Repulsive guidance of axons of spinal sensory neurons in Xenopus laevis embryos: roles of Contactin and notochord-derived chondroitin sulfate proteoglycans

An immunoglobulin superfamily neuronal adhesion molecule, Contactin, has been implicated in axon guidance of spinal sensory neurons in Xenopus embryos. To identify the guidance signaling molecules that Contactin recognizes in tailbud embryos, an in situ binding assay was performed using recombinant Contactin-alkaline phosphatase fusion protein (Contactin-AP) as a probe. In the assay of whole-mount or sectioned embryos, Contactin-AP specifically bound to the notochord and its proximal regions. This binding was completely blocked by either digestion of embryo sections with chondroitinase ABC or pretreatment of Contactin-AP with chondroitin sulfate A. When the spinal cord and the notochord explants were co-cultured in collagen gel, growing Contactin-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Contactin antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Contactin on spinal sensory axons.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38–72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer Dil in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48–72 h of incubation, but not in cases with early otocyst removal (38–48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the devloping facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon. © 1992 John Wiley & Sons, Inc.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo.

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38-72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer DiI in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48-72 h of incubation, but not in cases with early otocyst removal (38-48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the developing facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon.     

Reorganization of the Peripheral Projections of the Trigeminal Ganglion Following Neonatal Transection of the Infraorbital Nerve

Two different anatomical techniques were used to obtain evidence that transection of the infraorbital (IO) nerve on the day of birth would result in reorganization of the peripheral projections of the trigeminal nerve. In 14 of 19 neonatally nerve-damaged adult rats, injection of horseradish peroxidase (HRP) directly into the IO nerve, proximal to the point of the neonatal transection, resulted in labeled cells in the ophthalmic-maxillary portion of the ganglion and labeled fibers in mandibular sensory nerves. In an additional 28 neonatally nerve-damaged adult rats, double-labeling techniques were employed to document the reorganization suggested by the HRP tracing experiments. In these experiments, one fluorescent tracer, diamidino yellow (DY), was injected directly into the regenerate IO nerve, proximal to the point of the neonatal transection; a second tracer, true blue (TB), was deposited into peripheral ophthalmic and/ or mandibular fields. These combinations of injections invariably resulted in the demonstration of a small number (46-401) of double-labeled cells that were located in the ophthalmic-maxillary part of the ganglion. Identical combinations of injections in normal adult rats and the intact sides of nerve-damaged animals never produced more than 6 double-labeled cells per ganglion.

In two additional series of experiments, sequential double-labeling techniques were employed to demonstrate that the multiply projecting ganglion cells probably arose in at least two ways: (1) development of non-IO projections by ganglion cells that contributed axons to the IO nerve at the time of the lesion; (2) elaboration of IO axon branches by primary afferent neurons that had non-IO projections at the time of the lesion. A final two-stage double-labeling experiment demonstrated that approximately 75% of the ganglion cells that projected to the whisker pad at birth, and survived transection of the IO nerve on the first postnatal day, regenerated axons into this trigeminal branch.     

Neuronal expression of peripherin,a type III intermediate filament protein,in the mouse hindbrain

Peripherin is a 57 kDa Type III intermediate filament protein associated with neurite extension, neuropathies such as amyotrophic lateral sclerosis, and cranial nerve and dorsal root projections. However, knowledge of peripherin expression in the CNS is limited. We have used immunoperoxidase histochemistry to characterise peripherin expression in the mouse hindbrain, including the inferior colliculus, pons, medulla and cerebellum. Peripherin immunolabelling was observed in the nerve fibres and nuclei that are associated with all cranial nerves [(CN) V–XII] in the hindbrain. Peripherin expression was prominent in the cell bodies and axons of the mesenchephalic trigeminal nucleus and the pars compacta region of nucleus ambiguus, and in the fibres that comprise the solitary tract, the descending spinal trigeminal tract and the trigeminal and facial nerves. A small proportion of peripherin positive fibres in CN VIII likely arise from cochlear type II spiral ganglion neurons. Peripherin positive fibres were also observed in the inferior cerebellar peduncle and folia in the intermediate zone of the cerebellum. Antibody specificity was confirmed by absence of labelling in hindbrain tissue from peripherin knockout mice. This study shows that in the adult mouse hindbrain, peripherin is expressed in discrete neuronal subpopulations that have sensory, motor and autonomic functions.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. I. The initial nerve pathway to the CNS

