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Intraepithelial corneal nerves (ICNs) help protect the cornea as part of the blink reflex and by modulating tear production. ICNs are also thought to regulate the health and homeostasis of the cornea through the release of trophic factors. Disruption to these nerves can lead to vision loss. Despite their importance little is known about how corneal nerves function and even less is known about how the cornea is initially innervated during its embryonic development. Here, we investigated the innervation of the embryonic chicken cornea. Western blot and immunohistochemistry were used to characterize the localization of the synaptic vesicle marker SV2, a molecule thought to be involved in the release of trophic factors from sensory nerves. The data show that both SV2 and synaptotagmin co-localize to ICNs. Nerves in the conjunctiva also contained SV2 and synaptotagmin, but these were localized to below the basal layers of the conjunctiva epithelium. SV2 isolated from corneal epithelium migrates in western blot at a heavier weight than SV2 isolated from brain, which suggests a role in vesicle targeting, as the deglycosylating enzyme PnGase does not affect corneal SV2., (Copyright © 2018. Published by Elsevier Ltd.)

A Merkel cell-neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch-sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell-neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell-neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co-culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell-neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co-cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell-neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament-H-positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell-neurite complex can be observed under a microscope using our organotypic co-culture method., (© 2018 Japanese Society of Developmental Biologists.)

Vertebrates have succeeded to inhabit almost every ecological niche due in large part to the anatomical diversification of their jaw complex. As a component of the feeding apparatus, jaw muscles carry a vital role for determining the mode of feeding. Early patterning of the jaw muscles has been attributed to cranial neural crest-derived mesenchyme, however, much remains to be understood about the role of nonneural crest tissues in the evolution and diversification of jaw muscle morphology. In this study, we describe the development of trigeminal motor neurons in a parrot species with the uniquely shaped jaw muscles and compare its developmental pattern to that in the quail with the standard jaw muscles to uncover potential roles of nervous tissue in the evolution of vertebrate jaw muscles. In parrot embryogenesis, the motor axon bundles are detectable within the muscular tissue only after the basic shape of the muscular tissue has been established. This supports the view that nervous tissue does not primarily determine the spatial pattern of jaw muscles. In contrast, the trigeminal motor nucleus, which is composed of somata of neurons that innervate major jaw muscles, of parrot is more developed compared to quail, even in embryonic stage where no remarkable interspecific difference in both jaw muscle morphology and motor nerve branching pattern is recognized. Our data suggest that although nervous tissue may not have a large influence on initial patterning of jaw muscles, it may play an important role in subsequent growth and maintenance of muscular tissue and alterations in cranial nervous tissue development may underlie diversification of jaw muscle morphology., (Copyright © 2013 Wiley Periodicals, Inc.)

The trigeminal, the fifth cranial nerve of vertebrates, represents the rostralmost component of the nerves assigned to pharyngeal arches. It consists of the ophthalmic and maxillomandibular nerves, and in jawed vertebrates, the latter is further divided into two major branches dorsoventrally. Of these, the dorsal one is called the maxillary nerve because it predominantly innervates the upper jaw, as seen in the human anatomy. However, developmentally, the upper jaw is derived not only from the dorsal part of the mandibular arch, but also from the premandibular primordium: the medial nasal prominence rostral to the mandibular arch domain. The latter component forms the premaxillary region of the upper jaw in mammals. Thus, there is an apparent discrepancy between the morphological trigeminal innervation pattern and the developmental derivation of the gnathostome upper jaw. To reconcile this, we compared the embryonic developmental patterns of the trigeminal nerve in a variety of gnathostome species. With the exception of the diapsid species studied, we found that the maxillary nerve issues a branch (nasopalatine nerve in human) that innervates the medial nasal prominence derivatives. Because the trigeminal nerve in cyclostomes also possesses a similar branch, we conclude that the vertebrate maxillomandibular nerve primarily has had a premandibular branch as its dorsal element. The presence of this branch would thus represent the plesiomorphic condition for the gnathostomes, implying its secondary loss within some lineages. The branch for the maxillary process, more appropriately called the palatoquadrate component of the maxillary nerve (V(2)), represents the apomorphic gnathostome trait that has evolved in association with the acquisition of an upper jaw., (Copyright © 2013 Wiley Periodicals, Inc.)

Descriptions of the anatomy of the neural communications among the cranial nerves and their branches is lacking in the literature. Knowledge of the possible neural interconnections found among these nerves may prove useful to surgeons who operate in these regions to avoid inadvertent traction or transection. We review the literature regarding the anatomy, function, and clinical implications of the complex neural networks formed by interconnections among the lower cranial and upper cervical nerves. A review of germane anatomic and clinical literature was performed. The review is organized in two parts. Part I concerns the anastomoses between the trigeminal, facial, and vestibulocochlear nerves or their branches with any other nerve trunk or branch in the vicinity. Part II concerns the anastomoses among the glossopharyngeal, vagus, accessory and hypoglossal nerves and their branches or among these nerves and the first four cervical spinal nerves; the contribution of the autonomic nervous system to these neural plexuses is also briefly reviewed. Part I is presented in this article. An extensive anastomotic network exists among the lower cranial nerves. Knowledge of such neural intercommunications is important in diagnosing and treating patients with pathology of the skull base., (Copyright © 2013 Wiley Periodicals, Inc.)

Cranial placodes are thickenings of the embryonic head ectoderm that contribute to the paired sense organs and to the cephalic peripheral nervous system. Here we report the spatiotemporal expression pattern of transcription factor Pitx2c during Xenopus laevis cranial placode formation, focusing more specifically on key stages of trigeminal and profundal placode development. We also compare its expression to five genes that have been associated with development of these sensory placodes, namely Foxi1c, Islet1, NeuroD, Pax3, and Six1. We show that while initially expressed in both the trigeminal and profundal placodes, Pitx2c is later restricted to the prospective profundal ganglion, where it is co-expressed with Islet1, NeuroD and Pax3. This combination of factors defines a molecular signature for the characterization of the profundal versus trigeminal ganglia in Xenopus.

