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Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the transcription factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2021. Published by Elsevier Inc.)

In the vertebrate central nervous system, groups of functionally related neurons, including cranial motor neurons of the brainstem, are frequently organised as nuclei. The molecular mechanisms governing the emergence of nuclear topography and circuit function are poorly understood. Here we investigate the role of cadherin-mediated adhesion in the development of zebrafish ocular motor (sub)nuclei. We find that developing ocular motor (sub)nuclei differentially express classical cadherins. Perturbing cadherin function in these neurons results in distinct defects in neuronal positioning, including scattering of dorsal cells and defective contralateral migration of ventral subnuclei. In addition, we show that cadherin-mediated interactions between adjacent subnuclei are critical for subnucleus position. We also find that disrupting cadherin adhesivity in dorsal oculomotor neurons impairs the larval optokinetic reflex, suggesting that neuronal clustering is important for co-ordinating circuit function. Our findings reveal that cadherins regulate distinct aspects of cranial motor neuron positioning and establish subnuclear topography and motor function., Competing Interests: AK, GD, GW, JC, SG No competing interests declared, (© 2020, Knüfer et al.)

A long tortuous course of the abducens nerve (ABN) crossing a highly curved siphon of the internal carotid artery is of interest to neurosurgeons for cavernous sinus surgery. Although a "straight" intracavernous carotid artery in fetuses can change into an adult-like siphon in infants, there is no information on when or how the unique course of ABN is established. Histological observations of 18 near-term fetuses (12 specimens of frontal sections and 6 specimens of sagittal sections) demonstrated the following: (I) the ABN consistently took a straight course crossing the lateral side of an almost straight intracavernous carotid artery; (II) the straight course was maintained when sympathetic nerves joined; (III) few parasellar veins of the developing cavernous sinus separated the ABN from the ophthalmic nerve; and (IV) immediately before the developing tendinous annulus for a common origin of extraocular recti, the ABN bent laterally to avoid a passage of the thick oculomotor nerve. Since the present observations strongly suggested morphologies at birth and in infants, major angulations of the ABN as well as the well-known course independent of the other nerves in the cavernous sinus seemed to be established during childhood. In the human body, the ABN might be a limited example showing a drastic postnatal change in course. Consequently, it might be important to know the unique course of ABN before performing endovascular interventions and skull base surgery for petroclival and cavernous sinus lesions without causing inadvertent neurovascular injuries to neonates or infants.

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 Isl MN -GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Purpose: To spatially and temporally define ocular motor nerve development in the presence and absence of extraocular muscles (EOMs)., Methods: Myf5cre mice, which in the homozygous state lack EOMs, were crossed to an IslMN:GFP reporter line to fluorescently label motor neuron cell bodies and axons. Embryonic day (E) 11.5 to E15.5 wild-type and Myf5cre/cre:IslMN:GFP whole mount embryos and dissected orbits were imaged by confocal microscopy to visualize the developing oculomotor, trochlear, and abducens nerves in the presence and absence of EOMs. E11.5 and E18.5 brainstems were serially sectioned and stained for Islet1 to determine the fate of ocular motor neurons., Results: At E11.5, all three ocular motor nerves in mutant embryos approached the orbit with a trajectory similar to that of wild-type. Subsequently, while wild-type nerves send terminal branches that contact target EOMs in a stereotypical pattern, the Myf5cre/cre ocular motor nerves failed to form terminal branches, regressed, and by E18.5 two-thirds of their corresponding motor neurons died. Comparisons between mutant and wild-type embryos revealed novel aspects of trochlear and oculomotor nerve development., Conclusions: We delineated mouse ocular motor nerve spatial and temporal development in unprecedented detail. Moreover, we found that EOMs are not necessary for initial outgrowth and guidance of ocular motor axons from the brainstem to the orbit but are required for their terminal branching and survival. These data suggest that intermediate targets in the mesenchyme provide cues necessary for appropriate targeting of ocular motor axons to the orbit, while EOM cues are responsible for terminal branching and motor neuron survival.

Background: Precise anatomical data on the development of human oculomotor somatic nuclei (OSN) remain rare., Design/subjects: This study describes the histology of human OSN in 11 preterm and full-term infants aged 20-43 postmenstrual weeks who died of various causes. Celloidin-embedded serial sections were stained with the Klüver-Barrera and other conventional methods including silver impregnation. To evaluate the growth of OSN quantitatively, the author estimated the nuclear volume and the average neuronal area on morphometry., Results: Four subnuclei were identified at 20-21 weeks: the fascicular, principal, dorsal median, and ventral median nucleus. Early tigroid Nissl bodies appeared in presumed motoneurons by 27-28 weeks, then resembled adult Nissl bodies at birth. On silver impregnation, the oculomotor nerve roots, crossed or uncrossed fibers at the midline, and a plexus of efferent or afferent axons in the neuropil were observed at 20-21 weeks. Then, the plexus was elaborated to form a perineuronal net of thin axon terminals by 28-29 weeks. The nuclear volume of OSN exponentially increased with age over 20-43 weeks, while the average of neuronal profile areas linearly increased in each subnucleus; the coefficient of regression was largest in the principal nucleus, and the regression lines nearly overlapped among the other subnuclei. Statistical analysis confirmed that the average neuronal area was largest in the principal nucleus in older cases., Conclusion: This study suggests that four subnuclei can be distinguished in human OSN by mid gestation, and that the principal nucleus may be different in neuronal cytoarchitecture from the others.

