The unique axon trajectory of the accessory nerve is determined by intrinsic properties of the neural tube in the avian embryo.

The accessory nerve is a cranial nerve, composed of only motor axons, which control neck muscles. Its axons ascend many segments along the lateral surface of the cervical spinal cord and hindbrain. At the level of the first somite, they pass ventrally through the somitic mesoderm into the periphery. The factors governing the unique root trajectory are unknown. Ablation experiments at the accessory nerve outlet points have shown that somites do not regulate the trajectory of the accessory nerve fibres. Factors from the neural tube that may control the longitudinal pathfinding of the accessory nerve fibres were tested by heterotopic transplantations of an occipital neural tube to the cervical and thoracic level. These transplantations resulted in a typical accessory nerve trajectory in the cervical and thoracic spinal cord. In contrast, cervical neural tube grafts were unable to give rise to the typical accessory nerve root pattern when transplanted to occipital level. Our results show that the formation of the unique axon root pattern of the accessory nerve is an intrinsic property of the neural tube.

Occipital somites guide motor axons of the accessory nerve in the avian embryo.

The accessory nerve (nervus accessorius) displays a unique organization in that its axons ascend along the rostrocaudal axis after exiting the cervical spinal cord and medulla oblongata and thereafter project ventrally into the periphery at the first somite level. Little is known about how this organization is achieved. We have investigated the role of somites in the guidance of motor axons of the accessory nerve using heterotopic transplantations of somites in avian embryos. The formation of not only accessory nerve but also the vagal nerve was affected, when a more caudal occipital somite (somites 2-4) was grafted to the position of the first occipital somite. Our study reveals that only the first occipital somite permits the development of ventral projection of accessory axons, a process that is inhibited by more caudal occipital somites.

Surgical challenges associated with the morphology of the spinal accessory nerve in the posterior cervical triangle: functional or structural?

OBJECT: The spinal accessory nerve (SAN) has been reported to have a distinctly coiled appearance in its course through the posterior cervical triangle of the neck. As this is unusual compared with other peripheral nerves including the cranial nerves, the present histological analysis was performed to further elucidate the reason for this anatomy with potential application in nerve injury and repair. METHODS: Ten adult cadavers underwent dissection of the neck. The SAN was harvested proximally and within the posterior cervical triangle. For comparison with other cranial nerves within the neck, the cervical vagus and hypoglossal nerves were also harvested. All nerves underwent histological analysis. Additionally, 2 human fetuses (11 and 20 weeks' gestation) underwent examination of the SAN in the posterior cervical triangle, and 3 randomly selected specimens were submitted for electromicroscopy. RESULTS: All SANs were found to have a straight gross configuration proximal to the posterior triangle and a coiled appearance within this geometrical area. Histologically, no differences were identified for the SAN in these 2 locations (that is, proximal to and within the posterior cervical triangle). The histology of the SAN both with routine analysis and electron microscopy was similar in both regions and to nerves used as controls (for example, vagus and hypoglossal nerves). Interestingly, both fetal specimens were found to have coiled SANs in the posterior cervical triangle. CONCLUSIONS: Based on this study, it appears that the tortuous course of the SAN in the posterior triangle arises from functional as opposed to structural forces. It is hoped that this analysis will provide some insight into the nature behind the morphology observed in the SAN within the posterior cervical triangle and aid in future investigations regarding its injury. Moreover, such a coiled nature of this nerve may assist the neurosurgeon in identifying it during, for example, neurotization procedures.

UNC5C is required for spinal accessory motor neuron development.

In both invertebrates and vertebrates, UNC5 receptors facilitate chemorepulsion away from a Netrin source. Unlike most motor neurons in the embryonic vertebrate spinal cord, spinal accessory motor neuron (SACMN) cell bodies and their axons translocate along a dorsally directed trajectory away from the floor plate/ventral midline and toward the lateral exit point (LEP). We have recently shown that Netrin-1 and DCC are required for the migration of SACMN cell bodies, in vivo. These observations raised the possibility that vertebrate UNC5 proteins mediate the presumed repulsion of SACMN away from the Netrin-rich ventral midline. Here, we show that SACMN are likely to express UNC5A and UNC5C. Whereas SACMN development proceeds normally in UNC5A null mice, many SACMN cell bodies fail to migrate away from the ventral midline and inappropriately cluster in the ventrolateral spinal cord of mouse embryos lacking UNC5C. These results support an important role for UNC5C in SACMN development.

Forced expression of Phox2 homeodomain transcription factors induces a branchio-visceromotor axonal phenotype.