The trigeminal circuit relays somatosensory input from the face into the central nervous system. In central nuclei, the spatial arrangement of neurons reproduces the physical distribution of peripheral receptors, thus generating a somatotopic facial map during development. In mice, the ophthalmic, maxillary, and mandibular trigeminal nerve branches maintain a somatotopic segregation and generate spatially organized patterns of connectivity within hindbrain target nuclei. To investigate conservation of somatotopic organization, we compared trigeminal nerve organization in turtle, chick, and mouse embryos. We found that, in the turtle, mandibular and maxillary ganglion neuron rostrocaudal segregation and trigeminal tract somatotopy are similar to mouse. In contrast, chick mandibular ganglion neurons are located rostrally to maxillary neurons, with some intermingling, supporting previous observations (Noden [1980], J Comp Neurol 190:429-444). This organization results in an inversion of the relative positions and less precise axonal sorting of the maxillary and mandibular branches within the trigeminal tract, as compared to mouse and turtle. Moreover, using the turtle and chick orthologs of Drg11 in combination with Hoxa2 expression and axonal tracings from the periphery, we mapped the chick PrV nucleus position to rhombomere 1, confirming previous studies (Marin and Puelles [1995], Eur J Neurosci 7:1714-1738) and in contrast to mouse PrV, which mainly maps to rhombomere 2-3 (Oury et al. [2006], Science 313:1408-1413). Thus, somatotopy of trigeminal ganglion and nerve organization is only partially conserved through amniote evolution, possibly in relation to the modification of facial somatosensory structures and morphologies., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: Sensory trigeminal nerve growth cones innervate the cornea in a highly coordinated fashion. The purpose of this study was to determine if extracellular matrix glycosaminoglycans (ECM-GAGs), including keratan sulfate (KS), dermatan sulfate (DS), and chondroitin sulfate A (CSA) and C (CSC), polymerized in developing eyefronts, may provide guidance cues to nerves during cornea innervation., Methods: Immunostaining using antineuron-specific-β-tubulin and monoclonal antibodies for KS, DS, and CSA/C was performed on eyefronts from embryonic day (E) 9 to E14 and staining visualized by confocal microscopy. Effects of purified GAGs on trigeminal nerve growth cone behavior were tested using in vitro neuronal explant cultures., Results: At E9 to E10, nerves exiting the pericorneal nerve ring grew as tight fascicles, advancing straight toward the corneal stroma. In contrast, upon entering the stroma, nerves bifurcated repeatedly as they extended anteriorly toward the epithelium. KS was localized in the path of trigeminal nerves, whereas DS and CSA/C-rich areas were avoided by growth cones. When E10 trigeminal neurons were cultured on different substrates comprised of purified GAG molecules, their neurite growth cone behavior varied depending on GAG type, concentration, and mode of presentation (immobilized versus soluble). High concentrations of immobilized KS, DS, and CSA/C inhibited neurite growth to varying degrees. Neurites traversing lower, permissive concentrations of immobilized DS and CSA/C displayed increased fasciculation and decreased branching, whereas KS caused decreased fasciculation and increased branching. Enzymatic digestion of sulfated GAGs canceled their effects on trigeminal neurons., Conclusions: Data herein suggest that GAGs may direct the movement of trigeminal nerve growth cones innervating the cornea.

Introduction: There is limited published work on the abundant innervation of the human dura mater, its role and responses to injury in humans. The dura not only provides mechanical support for the brain but may also have other functions, including control of the outflow of venous blood from the brain via the dural sinuses. The trigeminal nerve supplies sensory fibres to the dura as well as the leptomeninges, intracranial blood vessels, face, nose and mouth. Its relatively large size in embryonic life suggests an importance in development; the earliest fetal reflexes, mediated by the trigeminal, are seen by 8 weeks. Trigeminal functions vital to the fetus include the coordination of sucking and swallowing and the protective oxygen-conserving reflexes. Like other parts of the nervous system, the trigeminal undergoes pruning and remodelling throughout development., Methods: We have investigated changes in the innervation of the human dura with age in 27 individuals aged between 31 weeks of gestation and 60 years of postnatal life. Using immunocytochemistry with antibodies to neurofilament, we have found significant changes in the density of dural innervation with age, Results: The density of innervation increased between 31 and 40 weeks of gestation, peaking at term and decreasing in the subsequent 3 months, remaining low until the sixth decade., Conclusions: Our observations are consistent with animal studies but are, to our knowledge, the first to show age-related changes in the density of innervation in the human dura. They provide new insights into the functions of the human dura during development.

In adults, the lateral pterygoid muscle (LPM) is usually divided into the upper and lower heads, between which the buccal nerve passes. Using sagittal or horizontal sections of 14 fetuses and seven embryos (five specimens at approximately 20-25 weeks; five at 14-16 weeks; four at 8 weeks; seven at 6-7 weeks), we examined the topographical relationship between the LPM and the buccal nerve. In large fetuses later than 15 weeks, the upper head of the LPM was clearly discriminated from the lower head. However, the upper head was much smaller than the lower head in the smaller fetuses. Thus, in the latter, the upper head was better described as an 'anterior slip' extending from the lower head or the major muscle mass to the anterior side of the buccal nerve. The postero-anterior nerve course seemed to be determined by a branch to the temporalis muscle (i.e. the anterior deep temporal nerve). At 8 weeks, the buccal nerve passed through the roof of the small, fan-like LPM. At 6-7 weeks, the LPM anlage was embedded between the temporobuccal nerve trunk and the inferior alveolar nerve. Therefore, parts of the LPM were likely to 'leak' out of slits between the origins of the mandibular nerve branches at 7-8 weeks, and seemed to grow in size during weeks 14-20 and extend anterosuperiorly along the infratemporal surface of the prominently developing greater wing of the sphenoid bone. Consequently, the topographical relationship between the LPM and the buccal nerve appeared to 'change' during fetal development due to delayed development of the upper head., (© 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.)