Proper movement of the vertebrate eye requires the formation of precisely patterned axonal connections linking cranial somatic motoneurons, located at defined positions in the ventral midbrain and hindbrain, with extraocular muscles. The aim of this research was to assess the relative contributions of intrinsic, population-specific properties and extrinsic, outgrowth site-specific cues during the early stages of abducens and oculomotor nerve development in avian embryos. This was accomplished by surgically transposing midbrain and caudal hindbrain segments, which had been pre-labeled by electroporation with an EGFP construct. Graft-derived EGFP+ oculomotor axons entering a hindbrain microenvironment often mimicked an abducens initial pathway and coursed cranially. Similarly, some EGFP+ abducens axons entering a midbrain microenvironment mimicked an oculomotor initial pathway and coursed ventrally. Many but not all of these axons subsequently projected to extraocular muscles that they would not normally innervate. Strikingly, EGFP+ axons also took initial paths atypical for their new location. Upon exiting from a hindbrain position, most EGFP+ oculomotor axons actually coursed ventrally and joined host branchiomotor nerves, whose neurons share molecular features with oculomotor neurons. Similarly, upon exiting from a midbrain position, some EGFP+ abducens axons turned caudally, elongated parallel to the brainstem, and contacted the lateral rectus muscle, their originally correct target. These data reveal an interplay between intrinsic properties that are unique to oculomotor and abducens populations and shared ability to recognize and respond to extrinsic directional cues. The former play a prominent role in initial pathway choices, whereas the latter appear more instructive during subsequent directional choices., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points., Results: We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes., Conclusions: We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.

Background: The trochlear nucleus, the smallest of the extraoculomotor nuclei, is unique or even curious, because the nerve roots emerge dorsally from the superior medullary velum after decussation. Little information is available on the developmental anatomy of this nucleus in humans., Design/subjects: We examined serial brain sections from 10 premature infants aged 20-39 weeks of gestation to document the histology and morphometry., Results: The trochlear nucleus was composed of three parts: the rostral tip, the main body, and the caudal division. The rostral tip was a rostral continuation of the main body, being closely related to the oculomotor nucleus; the main body was enveloped by a fibrous capsule; the caudal division was a small separate cluster of neurons in the medial longitudinal fasciculus or the root fibers with individual variations. Tigroid Nissl bodies first appeared at 28 weeks in presumed motoneurons. Various sizes of motoneurons were recognized; medium-sized to small motoneurons were preferentially accumulated in the rostral tip. Among the motoneurons, presumed non-motor neurons were infrequently scattered. Morphometric analysis showed that the nuclear volume exponentially increased with age, about 15 fold over 20-39 weeks, while the average profile area of the neurons linearly increased. Statistical analysis confirmed that cell area was smallest in the rostral tip among the three parts., Conclusion: Although the sample number is small in this study, it suggests that the human trochlear nucleus can be divided into three parts, and that the overall growth may be accelerated at about 30 weeks of gestation., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Development of the meninges in and around the plica ventralis encephali has not been well documented. A distinct mesenchymal structure, the so-called plica ventralis encephali, is sandwiched by the fetal mesencephalic flexure. We histologically examined paraffin-embedded sections from 18 human embryos and fetuses at 6-12 weeks of gestation. In the loose tissues of the plica, the first meninx appeared as a narrow membrane along the oculomotor nerve at 7-8 weeks. Subsequently, the plica ventralis evolved into 3 parts: bilateral lateral mesenchymal condensations and a primitive membranous meninx extending between. Notably, the topographical anatomy of the oculomotor, trochlear and trigeminal nerves did not change: the oculomotor nerve ran along the rostral aspect of the membranous meninx, the trigeminal nerve ran along the caudal side of the lateral mesenchymal condensation, and the trochlear nerve remained embedded in the lateral condensation. Up to 9-10 weeks, the lateral mesenchymal condensations became tongue-like folds; i.e., the primitive form of the tentorium cerebelli, while the membranous meninx became the diaphragma sellae. The falx cerebri seemed to develop from the tongue-like folds. Overall, the final tentorium cerebelli corresponded to the regressed plica ventralis, while the parasellar area originated from the base of the plica and other tissues along the ventral aspects of the basisphenoid and basioccipital., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Vertebrate eye movements depend on the co-ordinated function of six extraocular muscles that are innervated by the oculomotor, trochlear, and abducens nerves. Here, we show that the diffusible factors, stromal cell-derived factor-1 (SDF-1) and hepatocyte growth factor (HGF), guide the development of these axon projections. SDF-1 is expressed in the mesenchyme around the oculomotor nerve exit point, and oculomotor axons fail to exit the neuroepithelium in mice mutant for the SDF-1 receptor CXCR4. Both SDF-1 and HGF are expressed in or around the ventral and dorsal oblique muscles, which are distal targets for the oculomotor and trochlear nerves, respectively, as well as in the muscles which are later targets for oculomotor axon branches. We find that in vitro SDF-1 and HGF promote the growth of oculomotor and trochlear axons, whereas SDF-1 also chemoattracts oculomotor axons. Oculomotor neurons show increased branching in the presence of SDF-1 and HGF singly or together. HGF promotes the growth of trochlear axons more than that of oculomotor axons. Taken together, these data point to a role for both SDF-1 and HGF in extraocular nerve projections and indicate that SDF-1 functions specifically in the development of the oculomotor nerve, including oculomotor axon branch formation to secondary muscle targets. HGF shows some specificity in preferentially enhancing development of the trochlear nerve., (Copyright (c) 2010 Wiley Periodicals, Inc.)