What causes motor neurons to project into the periphery is not well understood. We here show that forced expression of the homeodomain protein Phox2b, shown previously to be necessary and sufficient for branchio-visceromotor neuron development, and of its paralogue Phox2a imposes a branchiomotor-like axonal phenotype in the spinal cord. Many Phox2-transfected neurons, whose axons would normally stay within the confines of the neural tube, now project into the periphery. Once outside the neural tube, a fraction of the ectopic axons join the spinal accessory nerve, a branchiomotor nerve which, as shown here, does not develop in the absence of Phox2b. Explant studies show that the axons of Phox2-transfected neurons need attractive cues to leave the neural tube and that their outgrowth is promoted by tissues, to which branchio-visceromotor fibers normally grow. Hence, Phox2 expression is a key step in determining the peripheral axonal phenotype and thus the decision to stay within the neural tube or to project out of it.

Molecular control of spinal accessory motor neuron/axon development in the mouse spinal cord.

Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

Targeted disruption of the homeobox gene Nkx2.9 reveals a role in development of the spinal accessory nerve.

The homeodomain-containing transcription factor Nkx2.9 is expressed in the ventralmost neural progenitor domain of the neural tube together with the related protein Nkx2.2 during early mouse embryogenesis. Cells within this region give rise to V3 interneurons and visceral motoneurons in spinal cord and hindbrain, respectively. To investigate the role of the Nkx2.9 gene, we generated a mutant mouse by targeted gene disruption. Homozygous mutant animals lacking Nkx2.9 were viable and fertile with no apparent morphological or behavioral phenotype. The distribution of neuronal progenitor cells and differentiated neurons in spinal cord was unaffected in Nkx2.9-deficient animals. This finding is in contrast to Nkx2.2-null mutants, which have been shown to exhibit ventral to dorsal transformation of neuronal cell fates in spinal cord. Our results suggest that specification of V3 interneurons in the posterior CNS does not require Nkx2.9, most probably because of functional redundancy with the co-expressed Nkx2.2 protein. In hindbrain, however, absence of Nkx2.9 resulted in a significantly altered morphology of the spinal accessory nerve (XIth), which appeared considerably shorter and thinner than in wild-type animals. Consistent with this phenotype, immature branchial motoneurons of the spinal accessory nerve, which normally migrate from a ventromedial to a dorsolateral position within the neural tube, were markedly reduced in Nkx2.9-deficient embryos at E10.5, while ventromedial motor column cells were increased in numbers. In addition, the vagal and glossopharyngeal nerves appeared abnormal in approximately 50% of mutant embryos, which may be related to the observed reduction of Phox2b expression in the nucleus ambiguus of adult mutant mice. From these observations, we conclude that Nkx2.9 has a specific function in the hindbrain as determinant of the branchial motoneuron precursor cells for the spinal accessory nerve and possibly other nerves of the branchial-motor column. Like other Nkx genes expressed in the CNS, Nkx2.9 seems to be involved in converting positional information into cell fate decisions.

Identification and characterization of a cell surface marker for embryonic rat spinal accessory motor neurons.

The developing mammalian spinal cord contains distinct populations of motor neurons that can be distinguished by their cell body positions, by the expression of specific combinations of regulatory genes, and by the paths that their axons take to exit the central nervous system (CNS). Subclasses of spinal motor neurons are also thought to express specific cell surface proteins that function as receptors which control the guidance of their axons. We identified monoclonal antibody (mAb) SAC1 in a screen aimed at generating markers for specific subsets of neurons/axons in the developing rat spinal cord. During early embryogenesis, mAb SAC1 selectively labels a small subset of Isl1-positive motor neurons located exclusively within cervical segments of the spinal cord. Strikingly, these neurons extend mAb SAC1-positive axons along a dorsally directed trajectory toward the lateral exit points. Consistent with the finding that mAb SAC1 also labels spinal accessory nerves, these observations identify mAb SAC1 as a specific marker of spinal accessory motor neurons/axons. During later stages of embryogenesis, mAb SAC1 is transiently expressed on both dorsally and ventrally projecting spinal motor neurons/axons. Interestingly, mAb SAC1 also labels the notochord and floor plate during most stages of spinal cord development. The mAb SAC1 antigen is a 100-kD glycoprotein that is likely to be the rat homolog of SC1/BEN/DM-GRASP, a homophilic adhesion molecule that mediates axon outgrowth and fasciculation.

Fasciculus intramedullaris accessorius in the development of the human embryonic spinal accessory nerve.

In embryos at stage 16 (37 days) the intramedullary roots of the spinal accessory nucleus ascend in the marginal layer of the spinal cord within one segment. These fibers form the intramedullary accessory fascicle of the XIth nerve. This fasciculus was observed in further developmental stages of investigated embryos.

The spinal accessory nerve in human embryos at 6th week (stages 16 and 17).