The infraorbital branch of the trigeminal nerve (ION) is essential in whisker-specific neural patterning ("barrelettes") in the principal nucleus of the trigeminal nerve (PrV). The barrelettes are formed by the ION terminal arbors, somata, and dendrites of the PrV cells; they are abolished after neonatal damage to the ION. Physiological studies show that disruption of the barrelettes is accompanied by conversion of functional synapses into silent synapses in the PrV. In this study, we used whole cell recordings with a paired-pulse stimulation protocol and MK-801 blocking rate to estimate the presynaptic release probability (Pr) of ION central trigeminal afferent terminals in the PrV. We investigated Pr during postnatal development, following neonatal ION damage, and determined whether conversion of functional synapses into silent synapses after peripheral denervation results from changes in Pr. The paired-pulse ratio (PPR) was quite variable ranging from 40% (paired-pulse depression) to 175% (paired-pulse facilitation). The results from paired-pulse protocol were confirmed by MK-801 blocking rate experiments. The nonuniform PPRs did not show target cell specificity and developmental regulation. The distribution of PPRs fit nicely to Gaussian function with a peak at ∼ 100%. In addition, neonatal ION transections did not alter the distribution pattern of PPR in their central terminals, suggesting that the conversion from functional synapses into silent synapses in the peripherally denervated PrV is not caused by changes in the Pr.

Somatosensation is the primary sensory modality employed by rodents in navigating their environments, and mystacial vibrissae on the snout are the primary conveyors of this information to the murine brain. The layout of vibrissae is spatially stereotyped and topographic connections faithfully maintain this layout throughout the neuraxis. Several factors have been shown to influence general vibrissal innervation by trigeminal neurons. Here, the role of a cell surface receptor, EphA4, in directing position-dependent vibrissal innervation is examined. EphA4 is expressed in the ventral region of the presumptive whisker pad and EphA4(-/-) mice lack the ventroposterior-most vibrissae. Analyses reveal that ventral trigeminal axons are abnormal, failing to innervate emerging vibrissae, and resulting in the absence of a select group of vibrissae in EphA4(-/-) mice. EphA4's selective effect on a subset of whiskers implicates cell-based signaling in the establishment of position-dependent connectivity and topography in the peripheral somatosensory system.

While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.

In the mouse trigeminal pathway, sensory inputs from distinct facial structures, such as whiskers or lower jaw and lip, are topographically mapped onto the somatosensory cortex through relay stations in the thalamus and hindbrain. In the developing hindbrain, the mechanisms generating such maps remain elusive. We found that in the principal sensory nucleus, the whisker-related map is contributed by rhombomere 3-derived neurons, whereas the rhombomere 2-derived progeny supply the lower jaw and lip representation. Moreover, early Hoxa2 expression in neuroepithelium prevents the trigeminal nerve from ectopically projecting to the cerebellum, whereas late expression in the principal sensory nucleus promotes selective arborization of whisker-related afferents and topographic connectivity to the thalamus. Hoxa2 inactivation further results in the absence of whisker-related maps in the postnatal brain. Thus, Hoxa2- and rhombomere 3-dependent cues determine the whisker area map and are required for the assembly of the whisker-to-barrel somatosensory circuit.

The prenatal development of neural circuits for rhythmical oral-motor behaviors used for feeding is essential for the survival of the newborn mammal. The N-methyl-D-aspartate (NMDA) receptor plays a critical role in brainstem circuits underlying postnatal oral-motor behaviors. To understand a role for the NMDA receptor in the emergence of sucking behavior we conducted physiological and immunohistochemical experiments using fetal rats. Physiology experiments examined the development of the NMDA dose response of the brainstem circuit responsible for generating rhythmical trigeminal activity by recording trigeminal motor outputs using an in vitro preparation. The high dose of NMDA agonist bath application affected the mean cycle duration of rhythmical trigeminal activity (RTA) at both embryonic day (E) 18-19 and E20-21 in comparison with standard concentration of NMDA agonist. NMDA receptor immunohistochemistry studies, using antibodies directed against subunits NR1, NR2A, NR2B, NR3A and NR3B were performed to determine the prenatal regulation of NMDA subunits in trigeminal motoneurons (Mo5), and mesencephalic trigeminal neurons (Me5) between E17 to E20. In Mo5, NR1, NR2A, NR2B and NR3A immunoreactivity was observed throughout the time frame sampled. NR3B immunoreactivity was not observed in Mo5 or Me5. In Mo5, there was a significant decrease in the percentage of NR2B immunoreactive neurons between E17 and E20, and a concurrent increase in the NR2A/NR2B ratio between E17 and E20. In Me5, NR1, NR2A and NR3A immunoreactivity was observed throughout the time frame sampled; a significant decrease in the percentage of NR2A immunoreactive neurons between E17 and E20, and NR3A immunoreactive neurons between E17 and E18 occurred. The timing of subunit changes between E17 and E18 is coincident with the prenatal emergence of rhythmical jaw movements, and in vitro rhythmical trigeminal activity, shown in earlier studies. Our data suggest that NMDA receptor plays an important role in the development and function of prenatal oral-motor circuits.