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed-end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti-Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes alpha2-chimaerin, a Rac guanosine triphosphatase-activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase alpha2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance alpha2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant alpha2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that alpha2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

Introduction: Isotretinoin (RA), used for the treatment of cystic acne, is a powerful teratogen, causing craniofacial dysmorphisms and neural tube defects. We present two patients with RA embryopathy and oculomotor nerve synkinesis., Methods: Retrospective review of patient records., Results: Two patients presented with third nerve synkinesis and fetal RA exposure. Both had marked elevation of the upper eyelids on adduction such that the lid fissures alternately opened and closed on gaze from side to side. Both patients showed typical dysmorphisms of RA embryopathy. The first patient had complete agenesis of the cerebellar vermix and died at 2 years. The second patient had restricted extraocular muscles in one eye and was exotropic and hypotropic., Discussion: Both patients demonstrated simultaneous innervation of the medial rectus and levator palpebrae muscles causing coincident lid elevation in adduction. This evidence of oculomotor nerve synkinesis is consistent with animal studies showing abnormalities in the formation of cranial nerve ganglia following fetal RA exposure., Conclusion: RA is a powerful teratogen. These patients provide additional clinical evidence of its influence on neural migration during early development.

The pattern of innervation of the extraocular muscles is highly conserved across higher vertebrate species and mediates sophisticated visuomotor processes. Defects in oculomotor development often lead to strabismus, a misalignment of the eyes that can cause partial blindness. Although it has been intensively studied from a clinical perspective, relatively little is known about how the system develops embryonically. We have therefore mapped the development of the oculomotor nerve (OMN) in chick embryos by using confocal microscopy. We show that OMN development follows a series of stereotyped steps that are tightly regulated in space and time. The OMN initially grows past three of its targets to innervate its distal target, the ventral oblique muscle, only later forming branches to the more proximal muscles. We have also investigated spatiotemporal aspects of the unusual contralateral migration of a subpopulation of oculomotor neurons by using molecular markers and have found the semaphorin axon guidance molecules and their receptors, the neuropilins, to be expressed in discrete subnuclei during this migration. Finally, we have created an embryological model of Duane retraction syndrome (DRS), a form of strabismus in which the OMN is believed to innervate aberrantly the lateral rectus, the normal target of the abducens nerve. By ablating rhombomeres 5 and 6 and hence the abducens, we have mimicked a proposed oculomotor deficit occurring in DRS. We find that the absence of the abducens nerve is not sufficient to produce this inappropriate innervation, so other factors are required to explain DRS., (Copyright 2004 Wiley-Liss, Inc.)

