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Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Fukushima Medical University

Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the tet regulatory elements also revealed doxycycline-dependent inducible GFP expression in the chicken embryo in ovo.

Regulated gene expression in the chicken embryo by using replication-competent retroviral vectors.

Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl-2 by a replication-competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl-2 expression did not affect the number of Islet-1/2-positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet-1/2-positive MNs was clearly increased in Bcl-2-transfected embryos compared with control and GFP-transfected embryos. Activation of caspase-3, which is normally observed in this early MN death, was also prevented by Bcl-2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl-2.

Bcl-2 rescues motoneurons from early cell death in the cervical spinal cord of the chicken embryo.

In the early stages, the dorsal root ganglion neurons extend their axons toward the dorsal spinal cord. We previously showed that surround repulsion by semaphorin 3A prevents sensory axons from straying from their paths. The finding, however, that sensory trajectories toward the dorsal spinal cord are almost normal in semaphorin 3A-deficient littermates raises the possibility that a chemoattraction-based mechanism also contributes to the formation of sensory axonal projections. By employing culture assays, we show that the dorsal spinal cord secretes chemoattractants for the dorsal root ganglion axons. Furthermore, we demonstrate that the activity of a dorsal spinal cord-derived cue is specific for early sensory axons. These results suggest that dorsal spinal cord-derived chemoattractants contribute to the formation of the initial trajectories of sensory axons.

Guidance cues from the embryonic dorsal spinal cord chemoattract dorsal root ganglion axons.

During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported the involvement of dorsal spinal cord-derived chemoattraction in this projection (Masuda et al. [ 2007] Neuroreport 18:1645-1649). However, the molecular nature of this attraction is not clear. Here we show that laminin-1 (alpha1beta1gamma1) is expressed strongly along the pathway of DRG axons and that its 67-kDa receptor (67LR) is present on DRG cells. This evidence suggests that laminin-1-67LR signaling may be involved in DRG axonal guidance. By employing culture assays, we show that laminin-1 or the YIGSR peptide, a soluble peptide of the laminin beta1 chain, promotes the DRG axonal response to dorsal spinal cord-derived chemoattraction. By using a function-blocking antibody against 67LR, we show that the anti-67LR antibody blocks the modulation of DRG axonal response by the YIGSR peptide in vitro. Furthermore, the in ovo injection of the anti-67LR antibody inhibits the DRG axonal growth toward the dorsal spinal cord. These results provide evidence that the YIGSR peptide promotes dorsal spinal cord-derived chemoattraction via 67LR to contribute to the formation of the initial trajectories of developing DRG axons.

Laminin peptide YIGSR and its receptor regulate sensory axonal response to the chemoattractive guidance cue in the chick embryo.

During early development, the ventral spinal cord expresses chemorepulsive signals that act on dorsal root ganglion (DRG) axons to help orient them toward the dorsolateral part of the spinal cord. However, the molecular nature of this chemorepulsion is mostly unknown. We report here that netrin-1 acts as an early ventral spinal cord-derived chemorepellent for DRG axons. In the developing mouse spinal cord, netrin-1 is expressed in the floor plate of the spinal cord, and the netrin receptor Unc5c is expressed in DRG neurons. We show that human embryonic kidney cell aggregates secreting netrin-1 repel DRG axons and that netrin-1-deficient ventral spinal cord explants lose their repulsive influence on DRG axons. In embryonic day 10 netrin-1 mutant mice, we find that DRG axons exhibit transient misorientation. Furthermore, by means of gain-of-function analyses, we show that ectopic netrin-1 in the dorsal and intermediate spinal cord prevents DRG axons from being directed toward the dorsal spinal cord. Together, these findings suggest that netrin-1 contributes to the formation of the initial trajectories of developing DRG axons as a repulsive guidance cue.

Netrin-1 acts as a repulsive guidance cue for sensory axonal projections toward the spinal cord.

During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported that this projection is achieved by dorsal spinal cord-derived chemoattraction. However, the molecular nature of the chemotrophic cue is not yet fully understood. To identify novel genes differentially expressed in the dorsal spinal cord in the embryonic day 10.5 mouse, we used the Kazusa cDNA array system comprising approximately 1700 mouse KIAA/FLJ (mKIAA/mFLJ) cDNA clones and laser capture microdissection (LCM) in combination with PCR-based cDNA amplification. We observed that a certain population of genes showed significantly increased expression in the dorsal spinal cord. In situ hybridization analysis verified the expression of mRNAs of 6 genes (Hip1r, Nav2, Fstl5, Cacna1h, Bcr, and Bmper) in the cells that constitute the dorsal spinal cord. The dorsal spinal cord-specific genes identified in this study provide a basis for studying the molecular nature of the neural development including the axonal guidance of DRG neurons. These results also demonstrate that the combined use of LCM coupled with the Kazusa cDNA array technology will be useful for the identification of large proteins expressed in the restricted small regions of embryos.

Laser capture microdissection and cDNA array analysis for identification of mouse KIAA/FLJ genes differentially expressed in the embryonic dorsal spinal cord.

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a 'waiting period' of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.

Netrin-1 signaling for sensory axons: Involvement in sensory axonal development and regeneration.

During development, dorsal root ganglion (DRG) neurons in higher vertebrates extend their axons centrally to the spinal cord through the dorsal root entry zone (DREZ) and peripherally to muscle and skin targets. After entering the spinal cord, DRG axons project into the dorsal mantle layer. In this review, we focus on evidence showing the role for netrin-1 in forming sensory axonal trajectories. Netrin-1 is a diffusible axonal guidance molecule that chemorepels developing DRG axons. When DRG axons project toward the DREZ, ventral spinal cord-derived netrin-1 prevents DRG axons from projecting aberrantly toward the ventral spinal cord. At later stages, the dorsal spinal cord cells transiently express netrin-1. This dorsal spinal cord-derived netrin-1 prevents DRG axons from invading the dorsal spinal cord during the waiting period. Together, the data reviewed provide strong evidence that netrin-1 plays a crucial role in sensory axon projection during development.

