Fetal facial nerve course in the ear region revisited.

PURPOSE: The aim of this study was to re-examine the structures that determine course of the facial nerve (FN) in the fetal ear region. MATERIALS AND METHODS: We used sagittal or horizontal sections of 28 human fetuses at 7-8, 12-16, and 25-37 weeks. RESULTS: The FN and the chorda tympani nerve ran almost parallel until 7 weeks. The greater petrosal nerve (GPN) ran vertical to the distal FN course due to the trigeminal nerve ganglion being medial to the geniculate ganglion at 7 weeks. Afterwards, due to the radical growth of the former ganglion, the GPN became an anterior continuation of the FN. The lesser petrosal nerve ran straight, parallel to the FN at 7 weeks, but later, it started to wind along the otic capsule, possibly due to the upward invasion of the tympanic cavity epithelium. Notably, the chorda tympanic nerve origin from the FN, and the crossing between the vagus nerve branch and the FN, was located outside of the temporal bone even at 37 weeks. The second knee of the FN was not evident, in contrast to the acute anterior turn below the chorda tympanic nerve origin. In all examined fetuses, the apex of the cochlea did not face the middle cranial fossa, but the tympanic cavity. CONCLUSION: Topographical relation among the FN and related nerves in the ear region seemed not to be established in the fetal age but after birth depending on growth of the cranial fossa.

Pharyngeal arch deficiencies affect taste bud development in the circumvallate papilla with aberrant glossopharyngeal nerve formation.

BACKGROUND: The pharyngeal arches (PAs) generate cranial organs including the tongue. The taste placodes, formed in particular locations on the embryonic tongue surface, differentiate into taste buds harbored in distinct gustatory papillae. The developing tongue also has a complex supply of cranial nerves through each PA. However, the relationship between the PAs and taste bud development is not fully understood. RESULTS: Ripply3 homozygous mutant mice, which have impaired third/fourth PAs, display a hypoplastic circumvallate papilla and lack taste buds, although the taste placode is normally formed. Formation of the glossopharyngeal ganglia is defective and innervation toward the posterior tongue is completely missing in Ripply3 mutant embryos at E12.5. Moreover, the distribution of neuroblasts derived from the epibranchial placode is severely, but not completely, atenuated, and the neural crest cells are diminished in the third PA region of Ripply3 mutant embryos at E9.5-E10.5. In Tbx1 homozygous mutant embryos, which exhibit another type of deficiency in PA development, the hypoplastic circumvallate papilla is observed along with abnormal formation of the glossopharyngeal ganglia and severely impaired innervation. CONCLUSIONS: PA deficiencies affect multiple aspects of taste bud development, including formation of the cranial ganglia and innervation to the posterior tongue.

Functional development of the vagal and glossopharyngeal nerve-related nuclei in the embryonic rat brainstem: optical mapping with a voltage-sensitive dye.

We investigated functional organization of the vagus nerve (N. X)- and glossopharyngeal nerve (N. IX)-related nuclei in the embryonic rat brainstem and compared their development and spatial distribution patterns, using multiple-site optical recording with a fast voltage-sensitive dye, NK2761. Intact brainstem preparations with N. X and N. IX attached were dissected from E13-E16 rat embryos, and electrical responses evoked by N. X/N. IX stimulation were optically recorded from many loci of the stained preparations. We analyzed optical waveforms and separated fast and slow optical signals corresponding to the antidromic/orthodromic action potentials and the excitatory postsynaptic potentials (EPSPs), respectively. We constructed contour line maps of signal amplitudes and identified motor and sensory nuclei of N. X and N. IX. In the N. X-related motor nucleus (the dorsal motor nucleus of the vagus nerve: DMNV), the fast signals were distributed in multiple-peak patterns, suggesting that the neurons and/or their activity are not distributed uniformly within the motor nuclei at early developmental stages. In the sensory nucleus (the nucleus of the tractus solitarius: NTS), the EPSPs were first detected from E15 in normal physiological solution for both N. X and N. IX. The N. IX-related NTS partially overlapped with the N. X-related NTS, but the peak locations were different between these two nerves. The results obtained in this study suggest that functional organization of the N. X- and N. IX-related nuclei changes dynamically with development in the embryonic rat brainstem.