Maternal cigarette use during pregnancy is associated with increased incidence of neural impairments in offspring, but nicotine's unique contribution to any neuropathology remains unclear, and nicotine's neurodevelopmental effects assessed in animal models vary with concentration. During ontogenesis, the chick oculomotor complex (OMN) is regulated by central nervous system (CNS) afferent-derived and target-derived trophic factors, allowing assessment of nicotine's potential interference in receptor-mediated CNS trophic phenomena, unconfounded by myriad other compounds in cigarette smoke. In the current study, 100 ng nicotine applied daily in ovo to yolk during embryonic days (E) 1-7 mimicked maternal plasma nicotine concentrations during fetal cranial nerve development. Nicotine-treated embryos exhibited a 15% decrease in whole body weight and 7% decrease in brain weight at E16. However, at E16, nicotine-treated embryos had 37% and 15% increases in the combined ventromedial+lateral (v) OMN motoneuron density and soma area, respectively, effects not observed in the optic tectum, in which nicotine cholinergic receptor expression is delayed until E8-12. Incorporation of tritiated thymidine into whole brain DNA demonstrated that the nicotine treatment did not cause increased rates of whole brain mitosis, suggesting that the dosage regimen did not elicit a cytotoxic, wound-healing, response of differentiating cells. As determined by DNA fragment-labeling assay during the normal period of cell death, vOMN apoptosis occurs maximally on E11 during a normal period of declining cell density, and a dose-response study demonstrated 78% E11 vOMN apoptotic suppression at approximately 0.30 microM cumulative yolk nicotine with an inhibition threshold between 0.10 and 0.20 microM. These results suggest that plasma nicotine concentrations resulting from tobacco use or nicotine replacement therapy (NRT) are sufficient to inhibit motoneuron apoptosis and enhance neuronal growth.

The chick ciliary ganglion is a neural crest-derived parasympathetic ganglion that innervates the eye. Here, we examine its axial level of origin and developmental relationship to other ganglia and nerves of the head. Using small, focal injections of DiI, we show that neural crest cells arising from both the caudal half of the midbrain and the rostral hindbrain contribute to the ciliary as well as the trigeminal ganglion. Precursors to both ganglia have overlapping migration patterns, moving first ventrolaterally and then rostrally toward the optic vesicle. At the level of the midbrain/forebrain junction, precursors to the ciliary ganglion separate from the main migratory stream, turn ventromedially, and condense in the vicinity of the rostral aorta and Rathke's pouch. Ciliary neuroblasts first exit the cell cycle at early E2, prior to and during ganglionic condensation, and neurogenesis continues through E5.5. By E3, markers of neuronal differentiation begin to appear in this population. By labeling the ectoderm with DiI, we discovered a new placode, caudal to the eye and possibly contiguous to the trigeminal placode, that contributes a few early differentiating neurons to the ciliary ganglion, oculomotor nerve, and connecting branches to the ophthalmic nerve. These results suggest for the first time a dual neural crest and placodal contribution to the ciliary ganglion and associated nerves.

Glial cell line-derived neurotrophic factor (GDNF) is the most potent motoneuron survival factor. We show here that in the chick oculomotor system, endogenous GDNF is derived largely from extraocular muscle but less from glial cells and not from muscle spindles. Increased levels of GDNF exclusively in the target rescued 30% of oculomotor neurons that would normally die during developmental cell death, a rate of rescue similar to that with systemic GDNF application. Thus, GDNF supports motoneuron survival in a retrograde, target-derived fashion, as opposed to a local paracrine route or an indirect route via sensory afferents. Persephin, another member of the GDNF family, did not increase survival with target delivery, despite its retrograde transport from the target. Unlike GDNF, however, persephin increased neurite outgrowth from oculomotor nuclei in vitro. Thus, one GDNF family member acts as a muscle-derived retrograde survival factor, whereas another one has distinct functions on neurite outgrowth.

The present study uses the embryonic chick to examine in vivo the mechanisms and regulation of Schwann cell programmed cell death (PCD) in spinal and cranial peripheral nerves. Schwann cells are highly dependent on the presence of axons for survival because the in ovo administration of NMDA, which excitotoxically eliminates motoneurons and their axons by necrosis, results in a significant increase in apoptotic Schwann cell death. Additionally, pharmacological and surgical manipulation of axon numbers also affects the relative amounts of Schwann cell PCD. Schwann cells undergoing both normal and induced PCD display an apoptotic-like cell death, using a caspase-dependent pathway. Furthermore, axon elimination results in upregulation of the p75 and platelet-derived growth factor receptors in mature Schwann cells within the degenerating ventral root. During early development, Schwann cells are also dependent on axon-derived mitogens; the loss of axons results in a decrease in Schwann cell proliferation. Axon removal during late embryonic stages, however, elicits an increase in proliferation, as is expected from these more differentiated Schwann cells. In rodents, Schwann cell survival is regulated by glial growth factor (GGF), a member of the neuregulin family of growth factors. GGF administration to chick embryos selectively rescued Schwann cells during both normal PCD and after the loss of axons, whereas other trophic factors tested had no effect on Schwann cell survival. In conclusion, avian Schwann cells exhibit many similarities to mammalian Schwann cells in terms of their dependence on axon-derived signals during early and later stages of development.

