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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.)

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.)

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.)

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.)

With in situ hybridization assays, we mapped the distribution of retinoid X receptor gamma (RXR gamma) gene transcripts in the central nervous system of the chicken embryo. Previous studies have demonstrated the presence of RXR gamma transcripts in migrating neural crest and in neural crest derivatives throughout the peripheral nervous system, implicating RXR gamma as an early pan-neural crest marker (Rowe et al. 1991. Development 111:771-778), and in the retina (Hoover et al. 1998. J Comp Neurol 391:204-213). Here we report the presence of RXR gamma transcripts in discrete regions of the developing neural tube, within the hindbrain, the cerebellar plate, the optic tectum, and the diencephalon. At stage 10, when migrating neural crest expresses RXR gamma transcripts, we detect no transcripts in the neural tube. By stage 13, RXR gamma transcripts accumulate to detectable levels along the midline of the posterior optic tectum, where the neural crest-derived sensory neurons of the mesencephalic trigeminal nucleus are located. By stage 15, RXR gamma transcripts also appear in an intermittent longitudinal cell column within the mantle zone of the alar plate of the hindbrain, eventually extending into the cerebellar plate rostrally and into the cervical spinal cord caudally, with a gap at about rhombomere 3. By stage 19, transcripts appear in a discrete population of cells within the diencephalon. Expression in these cell populations continues until at least stage 22.5, when many neuron populations have been generated in the hindbrain. The localization of the RXR gamma-positive cells to the mantle zone suggests that they are postmitotic and are probably neurons. Their specific alar locations indicate that they reside within sensory columns and potential downstream targets, evidently corresponding to some of the central components of the trigeminal system.

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.

The embryotoxic and dysmorphogenic effects of mercuric chloride (HgCl2) have been studied in mouse embryos cultured in vitro. In addition, the alterations induced in the developing branchial nerves and ganglia were analyzed. Mouse embryos with 6-8 pairs of somites were exposed for 26 hr to increasing concentrations (0, 12.5, 25, 50 microM) of HgCl2. After this period, a first set of embryos was removed and a second set of embryos transferred to culture medium without HgCl2 and remained in culture for an additional 22 hr. Both sets of embryos were examined for (1) survival, (2) presence of external dysmorphogenesis, (3) growth, and (4) differentiation. Dose-related alterations of these parameters were observed. The main target was the cephalic neural tube (mainly the forebrain), but several other systems were also affected (e.g., the turning of the embryos, the optic system). The 48-hr cultured embryos were immunostained using a monoclonal antineurofilament antibody to visualize defects in the development of branchial nerves and ganglia. HgCl2 induced a pronounced retardation in the differentiation of ganglion/nerve V and a slight retardation in the differentiation of ganglia/nerves VII and IX. The ganglia/nerves VIII and X were not retarded. In addition, hight percentages of abnormalities of ganglion/nerve V and fusions between ganglia/nerves IX and X were observed in these embryos. Disorganized fibers between ganglia/nerves VII-VIII and IX and between ganglia/nerves IX and X were also more frequently observed. At the highest concentration, asymmetric defects were induced by HgCl2 with a more pronounced effect observed on the right side of the embryos. These results demonstrate the usefulness of this approach in evaluating the susceptibility of the developing branchial nerves to the adverse effects of developmental toxicants.

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.

Fungiform taste buds in mature hamsters are less subject to neurotrophic influences than those of other species. This study evaluates taste-bud neurotrophism during development in hamsters by examining the relation between growing nerves and differentiating fungiform papillae. Chorda tympani (CT) or lingual (trigeminal) nerve (LN) fibers were labelled with Lucifer Yellow as they grew into (CT fibers) or around (LN fibers) developing taste buds. Developing fungiform papillae and taste pores were counted with the aid of a topical tongue stain. The tongue forms on embryonic days (E) 10.5-11 and contains deeply placed CT and LN fibers but no papillae. By E12, the tongue epithelium develops scattered elevations. These "eminences" selectively become innervated by LN fibers that grow to the epithelium earlier and in larger numbers than CT fibers. Definitive fungiform papillae form rapidly during E13-14 and become heavily innervated by LN fibers. Intraepithelial CT fibers, rare at E13, invariably innervate fungiform papillae containing nascent taste buds at E14. During E14-15 (birth = E15-16), most papillae contain taste buds with pores, extensive perigemmal LN innervation, and extensive intragemmal CT innervation. At birth, numbers of fungiform papillae and taste pores are adultlike. The results show that fungiform eminences begin forming in the absence of innervation. The subsequent differentiation of definitive fungiform papillae and their innervation by LN fibers occur synchronously, prior to the differentiation of taste buds and their CT innervation. The hamster is precocious (e.g., compared to rat) in terms of LN development and the structural maturity of the anterior tongue at birth.