The founding of the first nerve path of the grasshopper metathoracic leg was examined at the level of identified neurons, using intracellular dye fills, immunohistochemistry, Nomarski optics, and scanning and transmission electron microscopy. The embryonic nerve is established by the axonal trajectory of a pair of afferent pioneer neurons, the tibial 1 (Ti1) cells. Following a period of profuse filopodial sprouting, the Ti1 axonal growth cones, possessing 75- to 100-microns-long filopodia, navigate a stereotyped path across the limb bud epithelium to the base of the appendage and into the CNS. The Ti1 axons grow from cell to cell along a chain of preaxonogenesis neurons spaced at intervals along the pathway, forming dye-passing junctions with them. The contacted neurons subsequently undergo axonogenesis and follow the pioneer axons into the CNS. Later arising neurons project their axons onto the cell bodies of the chain, thereby establishing the principal branch points of the nerve. Among the later arising afferents are the sensory neurons of the femoral chordotonal and subgenual organs. The morphology of the adult nerve appears to be determined by the stereotyped positioning of neurons in the differentiating limb bud and by the resultant axonal trajectories established during the first 10% of peripheral neurogenesis.     

Chondroitin sulphate-binding molecules may pattern central projections of sensory axons within the cranial mesenchyme of the developing mouse

During mammalian hindbrain development, sensory axons grow along highly stereotyped routes within the cranial mesenchyme to reach their appropriate entry points into the neuroepithelium. Thus, trigeminal ganglion axons always project to rhombomere (r)2, whilst facial/acoustic ganglia axons always project to r4. Axons are never observed to enter the mesenchyme adjacent to r3, raising the possibility that r3 mesenchyme contains an axon growth-inhibitory activity. Conversely, in mice which lack the erbB4 receptor (normally expressed in r3), trigeminal and facial/acoustic ganglia axons misproject into r3 mesenchyme, suggesting that the putative axon barrier is absent. To investigate this hypothesis, we have developed an in vitro model in which dissociated wild-type embryonic trigeminal ganglion neurons are cultured on longitudinal cryosections of embryonic mouse head. We observed that on wild-type embryonic day 10 (E10) cryosections, neurites generally failed to grow into r3 mesenchyme from the adjacent r2 or r4 mesenchyme. This barrier was removed if cryosections were pretreated with chondroitinase or were washed with excess chondroitin 6-sulphate or hypertonic saline. By contrast, when trigeminal neurons were seeded onto cryosections of E10 erbB4 -/- embryo heads their neurites readily entered mutant r3 mesenchyme. Immunohistochemical analysis demonstrated chondroitin-sulphated proteoglycans throughout the cranial mesenchyme in both wild-type and erbB4 -/- embryos. We propose that trigeminal axons are excluded from wild-type r3 mesenchyme by a growth-inhibitory activity which associates with chondroitin-sulphated proteoglycans and that the synthesis of this activity may rely on signals transduced by erbB receptors.     

Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons

Trigeminal sensory axons project to several epithelial targets, including those of the maxillary and mandibular processes. Previous studies identified a chemoattractant activity, termed Maxillary Factor, secreted by these processes, which can attract developing trigeminal axons in vitro. We report that Maxillary Factor activity is composed of two neurotrophins, neurotrophin-3 (NT-3) and Brain-Derived Neurotrophic Factor (BDNF), which are produced by both target epithelium and pathway mesenchyme and which are therefore more likely to have a trophic effect on the neurons or their axons than to provide directional information, at least at initial stages of trigeminal axon growth. Consistent with this, the initial trajectories of trigeminal sensory axons are largely or completely normal in mice deficient in both BDNF and NT-3, indicating that other cues must be sufficient for the initial stages of trigeminal axon guidance.     