Role for netrin-1 in sensory axonal guidance in higher vertebrates.

The avian cervical spinal cord includes motoneurons (MNs) that send their axons through the dorsal roots. They have been called dorsal motoneurons (dMNs) and assumed to correspond to MNs of the accessory nerve that innervate the cucullaris muscle (SAN-MNs). However, their target muscles have not been elucidated to date. The present study sought to determine the targets and the specific combination of transcription factors expressed by dMNs and SAN-MNs and to describe the detailed development of dMNs. Experiments with tracing techniques confirmed that axons of dMNs innervated the cucullaris muscle. Retrogradely labeled dMNs were distributed in the ventral horn of C3 and more caudal segments. In most cases, some dMNs were also observed in the C2 segment. It was also demonstrated that SAN-MNs existed in the ventral horn of the C1-2 segments and the adjacent caudal hindbrain. Both SAN-MNs and dMNs expressed Isl1 but did not express Isl2, MNR2, or Lhx3. Rather, these MNs expressed Phox2b, a marker for branchial motoneurons (brMNs), although the intensity of expression was weaker. Dorsal MNs and SAN-MNs were derived from the Nkx2.2-positive precursor domain and migrated dorsally. Dorsal MNs remain in the ventral domain of the neural tube, unlike brMNs in the brainstem. These results indicate that dMNs and SAN-MNs belong to a common MN population innervating the cucullaris muscle and also suggest that they are similar to brMNs of the brainstem, although there are differences in Phox2b expression and in the final location of each population. J. Comp. Neurol. 521: 2987-3002, 2013. © 2013 Wiley Periodicals, Inc. 

Elucidation of target muscle and detailed development of dorsal motor neurons in chick embryo spinal cord.

The optic tectum comprises multiple layers, which are formed by radial and tangential migration during development. Here, we report that Neuropilin 1 (NRP1)-mediated Sema3A signals are involved in the process of tectal laminar formation, which is elaborated by tangential migration. In the developing chick tectum, NRP1, a receptor for Sema3A, is expressed in microtubule-associated protein 2 (MAP2)-positive intermediate layers IV and V. Sema3A itself is a diffusible guidance factor and is expressed in the overlying layer VI. Using stable fluorescent labeling of tectal cells, we show that MAP2-positive intermediate layers are formed by the neurons that have been dispersed by tangential migration along the tectal efferent axons. When Sema3A was mis-expressed during laminar formation, local Sema3A repelled the tangential migrants, thus eliminating MAP2-positive neurons that expressed NRP1. Furthermore, in the absence of the MAP2-positive neurons, tectal layers were disorganized into an undulated form, indicating that MAP2-positive intermediate layers are required for proper laminar formation. These results suggest that NRP1-mediated Sema3A signals provide repulsive signals for MAP2-positive neurons to segregate tectal layers, which is important in order to coordinate laminar organization of the optic tectum. 

NRP1-mediated Sema3A signals coordinate laminar formation in the developing chick optic tectum.

Elongation of the efferent fibers of dorsal root ganglion (DRG) neurons toward their peripheral targets occurs during development. Attractive or permissive systems may be involved in this elongation. However, the molecular mechanisms that control it are largely unknown. Here we show that class 5 semaphorin Sema5A had attractive/permissive effects on DRG axons. In mouse embryos, Sema5A was expressed in and around the path of DRG efferent fibers, and cell aggregates secreting Sema5A attracted DRG axons in vitro. We also found that ectopic Sema5A expression in the spinal cord attracted DRG axons. Together, these findings suggest that Sema5A functions as an attractant to elongate DRG fibers and contributes to the formation of the early sensory network. 

Attractive and permissive activities of semaphorin 5A toward dorsal root ganglion axons in higher vertebrate embryos.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1 MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1 MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1 MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1 MNs committed to LMC neurons, depending on the Hox expression pattern.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

During embryonic brain development, groups of particular neuronal cells migrate tangentially to participate in the formation of a laminated structure. Two distinct types of tangential migration in the middle and superficial layers have been reported in the development of the avian optic tectum. Here we show the dynamics of tangential cell movement in superficial layers of developing chick optic tectum. Confocal time-lapse microscopy in organotypic slice cultures and flat-mount cultures revealed that vigorous cell migration continued during E6.5-E13.5, where horizontally elongated superficial cells spread out tangentially. Motile cells exhibited exploratory behavior in reforming the branched leading processes to determine their pathway, and intersected with each other for dispersion. At the tectal peripheral border, the cells retraced or turned around to avoid protruding over the border. The tangentially migrating cells were eventually distributed in the outer stratum griseum et fibrosum superficiale and differentiated into neurons of various morphologies. These results revealed the cellular dynamics for widespread neuronal distribution in the superficial layers of the developing optic tectum, which underline a mode of novel tangential neuronal migration in the developing brain.

Chie Sakuma

Department of Neuroanatomy and Embryology,
School of Medicine,
Department of Neuroanatomy and Embryology, School of Medicine

Fukushima Medical University

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Chie Sakuma

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neuroanatomy and Embryology,School of Medicine,Department of Neuroanatomy and Embryology, School of Medicine

Fukushima Medical University

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Department of Neuroanatomy and Embryology,
School of Medicine,
Department of Neuroanatomy and Embryology, School of Medicine