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.

The objective of this study is to define the development of all components in the chick embryonic trigeminal primary sensory-motor complex, from their first appearance through the formation of central and peripheral axonal projections up to stage 34 (8 days of incubation). This was accomplished by two labeling procedures: application of the monoclonal antibody HNK-1, which binds to the precursors of all these components except the placode-derived neurons, and application of HRP to axons cut immediately distal to the trigeminal ganglion. Single immunopositive motor neuron precursors are present at stage 12. These accumulate in the transient medial motor column, whose neurons initiate axon outgrowth by stage 13-14, concomitant with the onset of translocation of their somata to form the definitive trigeminal lateral motor column (LMC). Initially these translocating somata accumulate on the medial margin of the LMC. Beginning on incubation day 5, axons growing from newly formed motor neurons pass beside the lateral margin of the LMC, and the nuclei of these cells subsequently follow this pathway. These events follow a rostral-to-caudal sequence, and this phase of motor nucleus formation is complete by day 8. The lateral translocation of some caudally located nuclei is arrested beginning on day 5. This cessation, which proceeds rostrally, demarcates neurons that form the dorsal motor nucleus of the trigeminal complex. Sensory neurite formation is initiated in ophthalmic placode-derived cells at stage 14.5, one stage later by maxillomandibular neurons, and from mesencephalic V cells at stage 15. Neural crest cells do not initiate axon formation until at least day 4 to 5. Following application of HRP distal to the condensing ganglion at stage 16, labeled ophthalmic nerve projections appear in contact with the wall of the hindbrain centrally and overlying the optic vesicle peripherally. Fibers forming the descending tract elongate rapidly, reaching the level of the VIIth nerve root (200 microns caudal to the trigeminal root) by stage 18 and the cervical cord by stage 22. Labeled terminal arborizations of descending trigeminal afferents are first visible at stage 22 and are evident along the entire descending and proximal ascending tracts by stage 27. Later-developing descending axons grow in close association with existing trigeminal fibers, though a few growth cones are consistently evident superficial to the other fibers. No projections different from those reported in adult birds are seen, nor are there any contralateral afferent projections.(ABSTRACT TRUNCATED AT 400 WORDS)

The previous study in this series demonstrated that the ingrowth of the central axons of the trigeminal (V) ganglion is prerequisite to V motor axon outgrowth and somatic translocation. In the present experiment we determined whether further interactions with V ganglion cell bodies were required by V motoneurons after the V ganglion innervates the brainstem. Soon after the ganglion axons had penetrated the brainstem they were severed, and a barrier, either permeable or impermeable, was placed between the ganglion cell bodies and the metencephalon. V motor axons grew along aberrant pathways to circumvent the impermeable barriers, many rerouting to reach the V ganglion. Only those V motor nerves which contacted the V ganglion distal to the barrier reached their target musculature in the mandible. The pattern of migration of V motoneurons was normal regardless of the V motor nerve trajectory, but the cell bodies of those axons which did not reach a muscle were not fully differentiated. When permeable barriers (Millipore filters) were implanted, the nerves followed two types of trajectories. If the pore size of the filter was small (0.45 and 0.025 microns), the V motor nerves grew identically to those observed in embryos in which impermeable barriers had been implanted. If the pore size of the filter was large (8.0 and 0.08 microns), the V motor nerve grew along its normal path directly to the barrier. Small axonal bundles from these nerves frequently grew into the filter toward the distal V ganglion. These results indicate that V motor axons preferentially grow to the V ganglion perikarya after exiting from the brainstem. Contact with the V ganglion always results in V motor nerve growth to the mandible while growth of the V motor axons to aberrant target sites only occurs when the axons fail to contact the V ganglion cells distal to the barrier.