Basic helix-loop-helix (bHLH) transcription factors are known to play important roles in neuronal determination and differentiation. However, their exact roles in neural development still remain to be determined because of the functional redundancy. Here, we examined the roles of neural bHLH genes Mash1 and Math3 in the development of trigeminal and facial branchiomotor neurons, which derive from rhombomeres 2-4. In Math3-null mutant mice, facial branchiomotor neurons are misspecified, and both trigeminal and facial branchiomotor neurons adopt abnormal migratory pathways. In Mash1;Math3 double-mutant mice, trigeminal and facial branchiomotor neurons are severely reduced in number partly because of increased apoptosis. In addition, neurons with migratory defects are intermingled over the midline from either side of the neural tube. Furthermore, oligodendrocyte progenitors of rhombomere 4 are reduced in number. In the absence of Mash1 and Math3, expression of Notch signaling components is severely downregulated in rhombomere 4 and neural progenitors are not properly maintained, which may lead to intermingling of neurons and a decrease in oligodendrocyte progenitors. These results indicate that Mash1 and Math3 not only promote branchiomotor neuron development but also regulate the subsequent oligodendrocyte development and the cytoarchitecture by maintaining neural progenitors through Notch signaling.

Background: Trigeminal sensory neurons detect thermal and mechanical stimuli in the skin through their elaborately arborized peripheral axons. We investigated the developmental mechanisms that determine the size and shape of individual trigeminal arbors in zebrafish and analyzed how these interactions affect the functional organization of the peripheral sensory system., Results: Time-lapse imaging indicated that direct repulsion between growing axons restricts arbor territories. Removal of one trigeminal ganglion allowed axons of the contralateral ganglion to cross the midline, and removal of both resulted in the expansion of spinal cord sensory neuron arbors. Generation of embryos with single, isolated sensory neurons resulted in axon arbors that possessed a vast capacity for growth and expanded to encompass the entire head. Embryos in which arbors were allowed to aberrantly cross the midline were unable to respond in a spatially appropriate way to mechanical stimuli., Conclusions: Direct repulsive interactions between developing trigeminal and spinal cord sensory axon arbors determine sensory neuron organization and control the shapes and sizes of individual arbors. This spatial organization is crucial for sensing the location of objects in the environment. Thus, a combination of undirected growth and mutual repulsion results in the formation of a functionally organized system of peripheral sensory arbors.

In this study, we investigate the possible role of ephrin-Eph signaling in trigeminal motor axon projections. We find that EphA receptors are expressed at higher levels by rhombomere 2 (r2) trigeminal motor neurons than by r3 trigeminal motor neurons in the chick embryo. Mapping of rhombomere-specific axon projections shows that r2 and r3 trigeminal motor neurons project to different muscle targets, including the mandibular adductor and the intermandibularis muscles respectively. Ephrin-A5 is expressed in these muscles, especially in some regions of the intermandibularis muscle, and can cause growth cone collapse of both r2 and r3 motor axons in vitro. We demonstrate that in vivo overexpression of ephrin-A5 in the intermandibularis muscle, or overexpression of dominant-negative EphA receptors in trigeminal motor neurons leads to a reduction in branching of r3-derived motor axons specifically. Overexpression of full-length EphA receptors impairs the formation of r3 projections to the intermandibularis muscle. These findings indicate that ephrins and their Eph receptors play a role in trigeminal motor axon topographic mapping and in rhombomere 3-derived projections in particular.

The Netrins form a small, conserved family of laminin-related signaling proteins regulating axon guidance in the developing nervous system. Here, we analyzed the roles of Netrin-1 and -3 in trigeminal axon guidance to the first lower molar of the embryonic mouse. Netrin-1 showed a restricted epithelial expression domain buccal to the tooth germ, toward which the pioneer tooth axons initially appear to navigate. Later, before birth, transcripts were colocalized with nerve fibers around the bell stage tooth germ. Analysis of Netrin-1-deficient mice, however, did not reveal any obvious disturbances in the axon growth or pattern of tooth innervation. In contrast, Netrin-3 showed a prominent, distinct expression in the axon pathway and target field mesenchyme around the tooth. Hence, it is possible that Netrin-3 may regulate pioneer axon growth toward and within the embryonic tooth target field., (Copyright 2003 Wiley-Liss, Inc.)

We have examined the roles of signaling molecules in the mechanisms underlying the induction of neurogenin (ngn)-1 expression. ngn-1 is a basic helix-loop-helix (bHLH) transcription factor, which is essential for the specification of trigeminal sensory neurons. Semiquantitative reverse transcriptase-polymerase chain reaction using cranial explants in organ cultures showed that sonic hedgehog (Shh) promotes ngn-1 expression. This promoting activity was not observed in other signaling molecules examined. The promotion of ngn-1 expression by Shh, furthermore, was inhibited by cyclopamine, a specific inhibitor of Shh signaling. Shh did not affect the expression of ngn-2, a bHLH transcription factor that plays an important role in the specification of epibranchial placode-derived sensory neurons. The expression levels of ngn-1 and ngn-2 decreased after fibroblast growth factor-2 treatment. These results suggest that Shh induces ngn-1 expression specifically and that expression of ngn-1 and ngn-2 is regulated by different mechanisms. The induction of ngn-1 expression by Shh suggests that this signaling molecule participates in the specification of trigeminal sensory neurons. We therefore examined the effect of Shh on the development of these neurons. Immunostaining using anti-ngn-1 demonstrated that Shh promotes ngn-1 expression in trigeminal neural crest cells. Trigeminal neural crest cells are derived from the posterior mesencephalon and the most-anterior rhombencephalon, and they contain a subset of precursors of trigeminal sensory neurons. Moreover, a subpopulation of trigeminal neural crest cells expressed the Shh receptor Patched. The number of cells that express Brn3a, a POU-domain transcription factor that plays an important role in differentiation of sensory neurons, also increased with Shh treatment. Our data suggest that Shh signaling is involved in the specification of trigeminal sensory neurons through the induction of ngn-1 expression. Furthermore, Shh promotes the differentiation of neural crest cells into trigeminal sensory neurons., (Copyright 2003 Wiley-Liss, Inc.)