The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) participates in a variety of developmental processes, including axonal guidance and cell migration. PSA's function in these contexts stems from its ability to reduce cell interactions. The present study examines the regulation of PSA expression during formation of the calyciform synapse by the oculomotor axons on chick ciliary neurons. Prior to synaptogenesis, PSA is abundantly and uniformly expressed on the surface of the ciliary neuron body. However, at the time synaptic bonds start to form, as reflected in the localized accumulation of synaptic vesicles, PSA is lost from the point of synaptic contact. Thereafter, PSA is progressively lost from the ciliary neuron surface as the calyx grows. The dense mats of pseudodendritic-like somatic spines, which extend from the postsynaptic cell body, form an exception. These spines, which are known to undergo morphological remodeling, retain PSA expression until the end of embryogenesis. The experimental removal of PSA did not affect synaptogenesis itself, in that no significant changes were observed in the surface covered by the calyx, the number of spine aggregates, the size of acetylcholine receptor clusters, the cell surface area covered by these receptors, or the ultrastructure of the calyx, spine mats, and active zones. Together, these observations suggest that the synapse eliminates PSA as a part of its normal development and that the loss of PSA from the site of axon-target interaction may serve to stabilize structures formed during synaptogenesis., (Copyright 2002 Wiley-Liss, Inc.)

Several trophic factors support the survival of developing motoneurons, but it is not known whether these factors act in a retrograde fashion from the motoneuron target muscle or are derived from other sources. Cardiotrophin-1 (CT-1) and the insulin-like growth factors (IGFs) are candidate target-derived motoneuron survival factors as both are expressed in muscle during naturally occurring motoneuron death and, applied systemically, support the survival of developing motoneurons. By using the embryonic chick oculomotor system, we show that CT-1 and IGF-I promote neurite outgrowth from E13-derived oculomotor explants and are retrogradely transported from muscle to nerve cell body in vivo, and injection of CT-1 or IGF-I into eye muscles increases motoneuron survival by 20 and 30%, respectively, as evidenced by calibrated stereological counting techniques. Pharmacological depletion of endogenous target-derived IGF-I in vivo reduces oculomotor neuron survival by up to 30% in a dose-dependent manner. These results significantly extend previous studies using systemic administration of trophic factors and are the first to demonstrate a target-derived retrograde mechanism of developing motoneuron survival factors., (©2002 Elsevier Science.)

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target., (Copyright 1999 John Wiley & Sons, Inc.)

Investigations were made on serial sections of human embryos at developmental stages 16 and 17 (37-41 days). The oculomotor nucleus in embryos at these stages is located at the cephalic flexure of the neural tube. It consists of paired lateral nucleus which is formed by well defined group of large neurons in the basal plate of the midbrain. Within the lateral nucleus two groups of cells (medial and lateral) can be distinguished. Posteriorly these groups unite and form a single nucleus.

Investigations were made on serial sections of human embryos at developmental stage 23 (56 days). The oculomotor complex in embryos at this stage consists of lateral, median, and accessory nuclei. The lateral (chief) nucleus forms the greatest part of the oculomotor complex and it is subdivided into ventromedial and dorsolateral groups. Between the posterior portions of lateral nuclear groups is the median nucleus. Dorsally and medially to the lateral nucleus is the accessory (parasympathetic) nucleus.

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.

Wnt-1 null mutant mice lack midbrain somatic motor nuclei. Primordial migration and spatial patterning of the extraocular muscles, however, was preserved, but myogenesis was disrupted in aneural muscles. Some muscles normally innervated by oculomotor and trochlear nuclei received aberrant innervation, which proved sufficient to maintain prenatal stages of myogenesis. The absence of motoneurons followed by innervation from inappropriate motoneuron pools is a viable candidate mechanism in ocular motility disorders, including Duane retraction syndrome and congenital fibrosis of extraocular muscle.

Dbx, a divergent homeobox gene, is expressed in a regionally restricted pattern in the developing mouse central nervous system (CNS). In order to understand its spatial regulation, we have isolated a cis-regulatory region using a reporter gene analytical approach in transgenic mice. A 5.7 kb DNA fragment that contains the transcriptional start site of the Dbx gene is sufficient to direct the expression of the transgene to various domains of the CNS in a temporally regulated fashion. The transgene expression can be detected between 9.5 and 15.5 days post coitum in embryos in the fore-, mid- and hindbrain and spinal cord in regions where the endogenous gene is expressed. Additionally, transgene expression can also be detected in the oculomotor nerve (cranial nerve III). The expression of the transgene closely resembles that of the Dbx gene with minor, but interesting differences. These results suggest that major cis-acting elements reside within a 5.7 kb DNA fragment located 5' of the Dbx gene. Further deletion analysis shows that at least two independently regulated elements are present within this DNA fragment: an element that directs expression to the brain and spinal cord and a second element that directs expression to the oculomotor nerve.