The formation of the superior and jugular ganglia: insights into the generation of sensory neurons by the neural crest.

The superior and jugular ganglia (S/JG) are the proximal ganglia of the IXth and Xth cranial nerves and the sensory neurons of these ganglia are neural crest derived. However, it has been unclear the extent to which their differentiation resembles that of the Dorsal Root Ganglia (DRGs). In the DRGs, neural crest cells undergo neuronal differentiation just after the onset of migration and there is evidence suggesting that these cells are pre-specified towards a sensory fate. We have analysed sensory neuronal differentiation in the S/JG. We show, in keeping with previous studies, that neuronal differentiation initiates long after the cessation of neural crest migration. We also find no evidence for the existence of migratory neural crest cells pre-specified towards a sensory phenotype prior to ganglion formation. Rather our results suggest that sensory neuronal differentiation in the S/JG is the result of localised spatiotemporal cues.

Embryogenetic expression of glossopharyngeal and vagal excitability in the chick brainstem as revealed by voltage-sensitive dye recording.

Using voltage-sensitive dye recording, we traced the ontogenetic expression of neural excitability related to the glossopharyngeal nerve (N. IX) and the vagus nerve (N. X) in the embryonic chick brainstem. At the 3.5-day embryonic stage, by averaging optical signals, we succeeded in recording very small action potential-related optical responses (DeltaI/I<10(-4)) induced by N. IX stimulation in the nucleus of the glossopharyngeal nerve (motor nucleus). This suggests that glossopharyngeal excitability in the motor nucleus is first generated no later than this developmental stage. On the other hand, action potential-related optical responses induced by N. X stimulation were first detected at the 4-day embryonic stage. Comparison with morphogenesis indicated that glossopharyngeal and vagal motoneurons acquire their excitability and send their axons to the periphery soon after they leave the proliferative pool.

Development of the specialized AMPA receptors of auditory neurons.

At maturity, the AMPA receptors of auditory neurons exhibit very rapid desensitization kinetics and high permeability to calcium, reflecting the predominance of GluR3 flop and GluR4 flop subunits and the paucity of GluR2. We used mRNA analysis and immunoblotting to contrast the development of AMPA receptor structure in the chick cochlear nucleus [nucleus magnocellularis (NM)] with that of the slowly desensitizing and calcium-impermeable AMPA receptors of brainstem motor neurons in the nucleus of the glossopharyngeal/vagal nerves. The relative abundance of transcripts for GluRs 1-4 changes substantially in auditory (but not motor) neurons after embryonic day (E)10, with large decreases in GluR2 and increases in GluR3 and GluR4. Relative to the motor neurons, NM neurons show a higher abundance of flop isoforms of GluRs 2-4 at E10, suggesting that auditory neurons are already biased toward expression of flop isoforms before the onset of synaptic function at E11. Immunoreactivities in NM show very distinct developmental patterns from E13 onward: GluR2 declines by >90%, GluR3 increases threefold, and GluR4 remains relatively constant. Our results show that there are a series of critical points during normal development, most occurring after the onset of function, when rapid changes in receptor structure (occurring via both transcriptional and post-transcriptional control mechanisms) produce the specialized AMPA receptor functions that enable auditory neurons to accurately encode acoustic information.

Developmental organization of the glossopharyngeal nucleus in the embryonic chick brainstem slice as revealed by optical sectioning recording.

We traced developmental changes in the ventro-dorsal distribution pattern of glossopharyngeal nerve (N. IX) responses by applying an optical sectioning method to thick slice preparations dissected from E4 to E8 chick embryos. We identified the motor and sensory nuclei related to the glossopharyngeal nerve in the rostral and caudal focal planes, respectively. In the E4 and E5 preparations, the motoneuronal responses appeared on the central part of the stimulated side of the brainstem. As development proceeded to E6, the response area became localized on the dorsal region. The change in the ventro-dorsal distribution pattern was similar to that observed in the vagus nerve-related nuclei, suggesting that there might be an essential process underlying functional organization of the brainstem nuclei.