Spontaneous electrical activity synchronized among groups of related neurons is a widespread and important feature of central nervous system development. Among the many places from which spontaneous rhythmic activity has been recorded early in development are the cranial motor nerve roots that exit the hindbrain, the motor neuron pool that, at birth, will control the rhythmic motor patterns of swallow, feeding and the oral components of respiratory behaviour. Understanding the mechanism and significance of this hindbrain activity, however, has been hampered by the difficulty of identifying and recording from individual hindbrain motor neurons in living tissue. We have used retrograde labelling to identify living cranial branchiomeric motor neurons in the hindbrain, and [Ca2+]i imaging of such labelled cells to measure spontaneous activity simultaneously in groups of motor neuron somata. We find that branchiomeric motor neurons of the trigeminal and facial nerves generate spontaneous [Ca2+]i transients throughout the developmental period E9.5 to E11.5. During this two-day period the activity changes from low-frequency, long-duration events that are tetrodotoxin insensitive and poorly coordinated among cells, to high-frequency short-duration events that are tetrodotoxin sensitive and tightly coordinated throughout the motor neuron population. This early synchronization may be crucial for correct neuron-target development.

Axons of the trigeminal ganglion convey sensory information from mechanoreceptors, thermoreceptors, and nociceptors in the face and nasal mucosa, then terminate on several groups of neurons including the principal sensory nucleus and the nuclei of the spinal trigeminal tract. To understand guidance mechanisms during the development of trigeminal sensory axons (TA) in the embryonic brain, we first investigated the growth pattern of TA in relation to organization in the hindbrain using flat whole-mount preparation from rat. We found that the primary TA from the trigeminal ganglion entered the brainstem and grew longitudinally within the hindbrain. Whereas descending axons ran just medial to the primary vestibular axons to innervate the spinal nucleus, ascending axons stayed near the entry point. In flat whole-mount culture, the TA extended both ascending and descending branches as they do in vivo. Rostral hindbrain was found to be a less permissive substrate for the TA compared to caudal hindbrain. In addition, the nonpermissive property of the ventral hindbrain substrate restricted the invasion of TA along the entire length of the hindbrain. Thus, cooperation of absolute and relative permissiveness of the substrate plays important roles in the guidance of TA to their targets., (Copyright 2003 Wiley-Liss, Inc.)

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.

Using immunohistochemistry in combination with confocal laser scanning microscopy, we studied the ontogeny of neuropeptide Y-Y1 receptor (Y1-R) expression in the trigeminal system of the rat. The study was limited to the nerve fibers innervating the mystacial pad and the trigeminal ganglia. In the trigeminal ganglia, Y1-R-immunoreactive (IR) neurons were first observed at E16.5. At this same stage some nerve fibers in the trigeminal ganglia also exhibited Y1-R-like immunoreactivity (LI). Strongly Y1-R-IR nerve fibers innervating the follicles of the mystacial vibrissae were first observed at E18. After double labeling, the Y1-R-LI was found to be colocalized with the neuronal marker protein gene product 9.5. At P1 only weak labeling for the Y1-R was found around the vibrissae follicles, whereas the neurons in the trigeminal ganglia were intensely labeled. The same was true for the adult rat, but at this stage no Y1-R labeling at all was observed in nerve fibers around the vibrissal follicles. These results strongly support an axonal localization of the Y1-R at this developmental stage. The transient expression of the Y1-R during prenatal mystacial pad development suggests a role for the Y1-R in the functional development of the vibrissae., (Copyright 2000 Wiley-Liss, Inc.)

The trigeminal nerve is the largest and most complex of twelve cranial nerves. Its vast size and influence are greatly appreciated when one attempts to diagnose and treat patients suffering from orofacial pain and temporomandibular joint disorders. Without a thorough knowledge of the trigeminal nerve, the efficacy of diagnostic and therapeutic procedures will be very disappointing. This is the first of a four-part series of articles about the trigeminal nerve, a basic over-view of both the gross and neuroanatomical structures is presented.

We examined the effects of neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) on trigeminal axon growth patterns. Embryonic (E13-15) wholemount explants of the rat trigeminal pathway including the whisker pads, trigeminal ganglia, and brainstem were cultured in serum-free medium (SFM) or SFM supplemented with NGF or NT-3 for 3 days. Trigeminal axon growth patterns were analyzed with the use of lipophilic tracer DiI. In wholemount cultures grown in SFM, trigeminal axon projections, growth patterns, and differentiation of peripheral and central targets are similar to in vivo conditions. We show that in the presence of NGF, central trigeminal axons leave the tract and grow into the surrounding brainstem regions in the elongation phase without any branching. On the other hand, NT-3 promotes precocious development of short axon collaterals endowed with focal arbors along the sides of the central trigeminal tract. These neurotrophins also affect trigeminal axon growth within the whisker pad. Additionally, we cultured dissociated trigeminal ganglion cells in the presence of NGF, NT-3, or NGF+NT-3. The number of trigeminal ganglion cells, their size distribution under each condition were charted, and axon growth was analyzed following immunohistochemical labeling with TrkA and parvalbumin antibodies. In these cultures too, NGF led to axon elongation and NT-3 to axon arborization. Our in vitro analyses suggest that aside from their survival promoting effects, NGF and NT-3 can differentially influence axon growth patterns of embryonic trigeminal neurons., (Copyright 2000 Wiley-Liss, Inc.)

Contactin is a glycosylphosphatidylinositol-anchored immunoglobulin-like neuronal cell adhesion molecule that has been implicated in cellular interaction during development of the vertebrate central nervous system. Here we report evidence for an essential role of contactin in development of the Xenopus nervous system. Contactin mRNA is detectable by in situ hybridization in subsets of neurons in the brain, primary sensory neurons in the spinal cord, and cells along the trigeminal nerves of tailbud embryos. Contactin immunoreactivities preferentially distribute on axon tracts of the brain, the spinal cord, and the trigeminal sensory nerves. Most prominently, cell bodies and peripheral and spinal axons of primary sensory neurons, Rohon-Beard (RB) cells, are strongly contactin positive. Injection of the contactin overexpression vector into one blastomere of two-cell stage embryos leads to misdirected elongation of the peripheral axons of RB neurons in the injected half. Overexpression of antisense transcript causes depletion of contactin mRNA accumulation and abnormal development of RB neurons. In 52.3% of the injected embryos, RB neurons decrease in number and their peripheral axons in dorsal lateral tracts are defasciculated. These results demonstrate that contactin plays an essential role in development of the Xenopus primary sensory system., (Copyright 2000 Academic Press.)