The trigeminal region of the chick was studied with indirect immunofluorescence in order to determine whether extracellular matrix components might be distributed in such a way as to guide trigeminal axons to their peripheral targets in the mandibular arch. Tissue sections from stages 13-15 and 21/22 were immunolabeled indirectly with affinity-purified antibodies raised against fibronectin and laminin, two extracellular matrix glycoproteins that support axon growth in vitro. Fibronectin was distributed ubiquitously throughout the head mesenchyme prior to and during initial axon growth from the brainstem (stages 13-15). Shortly after trigeminal axons reached their target tissues (stage 21/22), fibronectin immunolabeling was distributed throughout the head mesenchyme, but was present only at low levels in the trigeminal ganglion and motor nerve. Laminin immunolabeling was distributed in the lateral head mesenchyme at stage 13 as small specks and patches. At stage 14, when the motor axons first exit from the brainstem, short, linear arrays of laminin immunostaining were present from the basement membrane of the neural tube to the core of the mandibular arch, and many were parallel to the direction of axon growth. By stage 21/22 the trigeminal ganglion and motor root showed intense antilaminin immunofluorescence as did the central core of the mandibular arch. These studies suggest that the distribution of fibronectin within the head mesenchyme cannot give directional information to the growing trigeminal axons because of its homogeneous distribution. However, the initial distribution of laminin during the earliest stages of axon outgrowth may provide an extracellular matrix pathway that permits trigeminal axons to reach their targets.

The development of the trigeminal motor nucleus in the chick embryo was studied using autoradiographic, cell staining, fiber staining, and axonal transport techniques. It was found that this nucleus arises very early in neurogenesis, with the first cells produced at 48 hours of incubation (stage 12), peak cell production at 50--56 hours (stage 15), and neuroblast proliferation completed by 72 hours (stage 18). As has been described in mammalian embryos, the primordial trigeminal cells move from the ventricular layer to accumulate as part of the common medial column, and later migrate in a ventrolateral direction to form the definitive lateral motor nucleus. The first identifiable component of the trigeminal system is the semilunar ganglion, which flanks the neural tube at stage 12, and sends afferents into the metencephalon by stage 13. By stage 12-13, the medial column cells are first apparent, and at stage 14, a few of these medial column cells have moved to begin formation of a lateral nucleus. At this time, a thin motor root can be seen exiting the brainstem. During subsequent stages, migratory traffic from medial to lateral regions increases, with cells frequently moving in association with fiber processes in the marginal zone. These fibers are presumed to emanate from secondary sensory, reticular, and medial column neuroblasts. By day 5, the medial column is greatly depleted and by day 6--7, the definitive lateral motor nucleus is formed. Beginning at 5 days, the dorsal motor nucleus can be detected, with cells from the lateral nucleus appearing to stream in a dorsomedial direction for its formation. Injections of horseradish peroxidase (HRP) into the mandibular process of the first visceral arch resulted in retrograde labeling of lateral nucleus cells as early as 3.5 days of incubation. In addition, migrating cells, intermediate between medial column and lateral nucleus, were similarly labeled. These observations indicate that processes of the lateral nucleus cells and those of migrating cells are well into their peripheral field at this age, but we cannot conclude that neuromuscular affiliations have been established, due to the possibility of HRP diffusion and growth cone uptake.

An aim of this study was to define precisely the period in the developmental history of primary sensory neurons during which nerve growth factor (NGF) exerts its growth-promoting effect. The mouse trigeminal ganglion and its peripheral projection were studied at closely staged intervals throughout development using light and electron microscopy, and the influence of NGF and anti-NGF antiserum on neurite outgrowth from ganglion explants was investigated at corresponding stages in culture. By embryonic day 9.5 (E9.5) peripheral fibers were first visible and increased in number until E13. Throughout this period in vitro neurites grew in the presence of anti-NGF. Peripheral fibers initially contacted the epithelium of the mandibular process by E10.5 and the maxillary process by E11. This coincided with the stage in vitro during which the magnitude of neurite outgrowth was significantly increased by NGF. The development of this response was independent of target encounter since it occurred in neurons which had not contacted their targets prior to explantation. There was an approximate one-to-one relationship between the number of neurons in the ganglion and peripheral fibers throughout development. A peak of some 44,400 fibers and 42,600 neurons was reached by E13 and fell to 20,800 and 19,000, respectively, by birth. Neurite outgrowth was elicited by NGF throughout the period of neuronal death. It is argued that the time course of the influence of NGF is consistent with a role as a selective maintenance factor but not as an agent directing initial outgrowth.