Optical approaches to functional organization of glossopharyngeal and vagal motor nuclei in the embryonic chick hindbrain.

We investigated the functional organization of the glossopharyngeal and vagal motor nuclei during embryogenesis using multiple-site optical recording with a fast voltage-sensitive dye. Intact brain stem preparations with glossopharyngeal and vagus nerves were dissected from 4- to 8-day-old chick embryos. Electrical responses evoked by glossopharyngeal/vagus nerve stimulation were optically recorded from many loci of the stained preparations. In 4- to 6-day-old preparations, action potential-related fast spikelike signals were detected from the nucleus of the glossopharyngeal nerve and the dorsal motor nucleus of the vagus nerve. Contour line maps of the signal amplitude showed multiple-peak patterns, suggesting that the neurons and/or their activity were not uniformly distributed within the nuclei at early developmental stages. As development proceeded from 4 to 6 days, the peaks fused with each other and the number of peaks decreased gradually. In most 7- and 8-day-old preparations, only a single peak was identified in the nuclei, and the distribution of the signal amplitude formed a layered pattern surrounding the peak-signal area. These results suggest that functional organization of the motor nuclei in the embryonic hindbrain changes dynamically with development, resulting in a rearrangement of functional nuclear cores from multiple-peaks to a single peak.

Disruption of semaphorin III/D gene causes severe abnormality in peripheral nerve projection.

The molecules of the collapsin/semaphorin gene family have been thought to play an essential role in axon guidance during development. Semaphorin III/D is a member of this family, has been shown to repel dorsal root ganglion (DRG) axons in vitro, and has been implicated in the patterning of sensory afferents in the spinal cord. Although semaphorin III/D mRNA is expressed in a wide variety of neural and nonneural tissues in vivo, the role played by semaphorin III/D in regions other than the spinal cord is not known. Here, we show that mice homozygous for a targeted mutation in semaphorin III/D show severe abnormality in peripheral nerve projection. This abnormality is seen in the trigeminal, facial, vagus, accessory, and glossopharyngeal nerves but not in the oculomotor nerve. These results suggest that semaphorin III/D functions as a selective repellent in vivo.

Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization.

The COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. Multiple COUP-TF members have been cloned and they share a high degree of sequence homology between species as divergent as Drosophila and humans, suggesting a conservation of function through evolution. The COUP-TFs are highly expressed in the developing nervous systems of several species examined, indicating their possible involvement in neuronal development and differentiation. In the mouse, there are two very homologous COUP-TF genes (I and II) and their expression patterns overlap extensively. To study the physiological function of mCOUP-TFI, a gene-targeting approach was undertaken. We report here that mCOUP-TFI null animals die perinataly. Mutant embryos display an altered morphogenesis of the ninth cranial ganglion and nerve. The aberrant formation of the ninth ganglion is most possibly attributable to extra cell death in the neuronal precursor cell population. In addition, at midgestation, aberrant nerve projection and arborization were oberved in several other regions of mutant embryos. These results indicate that mCOUP-TFI is required for proper fetal development and is essential for postnatal development. Furthermore, mCOUP-TFI possesses vital physiological functions that are distinct from mCOUP-TFII despite of their high degree of homology and extensive overlapping expression patterns.

Segmental and neuronal architecture of the hindbrain of Krox-20 mouse mutants.