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.

We examined axon-target interactions in cocultures of embryonic rat trigeminal, dorsal root, nodose, superior cervical ganglia or retina with a variety of native or foreign peripheral targets such as the whisker pad, forepaw, and heart explants. Axon growth into these peripheral target tissues was analyzed by the use of lipophilic tracer DiI. Embryonic day 15 dorsal root and trigeminal axons grew into isochronic normal and foreign cutaneous targets. Both axon populations avoided the same age heart tissue, but grew profusely into younger (embryonic day 13) or older (postnatal) heart explants. In contrast, embryonic day 15 superior cervical or nodose ganglion axons grew heavily into the same age heart and forepaw explants and to a lesser extent into the whisker pad explants. Embryonic day 15 retinal axons grew into all three peripheral targets used in this study. Primary sensory and sympathetic axons, but not retinal axons, formed target-specific patterns in the whisker pad and forepaw explants. DiI-labeling and immunostaining of primary sensory neurons in coculture revealed that these neurons retain their bipolar characteristics, and express class-specific markers such as parvalbumin, calcitonin gene-related peptide and TrkA receptors. In the whisker pad explants, axons positive for all three markers were seen to form patterns around the follicles. Our results indicate that developing peripheral targets can attract and support axon growth from a variety of sources. Whereas neurotrophins play a major role in attracting and supporting survival of subpopulations of sensory neurons, other substrate-bound or locally released molecules must regulate sensory neurite growth into specific peripheral and central targets.

Neurturin (NTN) is a recently characterized member of the glial cell line-derived neurotrophic factor (GDNF)-family which, like GDNF, can promote the survival of certain populations of neuronal cells in peripheral and central nervous systems. To elucidate the roles of NTN and a novel glycosyl-phosphatidylinositol (GPI)-linked receptor protein GFRalpha-3, a member of GDNF-family receptor alpha, in the regulation of peripheral trigeminal innervation and tooth formation, their expression patterns during mouse embryonic (E) and early postnatal (P) development (E10-P5) of the first branchial arch were analyzed by in situ hybridization. NTN mRNAs were observed in oral and cutaneous epithelia of the mandibular process at all studied stages and expression became gradually restricted to the suprabasal epithelial cells. In addition, transcripts were also detected in the epithelium of whisker follicles. In the developing first molar tooth germ, NTN showed a developmentally regulated, spatiotemporally changing expression pattern, which partially correlated with the development of innervation. During the initiation of tooth formation NTN mRNAs were expressed in dental epithelium and during later embryonic development transcripts appeared in the dental papilla mesenchyme. In addition, some transcripts were seen in the dental follicle. During postnatal development, NTN expression was restricted to the dental follicle of the incisor tooth germs. GFRalpha-3 mRNAs were not detected in teeth, but an intense expression was seen in non-neuronal cells surrounding trigeminal nerve fibers and in the trigeminal ganglia during E11-E15. Ganglion explant cultures showed that trigeminal neurons start to respond to exogenous NTN at E12, which correlates to the earlier reported appearance of the Ret-tyrosine kinase receptor in the trigeminal ganglion. Local application of NTN with beads on isolated dental mesenchyme did not stimulate cell proliferation or prevent apoptotic cell death. In addition, exogenous NTN had no effects on tooth morphogenesis in in vitro cultures. Taken together, because trigeminal neurons respond to NTN after first axons have reached their primary epithelial target fields, NTN is apparently not involved in the guidance of pioneer trigeminal nerves to their peripheral targets. However, our results show that NTN is a potent neuritogenic factor and, therefore, may act as a target-field-derived neurotrophic factor for trigeminal nerves during innervation of the cutaneous and oral epithelia as well as dental follicle surrounding the developing tooth. In addition, although NTN appears not to be directly involved in the regulation of tooth morphogenesis, it may have non-neuronal, organogenetic functions during tooth formation.

The cavum trigeminale (Meckel's cave) anatomy is still poorly understood. Many different descriptions are found in the literature. In order to clarify the relationship of trigeminal ganglion and its branches with dura and arachnoid, we underwent an embryological and adult microanatomical and histological study. Serial sections of human embryos and fetuses were used. For adult study, microdissections and histological serial sections were performed. We found that dura and arachnoid stop at the trigeminal ganglion and do not extend the three branches of the trigeminal nerve. These three branches are embedded into separate peripheral sheaths. These results are important for clear understanding of the anatomy of the parasellar lodge (cavernous sinus) lateral wall.

The localization of TrkB, a signal transducing receptor for brain-derived neurotrophic factor and neurotrophin-4, was studied in the rat mandibular molar pulp during development and following nerve injury. Sections were incubated with rabbit polyclonal antiserum against the catalytic part of the TrkB receptor, thus only binding to full-length TrkB receptors, and examined by immunofluorescence microscopy and EM immunocytochemistry. At embryonic day 17, strongly TrkB-positive fibers were located in mandibular nerve trunks, in nerve fibers of deeper mesenchyme in close spatial relation to sites of future tooth development and in subepithelial nerve plexa. At postnatal days 1-12 intensely TrkB-positive nerve fibers surrounded and eventually invaded the developing dental papillae and pulps. In the normal adult pulp TrkB-immunoreactive axons were seen extending through the radicular pulp into the coronal areas. Double labelling demonstrated a considerable overlap between TrkB-like immunoreactivity and low affinity neurotrophin receptor-like immunoreactivity in the pulp, although some low affinity neurotrophin receptor-positive nerve fibers lacked TrkB-like immunoreactivity. Immunogold electron microscopy showed TrkB-like immunoreactivity in myelinated as well as in unmyelinated axons. One week following inferior alveolar nerve injury there was a dramatic decrease in the level of TrkB-like immunoreactivity labelling in the pulp, which paralleled an increase in the expression of Schwann cell low affinity neurotrophin receptor-like immunoreactivity. The first signs of regenerating TrkB-positive nerve fibers were found at 4 weeks post-operative, and at 9 weeks the distribution of pulpal TrkB-like immunoreactivity had returned to normal. These data indicate that brain-derived neurotrophic factor and/or neurotrophin-4 could be target-derived factors involved in sensory trigeminal tooth pulp nerve fiber development, differentiation or regeneration.

