the peak population of 600,000-700,000 axons is reached. Thereafter, the number decreases rapidly: About 400,000 axons are lost between E-39 and E-53. In contrast, from E-56 until the second week after birth the number of axons decreases at a slow rate. Even as late as postnatal day 12 (P-12) the nerve contains an excess of up to 100,000 fibers. The final number of fibers--140,000-165,000--is reached by the sixth week after birth. Growth cones of retinal ganglion cells are present in the optic nerve from E-19 until E-39. At E-19 and E-23 they have comparatively simple shapes but in older fetuses they are larger and their shapes are more elaborate. As early as E-28 many growth cones have lamellipodia that extend outward from the core region as far as 10 microns. These sheetlike processes are insinuated between bundles of axons and commonly contact 10 to 20 neighboring fibers in single transverse sections. At E-28 growth cones make up 2.0% of the fiber population; at E-33 they make up about 1.0%; from E-36 to E-39 they make up only 0.3% of the population. Virtually none are present in the midorbital part of the nerve on or after E-44. At all ages growth cones are more common at the periphery of the nerve than at its center. This central-to-peripheral gradient increases with age: at E-28 the density of growth cones is two times greater at the edge than at the center but by E-39 the density is four to five times greater. Necrotic fibers are observed as early as E-28 in all parts of the nerve. Their axoplasm is dark and mottled and often contains dense vesiculated structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ipsi- and contralateral commissural growth cones react differently to the cellular environment of the ventral zebrafish spinal cord

Early commissural axons in the zebrafish spinal cord extend along a pathway consisting of a ventrally directed ipsilateral, a contralateral diagonal, and a contralateral longitudinal segment. The midline floor plate cell is one important cue at the transition from the ipsilateral to the contralateral pathway segments. In order to identify additional guidance cues, the interactions between commissural growth cones and their substrates were examined at the electron microscopic level in the different pathway segments. The growth cones extended near the superficial margin of the spinal cord, within filopodial reach of three bilateral longitudinal axon pathways that were ignored irrespective of whether other axons were already present. Ultimately the commissural growth cones pioneered an additional independent longitudinal pathway in the dorsolateral spinal cord. Neuroepithelial cells were extensively contacted in the lateral marginal zone of the dorsal spinal cord and are thus in a position to contribute to the establishment of the longitudinal commissural pathway segment. The extent of contact with neuroepithelial cells in the ventral spinal cord was dependent on whether commissural growth cones had already crossed the ventral midline: ipsilateral, but not contralateral, growth cones showed extensive contacts with neuroepithelial processes and minor contacts with the basal lamina. In marked contrast, commissural growth cones that had already crossed the ventral midline and entered the diagonal pathway segment showed major appositions to the basal lamina. Extensive contact with the basal lamina was first established in the ventral midline region, where crossing growth cones always inserted between the basal lamina and the base of the midline floor plate cells. This indicates that a change occurs in the response characteristics of commissural growth cones as they cross the ventral midline of the spinal cord. Such a change could help to explain why the growth cones extend first toward but then away from the ventral midline. 1994 Wiley-Liss. Inc.     

Visual system of the channel catfish (Ictalurus punctatus): III. Fiber order in the optic nerve and optic tract

In channel catfish the ganglion cell axons leave the retina via a ring of approximately 13 separate optic papillae. Each papilla serves an area of retina extending from the central zone of the retina to the periphery. Papillae located at a dorsal position in the ring serve exclusively dorsal retina. Ventrally located papillae, however, have an exaggerated peripheral retinal representation, so that they serve mostly ventral retina but also some areas of peripheral retina dorsal to the nasal and temporal poles. The ganglion cell axon bundles departing from the retina via individual papillae were labelled with horseradish peroxidase, and sections of the optic pathway were examined to reveal the topographic organization of the fibers. The topographic order of the optic nerve was dissimilar to that of cichlids and goldfish. Fibers from individual papillae remained together throughout the optic nerve. Close to the optic nerve head, the papillae were arranged as a continuum around the U-shaped optic nerve, without the discontinuity in the representation of the ventral retina seen in other fish. Fibers associated with the dorsal papillae were located at the tip of the caudolateral arm of the U, and fibers from ventral papillae were on the rostromedial arm. Fibers from nasally and temporally located papillae were found on the base of the U. By the level of the optic chiasm the U shape had flattened out but retained the relative ordering of the papillae. Rotation of the nerve as it became the optic tract brought the representation of the ventral papillae to the dorsal pole of the tract, and the dorsal papillae to the ventral tract. It was only in the optic tract that rearrangement of fibers became apparent. As described above, the axons of some ganglion cells in dorsal, peripheral retina left the retina and travelled through the optic nerve with axons from extreme ventral retina. In the optic tract, these dorsal fibers joined the main body of fibers from the dorsal retina. The significance of these observations for theories of fiber rearrangement is discussed.     

Pathfinding by growth cones of commissural interneurons in the chick embryo spinal cord: a light and electron microscopic study

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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anterior commissure of the wallaby (Macropus eugenii): Adult morphology and development