The vertebrate hindbrain is transiently segmented during its early development with the formation of reiterated bulges, the rhombomeres (r). The Krox-20 gene, which encodes a zinc finger transcription factor, has been shown previously to be implicated in the maintenance of r3 and r5 (Schneider-Maunoury, S., Topilko, P., Seitanidou, T., Levi, G., Cohen-Tannoudji, M., Pournin, S., Babinet, C. and Charnay, P. (1993) Cell 75, 1199-1214; Swiatek, P. J. and Gridley, T. (1993) Genes Dev. 7, 2071-2084. However, it was not clear from these analyses how extensive the deletion of r3 and r5 was and whether the overall segmentation and internal architecture of the hindbrain was affected. We have now reinvestigated these issues by analysis of rhombomere boundaries, using both morphological and molecular markers, and of the fate of specific motor neuron populations, using retrograde and anterograde carbocyanine dye tracing. We conclude that r3 and r5 and their derivatives are completely eliminated in Krox-20(-/-) embryos while overall hindbrain segmentation is maintained. In addition, we show that the disappearance of these territories has important consequences for even-numbered rhombomeres as well, in particular on axonal navigation: (i) a population of r6 motoneurons, presumably normally fated to join the glossopharyngeal nerve, has its axons misrouted toward the facial exit point in r4; (ii) the trigeminal motor axons are also misrouted, presumably because of the proximity of the trigeminal and facial exit points. They fasciculate with facial axons outside the neural tube and enter the second branchial arch instead of the first arch. This navigational error could explain the disappearance, at around 17.5 dpc, of the trigeminal motor nucleus in Krox-20(-/-) embryos by inadequate supply of essential, possibly arch-specific survival factors.

Initial innervation of embryonic rat tongue and developing taste papillae: nerves follow distinctive and spatially restricted pathways.

The rat tongue has an extensive, complex innervation from four cranial nerves. However, the precise developmental time course and spatial routes of these nerves into the embryonic tongue are not known, although this knowledge is crucial for studying mechanisms that regulate development and innervation of the lingual taste organs, gustatory papillae and resident taste buds. We determined the initial spatial course of nerves in the developing tongue and papillae, and tested the hypothesis that sensory nerves first innervate the tongue homogeneously and then retract to more densely innervate papillae and taste buds. Antibodies to GAP-43 and neurofilaments were used to label nerve fibers in rat embryo heads from gestational day 11 through 16 (E11-E16). Serial sagittal sections were traced and reconstructed to follow paths of each nerve. In E11 rat, geniculate, trigeminal and petrosal ganglia were labeled and fibers left the ganglia and extended toward respective branchial arches. At E13 when the developing tongue is still a set of tissue swellings, the combined chorda/lingual, hypoglossal and petrosal nerves approached the lingual swellings from separate positions. Only the chorda/lingual entered the tongue base at this stage. At E14 and E15, the well-developed tongue was innervated by all four cranial nerves. However, the nerves maintained distinctive entry points and relatively restricted mesenchymal territories within the tongue, and did not follow one another in common early pathways. Furthermore, the chorda/lingual and glossopharyngeal nerves did not set up an obvious prepattern for gustatory papilla development, but rather seemed attracted to developing papillae which became very densely innervated compared to surrounding epithelium at E15. To effect this dense papilla innervation, sensory nerves did not first innervate the tongue in a homogeneous manner with subsequent retraction and/or extensive redirection of fibers into the taste organs. Results contribute to a set of working principles for development of tongue innervation. Points of entry and initial neural pathways are restricted from time of tongue formation through morphogenesis, suggesting distinctive lingual territories for each nerve. Thus, sensory and motor nerves distribute independently of each other, and sensory innervation to anterior and posterior tongue remains discrete. For taste organ innervation, gustatory papillae are not induced by a prepatterned nerve distribution. In fact, papillae might attract dense sensory innervation because neither chorda/lingual nor glossopharyngeal nerve grows homogeneously to the lingual epithelium and then redistributes to individual papillae.

Hox group 3 paralogous genes act synergistically in the formation of somitic and neural crest-derived structures.

Hox genes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multiple Hox genes is beginning to reveal a complex network of interactions among these closely related genes which is responsible for directing the formation of spatially restricted tissues and structures. In this report we present an analysis of the genetic interactions between all members of the third paralogous group, Hoxa3, Hoxb3, and Hoxd3. Previous analysis has shown that although mice homozygous for loss-of-function mutations in either Hoxa3 or Hoxd3 have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of the Hoxb3 gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant for Hoxa3 and Hoxd3 or Hoxb3 and Hoxd3 show an indistinguishable defect, loss of the entire atlas. This suggests that the identity of the specific Hox genes that are functional in a given region may not be as critical as the total number of Hox genes operating in that region.

Cranial nerves and ganglia are altered after in vitro treatment of mouse embryos with valproic acid (VPA) and 4-en-VPA.