Zebrafish collapsin response mediator protein (CRMP)-2 is expressed in developing neurons

The collapsin response mediator proteins (CRMPs) are highly expressed in the vertebrate nervous system. CRMP2 has been shown to function in Semaphorin and lysophosphatidic acid induced growth cone collapse. Correspondingly, the highest levels of CRMP2 protein are found in the distal portion of growing axons. To understand the role of CRMP2 during embryonic development we have documented its expression pattern in zebrafish embryos at multiple stages. We find that CRMP2 is expressed in the major neural clusters of the embryonic brain during the primary stages of neurogenesis. From 20 somites through 30 hpf CRMP2 is expressed in the dorsal rostral cluster of the telencephalon, the ventral rostral cluster of the diencephalon, the ventral caudal cluster of the mesencephalon, and the hindbrain clusters. CRMP2 is also expressed in the trigeminal sensory ganglia and the Rohon Beard cells of the neural tube from 15 somites. By 48 hpf, we find expression of CRMP2 throughout the developing brain, trigeminal sensory ganglia, and Rohon Beard cells. CRMP2 is also detected in the retinal ganglion cell layer of the eye, and in the otic vesicle. Finally, we have compared the expression of CRMP2 to PlexinA4, a Semaphorin receptor expressed in sensory neurons, and find that their expression partially overlaps.     

Early axonal contacts during development of an identified dendrite in the brain of the zebrafish

We have identified the initial synaptic contacts made onto the Mauthner (M) cell, an identified neuron that arises during early development of the zebrafish hindbrain. The contacts are made by a small bundle of pioneering trigeminal sensory axons onto the M cell soma before it forms dendrites. The sensory bundle is then partially enveloped by the M cell. The lateral dendrite appears at about the site of the contact, and eventually the trigeminal inputs are shifted to its trunk. As the dendrite elongates, other sensory contacts are made on its distal regions, sequentially from the acoustico-vestibular nerve and the lateral line nerves. To learn whether the earliest inputs induce the initial outgrowth of the M cell dendrite, we ablated the trigeminal neurons by laser irradiation before they contacted the M cell. Morphogenesis of the M cell, including its dendrite, appeared normal.     

BDNF promotes target innervation of <Emphasis Type="Italic">Xenopus</Emphasis> mandibular trigeminal axons <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland.     

Intrinsic properties guide proximal abducens and oculomotor nerve outgrowth in avian embryos

Proper movement of the vertebrate eye requires the formation of precisely patterned axonal connections linking cranial somatic motoneurons, located at defined positions in the ventral midbrain and hindbrain, with extraocular muscles. The aim of this research was to assess the relative contributions of intrinsic, population-specific properties and extrinsic, outgrowth site-specific cues during the early stages of abducens and oculomotor nerve development in avian embryos. This was accomplished by surgically transposing midbrain and caudal hindbrain segments, which had been pre-labeled by electroporation with an EGFP construct. Graft-derived EGFP+ oculomotor axons entering a hindbrain microenvironment often mimicked an abducens initial pathway and coursed cranially. Similarly, some EGFP+ abducens axons entering a midbrain microenvironment mimicked an oculomotor initial pathway and coursed ventrally. Many but not all of these axons subsequently projected to extraocular muscles that they would not normally innervate. Strikingly, EGFP+ axons also took initial paths atypical for their new location. Upon exiting from a hindbrain position, most EGFP+ oculomotor axons actually coursed ventrally and joined host branchiomotor nerves, whose neurons share molecular features with oculomotor neurons. Similarly, upon exiting from a midbrain position, some EGFP+ abducens axons turned caudally, elongated parallel to the brainstem, and contacted the lateral rectus muscle, their originally correct target. These data reveal an interplay between intrinsic properties that are unique to oculomotor and abducens populations and shared ability to recognize and respond to extrinsic directional cues. The former play a prominent role in initial pathway choices, whereas the latter appear more instructive during subsequent directional choices.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