The trigeminal placode is a thickened region of ectodermal epithelium located along the side of the embryonic head. Mesenchyme escapes from the placode to form neurons of the trigeminal (V) ganglion. To further our knowledge of the morphogenesis of this escape, plastic thick sections were cut from mouse embryos and stained for light microscopy by using a technique which revealed escaping mesenchyme. The escape of trigeminal mesenchyme began at approximately 12 somites of age and was substantially complete by 30 somites. These results provided spatial/temporal orientation for a subsequent electron microscopic study. The first ultrastructural manifestation of escape was the penetration of an otherwise continuous basal lamina by small cell processes. The presence of longitudinally oriented microtubules within these processes suggests that mesenchymal cells escape through the basal lamina by using microtubules to direct/move their contents (e.g., the cell nucleus) into an enlarging process. Nuclei were distorted as they passed into these processes. This distortion suggests that basal lamina, together with a possible contribution from basal microfilaments, forms a rigid obstruction which is disrupted in the region from which a process is formed. In some cases a collar of basal lamina was observed around the necks of processes, but their distal membranes were invariably lamina-free. This lamina-free membrane is possibly that which is newly formed to accommodate the growing process. In later stages of escape, instances were observed in which the lamina was completely absent beneath an escaping cell and partially degraded beneath adjacent cells as well. These instances suggest that enzymatic digestion may play a role in degrading the lamina during mesenchymal escape. Apical desmosomes were often retained beyond the initial stages of escape. Mechanisms involved in their disruption are thus not among those which initiate escape.

In the chick embryo the trigeminal (V) sensory ganglion cells send axons into the metencephalon a few hours before the V motoneurons migrate from the midline to form a lateral nucleus adjacent to the ingrowing sensory axons. This relationship suggests that the ganglion axons may influence the initiation and direction of V motoneuron migration. In the present experiment the development of the ganglion axons was retarded by removing the neural crest anlage of the V ganglion. Subsequently, V ganglion cells which were derived from the ectodermal placode anlage sent axons into the metencephalon up to 2 days later than normal. The lateral migration of the V motoneurons was similarly delayed, commencing only after the central axons from the placodal ganglia penetrated the metencephalon. This study demonstrates that the presence of V ganglion perikarya alone is not sufficient to guide the appropriate migration of V motoneurons. This migration occurs only after the axons from the V sensory ganglion cells have penetrated the brainstem.

The directed growth of axons to their peripheral targets during development may be influenced by a variety of intrinsic and environmental factors, the latter including the pattern of extracellular matrix components in the region through which they grow. We investigated the localization within the chick trigeminal mesenchyme of a variety of extracellular matrix molecules (laminin, heparan sulfate proteoglycan, entactin, collagen type IV) by using indirect immunofluorescence techniques. The trigeminal mesenchyme contained several of these molecules, but only laminin was specifically localized to trigeminal axon pathways. Double immunofluorescence localization of affinity-purified laminin antibodies and monoclonal antibodies directed against a neuron-specific beta-tubulin (to visualize growing axons and postmitotic V ganglion neurons) demonstrated that the V motor and sensory peripheral nerves confine their growth to two patches of laminin-rich mesenchyme--a ganglionic laminin patch colocalized with V ganglion neurons and their axons, and a mandibular laminin patch colocalized with the V motor axons. Thus, laminin pathways in the mesenchyme may help guide V axons along their appropriate routes in the periphery. Double immunofluorescence localization of these laminin antibodies and monoclonal antibodies that recognize neural crest cells (to visualize precursors of V ganglion neurons and glia) demonstrated that the majority of cells within the ganglionic laminin patch were neural crest and differentiating neurons derived from the trigeminal epidermal placode. Since these cells often were laminin-immunopositive, they might be a source of the ganglionic laminin matrix. The mandibular laminin patch contains nearly no neural crest cells, and probably contains the somitomeric precursors to the mandibular muscle mass. These results suggest that laminin, a matrix molecule implicated in the guidance of trigeminal peripheral axons, might be produced in localized patches by peripheral nervous system components and by muscle targets.

Detailed reconstructions of lengths of the embryonic mouse maxillary nerve were made from serial light and electron microscope sections to determine whether there is any correspondence between the arrangement of fasciculi in the developing nerve and the emerging pattern of whisker follicles on the snout. There was neither correspondence nor obvious pattern in the arrangement of fasciculi at either E11, when trigeminal nerve fibers first contact the presumptive whisker pad, or at E12, when the pattern of developing whisker follicles becomes apparent. Fasciculi merged and branched to form an intricate plexus. Furthermore, the ratio of the number of nerve fibers in one fasciculus to the number in another prior to their merger differed significantly from the ratio of fiber numbers in the two fasciculi after their separation, which indicates that nerve fibers are freely exchanged between fasciculi. Our findings suggest that the somatotopic representation of the whisker follicle pattern in the brainstem does not develop by nerve fiber growth augmenting an initially ordered pattern of fasciculi.