Prenatal valproic acid (VPA) exposure results in neural tube defects and in the fetal valproate syndrome (FVS), associated with developmental delay. In the present study we investigate the alterations induced by VPA and one of its metabolite, 4-en-VPA, on specific neural structures: branchial nerves and ganglia. This study was performed on 8-9 pairs of somites mouse embryos exposed in vitro for 24 h to 0.75 mM of VPA or 1 mM of 4-en-VPA. After an additional culture period of 20 h without drug, the embryos were processed for whole mount immunostaining using the monoclonal antibody 2H3, directed against the 155 kDa neurofilament protein. This technique makes it possible to visualise the branchial nerves/ganglia. VPA and 4-en-VPA induced a delay in the development of the trigeminal (V), glossopharyngeal (IX) and vagus (X) nerves/ganglia. The development of the facial (VII) nerve was delayed to a lesser extend. These treatments also induced defects in the four ganglia. The main abnormalities were a reduced dorsal component of ganglion V, the absence of the dorsal root of ganglion IX, a disorganised dorsal part of ganglion X and diffuse ventral fibres in nerves VII-VIII. In addition, scattered fibres were observed around and between ganglia. In conclusion, VPA and 4-en-VPA deeply altered the differentiation of branchial nerves/ganglia. The dorsal part of the ganglia, arising from the rhombencephalic neural crest, was particularly sensitive. The disorganisation of fibres could possibly be explained by alteration of the extracellular matrix.

Defects in the development of branchial nerves and ganglia induced by in vitro exposure of mouse embryos to mercuric chloride.

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.

Depolarizing stimuli induce high levels of dopamine synthesis in fetal rat sensory neurons.

To investigate the role of activity-dependent mechanisms in sensory transmitter development, we examined the effect of depolarizing stimuli on tyrosine hydroxylase expression and dopamine synthesis in cells of the fetal rat petrosal ganglion, a model of catecholaminergic sensory neurons. Although dopaminergic traits are normally detectable in only 10-20% of ganglion neurones, exposure to depolarizing concentrations of potassium chloride (40 mM) or veratridine (10 microM) in culture induced tyrosine hydroxylase expression in 100% of petrosal neurons and a 10-fold increase in dopamine content. Tyrosine hydroxylase expression remained elevated in a subset of neurons following return to control conditions, suggesting that chronic depolarization elicits a phenotypic switch in some cells. These data show for the first time that transmitter expression in developing sensory neurons can be regulated by activity-related cues.

Responses to glossopharyngeal stimulus in the early embryonic chick brainstem: spatiotemporal patterns in three dimensions from repeated multiple-site optical recording of electrical activity.

In an effort to assess the spatial patterning of glossopharyngeal responses in the early embryonic chick brainstem, we used a multiple-site optical recording system with a 12 x 12 element photodiode array and a voltage-sensitive merocyanine-rhodanine dye (NK2761) to monitor neural transmembrane voltage activities. Seven and 8 d old embryonic chick brainstems were sliced into 1400-1600 microns thick sections with the glossopharyngeal and vagal nerves attached, and then stained with the dye. Neural voltage-related optical signals were evoked by a positive brief (depolarizing) square current pulse applied to the glossopharyngeal nerve with a microsuction electrode, and then recorded simultaneously from many loci in the objective two-dimensional image plane of a compound microscope. In addition to the multiple-site optical recording technique, we tried to introduce an optical sectioning method by changing the focal plane of the microscope to obtain three-dimensional information. Thus, we have been able to assess semiquantitatively the three-dimensional profiles of two glossopharyngeal response areas corresponding to the nucleus of the glossopharyngeal nerve (nucleus nervi glossopharyngei) and the nucleus of the tractus solitarius. Furthermore, glutaminergic excitatory postsynaptic potentials were determined within the response area corresponding to the nucleus of the tractus solitarius. In addition, we also compared the glossopharyngeal and vagal response areas and found that the cores of the related nuclei are separated in three dimensions.