We investigated the potential role of rostral-caudal and dorsal-ventral subdivisions of the early rostral brain by relating these subdivisions to the early patterning of neuron cell bodies and their axon projections. The earliest neurons were mapped using the lipophilic axon tracers diI and diO on embryos fixed on embryonic days 9.5-10.5 (E9.5-E10.5); neuromeric boundaries were marked by diO. The tracts were small in number, were organized orthogonally (2 dorsal-ventral and 4 rostral-caudal), and originated from groups of cell bodies which we term "sources." Two parallel longitudinal axon systems, one dorsal (the tract of the postoptic commissure and the mesencephalic tract of the trigeminal nerve) and one ventral (the mammillotegmental tract and the medial longitudinal fasciculus), projected caudally from the prosencephalon into the rhombencephalon. We argue that the dorsal longitudinal pathway marked the boundary between the alar and basal plates along the entire neuraxis. The dorsal-ventral axons coursed circumferentially and either crossed the midline (forming the posterior and ventral tegmental commissures) or turned caudally without crossing the midline. The dorsal-ventral axons were not generally restricted to the interneuromeric boundaries, as others have suggested. Earlier, all neighboring neurons projected their axons together; later, nearby neurons projected into different pathways. Some tracts originated in single neuromeres, while other tracts had origins in two or more neuromeres. The dorsal longitudinal axons altered course at several of the borders, but the ventral longitudinal axons did not. In summary, the early subdivisions appeared to influence some, but not all, aspects of tract formation.

To determine when the calcium-binding protein parvalbumin appears during development, neurons in the chick Edinger Westphal nucleus were examined for parvalbumin immunoreactivity at a variety of embryonic stages. Parvalbumin immunoreactivity appeared on embryonic day 14 (E14, Hamburger and Hamilton stage 40) in predominantly lateral Edinger Westphal neurons. Cytochrome oxidase activity within the nucleus was examined throughout development, as an indicator of physiological activity, and expression of cytochrome oxidase was compared with that of parvalbumin. Cytochrome oxidase activity was found to be uniformly high in all parts of the Edinger Westphal nucleus throughout development. Either the Edinger Westphal nucleus in physiologically active quite early in its development or other energy demands mask the correlation of cytochrome oxidase with electrical activity. Cytochrome oxidase was expressed well before parvalbumin immunoreactivity appeared. Voltage-activated calcium currents were characterized in E12 Edinger Westphal neurons. In both amplitude and composition, E12 calcium currents resemble those of E16 neurons, excluding the possibility that calcium currents appear de novo during or just prior to the appearance of parvalbumin. Both cytochrome oxidase activity and calcium currents are observed in Edinger Westphal neurons well before the appearance of parvalbumin during development. These findings do not exclude the possibility that physiological activity affects the expression of parvalbumin since other factors such as changing patterns of synaptic activity or the appearance of calcium conducting NMDA receptors have yet to be examined. However, they raise the possibility that additional factors such as an intrinsic developmental program or a change in the neuron's basal intracellular calcium requirements may also be involved.

From so-called "dead-in-pipped-eggs" provided by the Poultry Breeding Center of Gifu Prefecture, we obtained two chick embryos with hypoplastic eyes on external examination, and studied the visual system and cranial nerve nuclei innervating the extraocular muscles. The upper bills of both embryos deviated toward one side when observed with the naked eye. In histological sections from the most severely affected eyeball of each case primordial retinal tissue in the poorly developed orbit seemed to be at the stage of optic vesicle formation; it had not differentiated into individual layers. The optic nerve was absent from these eyeballs and the neural retina had multiple folds. The ciliary ganglion was smaller on the more severely affected side than on the contralateral side which had only mild hypoplasia. The optic nerve, which was present on the contralateral side, bifurcated at the level of the chiasma close to the diencephalic base; one branch ran toward the ipsilateral optic tectum and the other toward the contralateral brain region. The oculomotor and trochlear nerve nuclei could be identified bilaterally in one of the embryonic brains but not in the other. These findings suggest that hypoplasia of the neural retina and optic nerve in an orbit leads to bifurcation of the contralateral optic nerve which, under normal conditions, should completely cross at the chiasma. This may also suggest that retinal afferents from the brain are also affected in the hypoplastic eyeball.

The development of the oculomotor nucleus in five species of salamanders and one anuran species was investigated with tracing techniques. The data presented support the hypothesis that oculomotor motoneurons innervating the superior rectus muscle migrate across the midline. In the salamander Pleurodeles waltl, only ipsilateral oculomotor motoneurons are labeled in early development. Later, these neurons extend dendrites toward the contralateral side into the ventral tegmental neuropil, after which there is displacement of their nuclei (neuronal somata) across the midline. Cell bodies can be observed directly at the midline. In adult Salamandra salamandra, motoneurons innervating the superior rectus muscle are seen occasionally at the midline and on the ipsilateral side, with dendrites toward the contralateral side. Motoneurons on the ipsilateral side do not display these features. In Pleurodeles, developmental brain processes are slowed down, and the sequence of development of the contralateral subnucleus, which can be clearly observed, supports the migration hypothesis. In Xenopus laevis and most other species of salamanders this process is accelerated.

