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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.)

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: 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.

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.

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.

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 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.

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.

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.

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.