Previous studies from this laboratory have demonstrated that prenatal damage to vibrissae follicles results in significant increases in the brainstem representations of the remaining vibrissae as demonstrated by staining for the mitochondrial enzyme cytochrome oxidase (CO). Because CO is primarily a postsynaptic marker, these results do not directly address the question of whether there were changes in the projections of primary afferent fibers. To address this issue, we made intra-axonal recordings from individual vibrissa-related primary afferents in rats that sustained damage to vibrissae follicles on embryonic day 17, and then injected horseradish peroxidase (HRP) into these axons to visualize their terminal arbors in the brainstem at the level of trigeminal subnucleus interpolaris (SpI). All vibrissae-related primary afferents responded to deflection of one and only one vibrissa, and the terminal arbors of axons (N = 47) recovered from animals that sustained fetal peripheral lesions were significantly larger than those (N = 23) from normal rats. Fibers from fetally damaged animals had increased total fiber lengths and numbers of branch points. These results indicate that reduced competition among primary afferent axons results in increases in the terminal arbors that remain. These increases occur without any significant alteration in their peripheral receptive fields.

To investigate if different neurotrophins regulate the survival of neurons at successive developmental stages, we studied the effect of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the survival of mouse trigeminal neurons at closely staged intervals in development. We show that during the earliest stages of target field innervation trigeminal neurons display a transitory survival response to BDNF and NT-3. This response is lost as the neurons become NGF-dependent shortly before neuronal death begins in the trigeminal ganglion. BDNF and NT-3 mRNAs are expressed in the peripheral trigeminal target field before the arrival of the earliest axons and the onset of NGF mRNA expression. The levels of BDNF and NT-3 mRNAs peak during the early stages of target field innervation and decline shortly after the loss of neuronal responsiveness to BDNF and NT-3. Our study provides the first clear evidence that different target-derived neurotrophins can act sequentially to promote the survival of developing neurons.

Expression of the c-fyn proto-oncogene was analysed in the developing and adult rat brain by subcellular fractionation and immunocytochemistry using polyclonal antibodies specific for its protein product (p59fyn). Immunoperoxidase staining revealed widespread localization of p59fyn in developing axonal tracts throughout the fetal (E18) rat brain. fyn immunoreactivity was not observed in most axon-rich regions of the adult brain, but continued to be expressed at elevated levels in the adult olfactory and vomeronasal systems. At other sites in the adult rat brain, fyn immunoreactivity was restricted to cell bodies of neuronal subpopulations, especially those in brain stem and hypothalamic nuclei, and to subpopulations of glial cells along axonal tracts in the medulla, optic nerve and white matter of the spinal cord. In the peripheral nervous system, fyn staining was also prominent in Schwann cells. Subcellular fractionation of fetal and adult rat brain confirmed the immunocytochemical localization, demonstrating an enrichment of p59fyn in membranes from a fetal brain fraction containing nerve growth cones, and lower levels in adult brain where there was only a small enrichment in synaptosomal membranes. The developmental regulation of p59fyn suggests that the fyn tyrosine kinase may serve separate cellular roles in axonal growth and specialized functions of mature neurons and glia.

Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined (somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)), only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double-labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types.

The mesencephalic nucleus of V was investigated in embryos of developmental stages 14 and 15. In stage 14 the nucleus is formed by a small group of cells lying a little rostrally to the sulcus limitans in the pontine flexure. In stage 15 the cells of the nucleus are also found in the anterior crus of the pontine flexure. The nucleus is composed of oval cells resembling those of the trigeminal ganglion.

The anterior ethmoidal nerve (a.e.n.) was examined in 10 human adults (40-89 years old) and 5 fetuses (7 months). A.e.n. arises from the naso-ciliary++ nerve, which is the first division of the trigeminal nerve. From this point to the crista galli, a.e.ns. were observed under a dissection microscope in relation to their direction and distribution. The following results were obtained: 1) The number of a.e.n. i. The number of a.e.n. was 1 to 3 (1.45 on average) when they arose from the naso-ciliary nerve in the adults. ii. In the fetuses, the number of a.e.n. was 1 to 2 (1.1 on average). 2) The length of a.e.n. i. The length was 12.0 mm to 24.0 mm (17.3 mm on average) in the adults. ii. In the fetuses, the length was 2.0 mm to 7.0 mm (5.0 mm on average). 3) The anterior ethmoidal branch (e.b.). The anterior ethmoidal branch (e.b.) supplies the mucous membrane of the ethmoidal sinus. i. The number of e.bs. was 20 on the right side and 22 on the left in 8 adult cases. In the upper wall of the ethmoidal sinus, the number of e.bs. was 1.0 on average. In the medial wall of the ethmoidal sinus, the number of e.bs. was also 1.0 on average. In the lateral wall of the ethmoidal sinus, the number of e.bs. was 0.2 on average. ii. The number of e.bs. was 3 on the right side and 2 on the left in 3 fetus cases. In the upper wall of the ethmoidal sinus, the number of e.bs. was 0.2 on average. In the medial wall of the ethmoidal sinus, the number of e.bs. was 0.3 on average. No e.bs. were found in the lateral wall. 4) The anterior ethmoidal foramen. The anterior ethmoidal foramen was examined to determine its positional relation to the angle of the crista galli. i. The angle was 40.0 degrees to 10.0 degrees and 30.0 degrees on average in the adults. ii. In the fetuses, the angle was 50.0 degrees to 20.0 degrees and 43.2 degrees on average.