The development of the oculomotor and trochlear nuclei (nIII and nIV) was studied with the horseradish peroxidase (HRP) and the cobalt labelling techniques in Xenopus laevis tadpoles. The earliest labelling of the oculomotor neuroblasts was observed at stage 32. The ipsi- and contralateral nuclei were found in two distinct groups on either side of the brainstem and the oculomotor commissure formed by crossing axons was present at this early stage. The fusion of the two nuclei began at the late larval stage when the axonal outgrowth had been presumably completed. The trochlear neuroblasts could be first labelled at stage 39 when the position of the nucleus and axonal pathway was similar to the adult form.

Following transplantation of an additional optic primordium into the orbital mesenchyme of chick embryos of approximately 2 days of incubation age, the changes in cell number in the ciliary ganglion, accessory oculomotor and trochlear nuclei were studied at various stages of development. Cell counts were made at 1-day intervals from days 9 through 15 for ciliary ganglion, and from days 13 through 15 for the accessory oculomotor and trochlear nuclei. Cell counts for the ciliary ganglion on days 9 and 11 were similar on the operated and control sides which suggests that grafting of an additional optic primordium, and thus enlarging the periphery, is not involved in the control of proliferation. Comparison of the number of cells for the ciliary ganglia and the accessory oculomotor nuclei at days 13 and 15 showed an increase on the affected side ranging from 8 to 27%, and 9 to 33% respectively. We interpret this increase on the experimental side as a reduction in the number of degenerating cells that occur in normal development, as a result of an enlargement of the peripheral field of innervation. Three cases showed an increase in the number of cells in the trochlear nucleus ranging from 9 to 29%. This increase was attributed to an increase in the size of the superior oblique muscle of the operated side as determined by volumetric measurements. On the basis of the evidence we conclude that an enlarged periphery acts by regulating the level of naturally occurring cell death by reducing the amount of cell loss, leading to a corresponding increase in final cell number.

Development of the vegetative component of the oculomotor nerve nuclei was studied in the main mammalia orders and in human ontogenesis. In phylogenesis parasympathetic nuclei of Jakubovitch--Edinger--Westphal and those of Perlia are formed later than somatic nuclei of the oculomotor nerve. The animals studied have, together with well developed somatic components of the oculomotor nerve nuclei, gomologues of parasympathetic nuclei, which are rather well developed in carnivores and monkeys. In human prenatal ontogenesis, somatic nuclei are formed first and then parasympathetic ones. Both parts of the oculomotor nerve nuclei are fully developed in adult man.

Groups of pregnant rats were injected with two successive daily doses of 3H-thymidine from gestational day E12 and 13 (E12 j3) until the day before parturition (E21 k2) in order to label in their embryos the proliferating precursors of neurons. At 60 days of age the proportion of neurons generated (no longer labeled) on specific embryonic days was determined quantitatively in 18 regions of the midbrain tegmentum. The neurons of the oculomotor and trochlear nuclei are generated concurrently on days E12 and 13. There was a mirror image cytogenetic gradient in these nuclei and this was interpreted as the dispersal of neurons derived from a common neuroepithelial source to the medial longitudinal fasciculus. Neurons in three other components of the tegmental visual system are produced in rapid succession after the motor nuclei. In the nucleus of Darkschewitsch peak production time was on day E12 and 13, extending to day E15; in the Edinger-Westphal nucleus the time span was the same but with a pronounced between days E13; finally, the neurons of the parabigeminal nucleus were produced between days E13 and E15 with a peak on day E14. The neurons of the periaqueductal gray were generated between days E13 and 17 with a pronounced ventral-to-lateral and lateral-to-dorsal gradient. In the red nucleus the neurons were produced on days E13 and E14 with a caudal-to-rostral gradient: the cells of the magnocellular division preceding slightly but significantly the cells of the parvocellular division. The neurons of the interpeduncular nucleus originated between days E13 and E15; the peak in its ventral portion was on day E13, in its dorsal portion on days E14 and E15. A ventral-to-dorsal gradient was seen also in both the dorsal and the median raphe nuclei in which neuron production occurred between days E13 and E15. The neurons of the pars compacta and pars reticulate of the substantia nigra were both produced between days E13 and E15 with a modified lateral-to-medial gradient. This gradient extended to the ventral tegmental area where neurons of the pars medialis were produced between days E14 and E16. With the exception of the central gray, neuron production was rapid and relatively early in the structures situated ventral to the midbrain tectum. A comparison of the cytogenetic gradients in the raphe nuclei of the lower and upper medulla, the pontine region, and the midbrain suggests that they originate from at least three separate neuroepithelial sources.