[Early development and cell differentiation of the parasympathetic vagus-glossopharyngeal nucleus in cattle. Light and electron microscopic studies]. / Frühentwicklung und Zelldifferenzierung des parasympathischen Vagus-Glossopharyngeus-Kernes beim Rind. Licht- und elektronenmikroskopische Untersuchungen.

The early development, differentiation of the cell and cell migration of the nucleus parasympathicus nervi vagi et glossopharyngei were examined by light microscope in 32 bovine embryos with crown-rump-lengths (CRL) ranging from 1 cm to 53 cm. During this period the nucleus is being enlarged 6 to 7 times and the size of the cell increases to 35-40 microns. The ultrastructure during the differentiation of the cell is shown electron microscopically in embryos with CRL of 2.5 cm and 3.6 cm. Several layers of matrix cells arise from the neuroepithelium of the neural tube by mitosis. They migrate in the shape of dark nucleated cells into the parasympathetic cell column. With advancing age of the embryos the number of cells with light nucleus increases. They represent the presumptive neurons. In embryos of 2.5 cm CRL their cytoplasm surrounds the nucleus on three sides in the shape of a narrow rim while on the fourth side it is enlarged into an outgrowing process. In this process a smaller number of organelles and their preliminary stages appears. Their number is significantly increased in embryos of 3.6 cm CRL and they can be seen throughout the growing process. In the following stages of maturity cytological development proceeds. In embryos of 53 cm CRL topographical and cytological data are comparable to those in adult animals.

Development of cranial nerves in the chick embryo with special reference to the alterations of cardiac branches after ablation of the cardiac neural crest.

Development of cranial nerve branches in the cardiac region was observed in whole-mount specimens which were stained with a monoclonal antibody, E/C8, after the ablation of the cardiac neural crest. In early embryos, nerve trunks of IX and X were lacking or only poorly developed, while the early development of pharyngeal branch primordia was normal. In day 5 embryos, the nerve trunks of IX-X were present in all the embryos, however; extensive communication was observed between X and XII. On day 6 and later, the spiral pattern of superior cardiac branches was disturbed, as were the blood vessels. Furthermore, the distal branches of XII passed within the superficial layer of cardiac outflow mesenchyme. Vagal branches passed within the deeper layer. There was no apparent change in the development of the sinal branch. Using quail--chick chimeras, it was found that the cardiac neural crest cells formed the Schwann cells of XII, and that they were also associated with the hypobranchial muscle primordium, suggesting that the absence of the cardiac neural crest not only disturbs the development of the cardiac outflow septation, but also affects the normal morphogenesis of the hypobranchial musculature and its innervation. Embryologically, the tongue is located close to the cardiac outflow tract, which is the migration pathway of the cardiac neural crest-derived cells.

Vagal communicating branches between the facial and glossopharyngeal nerves, with references to their occurrence from the embryological point of view.

A rare and hitherto not reported case in which a branch of the vagal nerve communicated simultaneously with the facial and the glossopharyngeal nerves was encountered in the body of a Japanese male cadaver in an anatomy class. This vagofacial-vagoglossopharyngeal (X.VII-X.IX) communicating branch was found to issue from the vagal nerve truck in close association with the pharyngeal branches (rami pharyngei nervi vagi), bifurcating soon into a vagofacial (X.VII) and a vagoglossopharyngeal division (X.IX). The X.VII division coursed forward and reached the posterior belly of the digastric muscle; after entering this muscle, this division broke up into filaments to communicate with the ramus digastricus of the facial nerve which was found to play an equivalent role in making the vagofacial ansa. The X.IX division, in contrast, took its course medially to reach the stylopharyngeal muscle. After entering this muscle, the X.IX division communicated with the stylopharyngeal branch of the glossopharyngeal nerve, which was found to be the equivalent to the X.IX division; these two form together the vagoglossopharyngeal ansa. Therefore, it could be concluded that the X.VII-X.IX communicating branch constitutes the vagal moieties of the vagofacial as well as the vagoglossopharyngeal ansae. The background of the appearance of the communicating branches observed in this report is discussed in the text from the developmental viewpoint on the basis of the findings obtained in chick embryos stained in whole mounts with anti-neurofilament protein antibody.