Dissociates from the metencephalic basal plate of early chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured on substrates conditioned with appropriate target-derived muscle conditioned medium (MCM), laminin (LAM), MCM with nerve growth factor (NGF) in the medium, and fibronectin (FN). Comparisons were made of neuronal survival, the number of neurons with processes, and the length of processes elaborated. It was found that both MCM and LAM significantly enhanced survival and neuritic production from this population when compared to controls grown on a collagen-polyornithine substrate, but MCM surpassed LAM in these measures. When the neurons were grown on an MCM-conditioned substrate with an NGF-supplemented medium, no improvements were produced over the MCM or the NGF conditions alone. Therefore, the two do not appear to act in synergy, as NGF and LAM have been shown to do. FN produced no enhancement of any of the measures taken from this population. An ELISA analysis revealed no detectable LAM in the early target MCM. These results indicate that the specific responsiveness of this early neural tube population to its target MCM is not mediated by LAM, but the growth-enhancing component acts in a similar manner, although its influence is more potent.

1. The development of the mouse trigeminal system is outlined and its advantages for studying the synthesis of low-abundance regulatory proteins are described. 2. The onset of NGF gene expression and NGF synthesis in the cutaneous target field of the trigeminal ganglion coincide with the arrival of the earliest nerve fibres. 3. The distribution and magnitude of NGF synthesis within the target field are related to its final innervation density. 4. NGF receptors are first detected on trigeminal neurons when their peripheral axons reach the target field. 5. Neither NGF synthesis nor NGF receptor expression are dependent on nerve-target contact but appear to occur as part of an intrinsic developmental program in the target field and neurons, respectively. 6. The time-course of NGF synthesis and NGF receptor expression indicate that NGF does not play a role in guiding axons to their target fields in development.

Development of the trigeminal nerve branches was studied in stage -17 to -27 chick embryos stained with an antibody to neurofilament protein. The following findings were obtained. 1) Ectopic ganglia transiently appeared in the ectoderm of the supraorbital region and were considered as remnant ophthalmic-placode-derived ganglia. 2) Most of the cutaneous sensory branches of the maxillomandibular nerve arose from a loosely arborized mass of neurites, provisionally termed the maxillomandibular reticulum, in which the fibers intermingled in a seemingly random fashion. 3) The growth of the trigeminal branches was mainly correlated with the development of the facial processes; however, irregular communications between different groups of branches were observed, suggesting that topographical organization of the peripheral branches is not rigid in early stages. 4) From the ophthalmic nerve around stage 23, transient dorsal rami developed and were distributed in the mesenchymal space, the cavum epiptericum, and passed near the ectoderm. Their homology with the rr. tentorii in human anatomy is suggested.

The migration and early development of trigeminal (V) motoneurons were studied in chick embryos in which two different populations of primary trigeminal sensory neurons had been removed prior to the birthdate of the V motoneurons. Ablation of mesencephalic neural crest cells, which eliminates monosynaptic sensory input, did not affect the migration, early development, or later differentiation of the V motoneurons. However, when the anlagen of the V ganglion were removed, the V motor root did not exit from the brainstem and the V motor nucleus did not develop. Although the neurons of the V ganglion do not innervate adult V motoneurons, these populations are related developmentally. In those embryos in which the V ganglion did not develop, medial column cells, which are midline, postmitotic, premigratory V motoneurons, and a few medial, elongated cells (possibly migratory) were present until days 5-6, but these cells did not complete their lateral migration to form the lateral nucleus of V. In cases where the ganglion anlagen were not completely removed, the number of postmigratory V motoneurons was positively correlated to the size of the ganglion remnant. There also was a correlation between the axial position of the postmigratory V motoneurons and the ganglion remnants. If a caudal remnant developed, only caudal V motoneurons, whose axons reached the ganglion, migrated; if a rostral remnant developed, only rostral V motoneurons, with axons reaching this remnant, migrated. Additionally, if the central axons of the ganglion remnant entered the metencephalon in either dorsal or ventral ectopic positions, the V motor nucleus was located in a corresponding aberrant position. Thus, some characteristic of the V ganglion cells appears to guide the motor axons and somas to their final brainstem position.

Horseradish peroxidase (HRP) has been injected into periocular, upper beak, or lower jaw tissues of chick embryos aged 6--8 days of incubation. Subsequent mapping of the distribution of labeled neurons in the trigeminal ganglion indicates that the somatotopic organization of neurons is essentially identical to that found after hatching. At these stages most labeled cells are in the distal parts of the bilobed ganglion. There is no indication that any of these neurons, most but not all of which are derived from epidermal placodes, establish any erroneus projections. Most of the proximo-central or core neurons, which are of neural creast origin, are immature at these stages. On the eighth day of development, by which time the embryo is responsive to tactile stimulation of the beak, few of these core cells have established projections to more distal (rostral) periocular or upper beak areas or to the lower jaw. In contrast, injection of HRP above the eye results in labeling of many of the core neurons. This suggests that there is an asymmetry in the time of neurite outgrowth from core neurons that is correlated with their peripheral projections but not with their time of terminal mitosis. Following injection of HRP into temporal periocular tissues, most of the core cells in the maxillo-mandibular lobe contain peroxidase. This is probably due to uptake by growth cones emerging along the maxillary ramus. Examination of contralateral trigeminal ganglia indicates that the transmedian projections of these sensory fibers form later in development. Branches of the mandibular motor nerve extend throughout the lower jaw and presumptive jaw-closing muscle anlagen, as evidenced by incorporation of HRP into motoneurons of the trigeminal nucleus.