In metatheria, all neo- and paleo-cortical commissural connections are made by the anterior commissure. We have examined the adult morphology of this commissure and its development in a diprotodontid metatherian, the wallaby (Macropus eugenii), at both the light and electron microscope level. The total number of axons in the adult anterior commissure was 21.7 million, of which 55–62% were myelinated. The dorsal two thirds of the commissure, containing neocortical commissural axons, showed a higher percentage of larger, myelinated axons than the ventral one third, which contains paleocortical commissural axons. The commissure also showed a topographical gradient, with cells in the dorsal cortex projecting through the dorsal region of the commissure, the fasciculus aberrans. In the rostrocaudal axis, axons from the frontal cortex tended to pass more anteriorly through the commissure and those from the occipital more posteriorly, but there was extensive overlap of projections from different areas. The gestation length of this wallaby is 28.3 days, and all commissural development occurs postnatally. The anterior commissure first appeared at P (postnatal day) 14, at which time commissural fibres were apparently derived from the external capsule exclusively. Commissural fibres passing through the internal capsule, and joining the anterior commissure via the fasciculus aberrans, were first noted at P18. By that age there were 94,000 to 161,000 axons. Peak axon counts of 50 to 63 million occurred between P100 and P150. The number of growth cones in a single midline section peaked at approximately P114 (480,000) and dropped to 0 by P170. The distribution of growth cones was analysed during the early stages of anterior commissure development (P18, P30, P82). At P18, growth cones were concentrated in the dorsal parts of the commissural bundle, suggesting a ventrodorsal sequence of addition of axons. There was no apparent preferential association of growth cones with the periphery of the commissure or glial structures at any of the three ages examined. The results show that axonal overproduction and regression in cortical commissural connections are features of development in diprotodontid metatheria, as in eutheria. © 1996 Wiley-Liss, Inc.     

Precocious invasion of the optic stalk by transient retinopetal axons

This study demonstrates that the fetal optic nerve contains a conspicuous population of transient retinopetal axons. Implants of the carbocyanine dye, DiI, were made into the retina or diencephalon of fetal ferrets to label the retinopetal axons retrogradely or anterogradely, respectively, and sections were immunostained for β-tubulin to label the early differentiating axons in the optic nerve. Dye implants into the optic nerve head, but not the retinal periphery, retrogradely labeled somata in the ventrolateral diencephalon, provided the implants were made before embryonic day (E) 30. When dye implants were made into the ventrolateral diencephalon, these same retinopetal axons were anterogradely labeled, coursing through the optic nerve but never invading the retina. The axons course as 2–5 fascicles from their cells of origin and turn laterally to enter the optic nerve where it joins the future hypothalamus. The retinopetal cells can be retrogradely labeled as early as E20, before optic axons have left the retina. The optic nerve and fiber layer are immunoreactive for β-tubulin on E24 and thereafter, whereas on E20 and E22, they are immunonegative. Yet at these early embryonic ages, immunopositive fascicles of axons course from the diencephalon into the optic stalk, confirming the precocious nature of the retinopetal projection. Implants of dye made into the future optic nerve head at these very early stages also retrogradely label retinopetal cells in the future chiasmatic region. These cells are distributed primarily on the side ipsilateral to the midline, but a few can be found contralateral to it. Both these, as well as the retinopetal axons arising from the ventrolateral diencephalon, may serve a transient guidance function for later developing optic axons. © 1995 Wiley-Liss, Inc.     

The environment of axonal migration in the developing chick retina: a scanning electron microscopic (SEM) study.

We have made a SEM study of the basal intercellular spaces of the retina in chick embryos of different developmental stages. Since this is the environment where optic axons grow, the structural characteristics of this region might play some role in the orientation of axonal migration towards the choroid fissure. The basal region of undifferentiated retinas is formed by the vitreal expansions of neuroepithelial cells. In pre-axonal stages, the intercellular spaces between these expansions do not show any preferential orientation towards the fissure. The growth cones of ganglion cell axons appear in an apicobasal direction and turn towards the fissure immediately beneath the vitreal surface. Fasciculation is an early event during development and, in the more advanced stages, the vitreal expansions from retinal cells are placed in rows following the same orientation as the axon bundles. These observations are discussed in relationship to current hypotheses on axonal migration and orientation.     

Chick wing innervation. II. Morphology of motor and sensory axons and their growth cones during early development

The development and distribution of neuronal projections to the developing chick wing was studied using anterograde transport of horseradish peroxidase (HRP). Small injections of HRP were made into motor or sensory neuronal populations in order to visualize individual axons and their associated growth cones. Motor growth cones were observed in different regions of the embryo at different stages, in a proximal-to-distal pattern of distribution which paralleled the process of axon outgrowth and nerve formation. Different growth cone morphologies were associated with differing regions of the developing projection. In the spinal nerves, axons destined for the limb were unbranched and terminated in simply shaped growth cones. As axons approached the developing limb and entered the plexus region, their growth cones became more complex and larger primarily because of widening, and they sometimes branched, producing processes which could extend tens of microns from a tricorne branch point on the parent axon. Both motor and sensory fibers showed similar morphological changes in the plexus region. A distinctively shaped growth cone expanded on its leading edge was observed, sequentially apparent in the distal spinal nerves, in the plexus region, in the loosely organized axonal sheets projecting to the uncleaved dorsal or ventral muscle masses, and where muscle nerves diverged from nerve trunks and within muscle nerves. It is likely that some of these are transitional growth cones preparing to branch, because complex and branched growth cones were also observed in these regions. Branched axons oriented along the anteroposterior axis were similarly observed in the plexus region and distal to the plexus when axons first projected to the limb bud. At somewhat older stages when the basic peripheral nerve branching pattern had formed, motor growth cones were observed in common nerve trunks and in individual muscle nerves, but they were no longer found in the plexus region. Branched axons were likewise restricted to these peripheral Imations. Taken together, these observations suggest that one of the ways in which axons navigate is by exploration in the form of growth cone widening, and in some cases terminal bifurcation which may produce axon branches. Selection of the most appropriately directed growth cone process and/or precocious axonal branches may be one of the ways in which axons respond to specific growth cues which guide axons into the limb bud. Alternatively, this precocious branching may be an early neurotrophic response to developing muscle and play no significant role in axon navigation. © 1995 Wiley-Liss, Inc.     

The formation of the axonal pattern in the embryonic avian retina

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.