Investigations were made on serial sections of human embryos at developmental stages 16 and 17 (37-41 days). The spinal nucleus of the accessory nerve presents well delimited cellular group in the dorsplateral part of the ventral horn of the spinal cord. The migration zone is still present. In embryos of stage 16 the spinal accessory nerve joints the fibers originating from the nucleus ambiguous. This takes place at the level of the inferior ganglion of the vagus nerve. In stage 17 the secondary rami of the accessory nerve develop. This is correlated with the differentiation of the sternocleidomastoid and trapezius muscles.

The development of the spinal accessory nerve in human embryos during 5th week (stages 14 and 15).

The spinal part of the accessory nerve was investigated in serially sectioned human embryos at developmental stages 14 and 15. It has been recognized that the spinal accessory nucleus extends through the upper 4 or 6 cervical segments of the spinal cord. The nucleus is formed by group of cells lying dorsolaterally to the primordium of the ventral horn. There is no continuation of the cells forming the spinal nucleus of the XIth nerve with primordium of the nucleus ambiguus. Extramedullary roots of the spinal accessory nucleus form a long trunk ascending into skull and uniting with vagus nerve. In one embryo at stage 15 the spinal accessory nerve separates at the level of the lower ganglion of the vagus.

The spinal nucleus of the accessory nerve in human embryos at stage 13.

In 8 serially sectioned human embryos the spinal accessory nucleus was traced. It was found that the spinal nucleus of the accessory nerve extends as continuation of the medial part of the nucleus ambiguus in the cervical segments of the spinal cord to the 5th segment. The nucleus forms small group of cells lying dorsolaterally in the ventral horns. Fibers from the nucleus leave the spinal cord close to the sulcus limitans.

Prenatal development of the human nucleus ambiguus during the embryonic and early fetal periods.

The ontogenetic development of the nucleus ambiguus was studied in a series of human embryos and fetuses ranging from 3 to 12.5 weeks of menstrual age (4 to 66 mm crown-rump length). They were prepared by Nissl and silver methods. Nucleus ambiguus neuroblasts, whose neurites extend towards and into the IXth and rostral Xth nerve roots, appear in the medial motor column of 4-6-week-old embryos (4.25-11 mm). These cells then migrate laterally (6.5 weeks, 14 mm) to a position near the dorsal motor nucleus of X. At 7 weeks (15 mm), nucleus ambiguus cells begin their migration, which progresses rostrocaudally, into their definitive ventrolateral position. The basic pattern of organization of the nucleus is established in its rostral region at 8 weeks (22.2-24 mm) and extends into its caudal region by 9 weeks (32 mm), when its nearly adult organization is evident. Cells having the characteristics of mature neurons first appear rostrally in the nucleus during the 8.5-9-week period (24.5-32 mm), gradually increase in number, and constitute the entire nucleus at 12.5 weeks (65.5 mm). Definitive neuronal subgroups first appear at 10 weeks (37.5 mm) in the large rostral nuclear region. These features suggest that the human nucleus ambiguus develops along a rostrocaudal temporospatial gradient. Evidence indicates that function of nucleus ambiguus neurons, manifested by fetal reflex swallowing, occurs after the cells migrate into their definitive position, establish the definitive nuclear pattern, and exhibit mature characteristics.

[Relations between the accessory nerve and the distal ganglion of the vagus nerve in sheep during the prenatal period]. / Das Verhältnis des Nervus accessorius zum Ganglion distale nervi vagi beim Schaf in der Pränatalperiode.

This contribution presents the results of morphological studies regarding the nervus accessorius and the ganglion distale nervi vagi of the sheep during the prenatal period. Special attention was paid to the roots of the brain and spinal medulla, which are implicated in the formation of the nervus accessorius, and the region of the spinal medulla in embryos at different stages of development. We investigated the relationship between the above nerve and the ganglion distale nervi vagi. The morphology of this ganglion was also investigated, and its external structure is described.

Development of the oculomotor nucleus, with special reference to the time of cell origin and cell death.

The developmental pattern of the oculomotor nucleus from day 7 of incubation through two weeks after hatching was studied in white Peking duck embryos. The neuroblasts comprising the nucleus complete their last phase of DNA synthesis on days 4 and 5 and the anlage first appears on day 7. The various subnuclei become identifiable as distinct cell groups on day 8 or 9. There is a cell migration between the ventral-most portions of the two ventromedial nuclei on days 9 through 11, and as a result a well-developed oculomotor commissure is established between these two subnuclei. The maximum number of cells in the nucleus is present on day 11. There is a normally occurring overall loss of approximately 43% of the cells during ontogenesis. Cell death appears to be random, without any gradient, and virtually all of it occurs between days 11 and 15. Although the duration of cell death is essentially similar in all subnuclei, great variations exist in its magnitude. For example, there is a cell loss of approximately 61% in the accessory nucleus, 38% in the dorsolateral nucleus, 40% in the dorsomedial nucleus and 33% in the ventromedial nucleus. Cell loss in the oculomotor nucleus is compared with that observed in the other two eye-muscle nuclei.