Development of leg chordotonal sensory organs in normal and heat shocked embryos of the cricket Teleogryllus commodus (Walker)

This paper describes the embryonic development of some parts of the sensory peripheral nervous system in the leg anlagen of the cricket Teleogryllus commodus in normal and heat shocked embryos. The first peripheral neurons appear at the 30% stage of embryogenesis. These tibial pioneer neurons grow on a stereotyped path to the central nervous system and form a nerve which is joined by the growth cones of axons that arise later, including those from the femoral chordotonal organ, subgenual organ and tympanal organ. The development of these organs is described with respect to the increase in number of sensory receptor cells and the shape and position of the organs. At the 100% stage of embryogenesis all three organs have completed their development in terms of the number of sense cells and have achieved an adult shape. To study the function of the tibial pioneer neurons during embryogenesis a heat shock was used to prevent their development. Absence of these neurons has no effect on the development of other neurons and organs proximal to them. However, the development of distal neurons and organs guided by them is impaired. The tibial pioneer neurons grow across the segmental boundary between femur and tibia early in development, and the path they form seems to be essential for establishing the correct connections of the distal sense organs with the central nervous system.     

The neuronal targets for GABAergic reticulospinal inhibition that stops swimming in hatchling frog tadpoles

In most animals locomotion can be started and stopped by specific sensory cues. We are using a simple vertebrate, the hatchling Xenopus tadpole, to study a neuronal pathway that turns off locomotion. In the tadpole, swimming stops when the head contacts solid objects or the water's surface meniscus. The primary sensory neurons are in the trigeminal ganglion and directly excite inhibitory reticulospinal neurons in the hindbrain. These project axons into the spinal cord and release GABA to inhibit spinal neurons and stop swimming. We ask whether there is specificity in the types of spinal neuron inhibited. We used single-neuron recording to determine which classes of spinal neurons receive inhibition when the head skin is pressed. Ventral motoneurons and premotor interneurons involved in generating the swimming rhythm receive reliable GABAergic inhibition. More dorsal inhibitory premotor interneurons are inhibited less reliably and some are excited. Dorsal sensory pathway interneurons that start swimming following a touch to the trunk skin do not appear to receive such inhibition. There is therefore specificity in the formation of descending inhibitory connections so that more ventral neurons producing swimming are most strongly inhibited.     

Histology and ultrastructure of the olfactory organ of the freshwater pulmonate Helisoma trivolvis

Summary The olfactory organ of Helisoma trivolvis is located on the surface of the body at the base of the cephalic tentacles. An evagination of skin, the olfactory plica, at the base of the tentacle extends over the olfactory organ dorsally. The epithelium of the olfactory organs contains unspecialized epithelial cells, ciliated epithelial cells, basal cells, mucous secretory cells, and sensory dendrites. The surface of the epithelium has a complex brush border of thick plasmatic processes, which branch to form several terminal microvillar twigs. Long slender cytoplasmic processes form a dense spongy layer among the plasmatic processes beneath the level of the terminal twigs. Bipolar primary sensory neurons clustered beneath the epithelium of the olfactory organ send dendrites through the epithelium to the free surface. Some sensory endings have a few short cilia, but most bear only microvilli. Cilia of sensory endings and epithelial cells extend beyond the brush border of the epithelium. Small axons arise from the perikarya of the sensory neurons and enter a branch of the olfactory nerve. HRP tracing indicates that the axons pass to the cerebral ganglion without interruption. Histochemical tests indicate that the sensory neurons are neither aminergic nor cholinergic.