Toluidine blue-stained semithin sections and Cajal-Castro preparations are used to study in rat fetuses whether oculomotor neuroblasts migrate across the midline at a certain period of development. In confirmation of previous studies, a group of oculomotor neuroblasts was detected which first grow cytoplasmic processes into the mesencephalic midline, and afterwards translocate their somata towards the midline, between the 12th and the 15th days of gestation. At this moment a midline mass of neuroblasts characterizes the meeting at this landmark of both left and right migrating neuroblastic groups. No crossing oculomotor axons yet are demonstrable with reduced silver techniques. In further stages of development the neuroblasts continue their migration until they arrive at the contralateral nucleus at the 16th and 17th day of gestation. At the midline the mass of neuroblasts disappears gradually and crossed oculomotor axons become visible. The electron microscope was then used to study ultrastructurally the migrating motoneurons. It was discovered that no preexisting structure guides their movement by contact. Their leading processes show no filopodial activity, and contain abundant microtubules and thick bundles of neurofilaments in eccentric position. The neuroblasts carry their axon across the midline as a trailing process.

The development of the oculomotor nuclear complex was studied in the Japanese quail. In hatchlings, this complex was found to consist of four subnuclei: The accessory, the dorsolateral, the dorsomedial, and the ventromedial. An arciform subnucleus or central subnucleus was not found in this species. All subnuclei were made up of homogeneous cell populations. The oculomotor primordia can first be recognized at day 3 of incubation, and a clear subdivision within this primordia is apparent on day 5 (total incubation period = 17 days). At this time the accessory subnucleus can be discerned. By day 6, a horizontally oriented dorsal cell mass, the anlagen of the dorsolateral and dorsomedial subnuclei, is seen, as is a vertically oriented ventral cell mass, the anlagen of the ventromedial subnucleus. The boundaries between the subnuclei are not yet distinct at this age, however. By day 7 of incubation, all four subnuclei can be detected the ventromedial subnuclei, are first seen on day 5, at which time the oculomotor commissure begins to appear. Migratory traffic is extensive during days 6 and 7 of incubation, with wide bands of migrating cells typically seen, spanning the entire dorsal-ventral extent of the oculomotor complex. These cells do not appear to be associated exclusively with the ventromedial subnuclei. In many instances, migratory cells appear to be affiliated with the dorsomedial cell group. By day 10 of incubation, migration has ceased and the oculomotor complex has attained its definitive configuration. These observations are discussed with comparative reference to earlier studies of chick and duck embryos and hatchings.

Unilateral removal of one eye primordium from Xenopus laevis embryos affects the number of axons in both oculomotor nerves: on the side of the operation, we observed a substantial decrease, and on the contralateral side an increase of over 30%. Mechanisms that may be invoked to explain this striking increase are: sprouting, rerouting of axons, decreased death of the motoneurones involved, and abnormal survival of ephemeral axon collaterals.

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.

In order to understand the significance of cell death in the formation of neural circuits, it is necessary to determine whether before cell death neurons have (a) sent axons to the periphery; (b) reached the proper target organs; and (c) have established synaptic connections with them. Axon counts demonstrated that, after sending out initial axons, ciliary cells sprouted numerous collaterals at the time of peripheral synapse formation. Subsequently, large numbers of axons were lost from the nerves, slightly later than the onset of ganglion cell death. A secondary loss of collaterals later occurred unaccompanied by cell death. Measurements of conduction velocity and axon diameters indicated that all ganglion cell axons grew down the proper pathways from the start, but it was not possible to determine whether all axons had actually formed proper synapses. This was ascertained, however, in the ganglion itself where preganglionic fibres were shown to synapse selectively with all ganglion cells before cell death. During this period, degenerating preganglionic synapses were observed on normal cells. It can therefore be inferred that at least some preganglionics established proper synapses before dying and that a single synapse is not sufficient to prevent cell death. In this system neither preganglionic nor ganglionic cell death seems designed to remove improper connections but rather to remove cells that have not competed effectively for a sufficient number of synapses, resulting in a quantitative matching up of neuron numbers.

The ontogenesis of the optic system in the brain has been studied with reference to the general biology of the embryo and to the cytoarchitecture of the optic tectum. 1. Sharks vary in terms of retinal structure (Franz 1931). Scyliorhinus shows densely packed and large cellular layers alternating with thin fiber layers. 2. The optic tracts, which are not excessively large, cross in the optic chiasm by approximately 95%. The optic fibers, forming a medial and a lateral tract, enter the tectum in zones 9 and 11. 3. The eye muscle nuclei do not differ from the general vertebrate pattern. The oculomotor nucleus represents the largest portion; the abducens nucleus is small. 4. The nucleus interstitialis of the fasciculus longitudinalis medialis is remarkably developed and consists of large neurons. Together with the interpreduncula nucleus it receives fibers of fasciculus retroflexus.