Chie Sakuma

Netrin-1 Acts as a Repulsive Guidance Cue for Sensory Axonal Projections toward the Spinal Cord

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.

Regulated Gene Expression in the Chicken Embryo by Using Replication-Competent Retroviral Vectors

ABSTRACT 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.

Netrin-1 signaling for sensory axons

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.

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

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

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.

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

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.

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

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 (α1β1γ1) 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 β1 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.

Distinct susceptibility of developing neurons to death following Bax overexpression in the chicken embryo

Bax is a proapoptotic protein that is required for programmed cell death (PCD) of many neuronal populations. Here we show that, during an early period of retinal PCD and in naturally occurring sensory and motor neuron (MN) death in the spinal cord, Bax delivery results in enhanced death of these neural populations. In contrast, Bax overexpression fails to enhance an early phase of MN death that occurs in the cervical spinal cord, although overexpressed Bax appears to be activated in dying MNs. Bax overexpression does not also affect the survival of immature neurons prior to the PCD period. Taken together, these data provide the first in vivo evidence suggesting that Bax appears to act selectively as an executioner only in neurons undergoing PCD. Furthermore, although Bax appears to mediate the execution pathway for PCD, the effect of Bax overexpression on susceptibility to death differs between different neuronal populations.

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

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.

Development of the dorsal ramus of the spinal nerve in the mouse embryo: Involvement of semaphorin 3A in dorsal muscle innervation

The spinal nerve, which is composed of dorsal root ganglion (DRG) sensory axons and spinal motor axons, forms the dorsal ramus projecting to the dorsal musculature. By using the free‐floating immunohistochemistry method, we closely examined the spatiotemporal pattern of the formation of the dorsal ramus and the relationship between its projection to the myotome/dorsal musculature and semaphorin 3A (Sema3A), which is an axonal guidance molecule. In embryonic day (E) 10.5–E11.5 wild‐type mouse embryos, we clearly showed the existence of a waiting period for the dorsal ramus projection to the myotome. In contrast, in E10.5–E11.5 Sema3A‐deficient embryos, the dorsal ramus fibers projected beyond the edge of the myotome without exhibiting the waiting period for projection. These results strongly suggest that the delayed innervation by dorsal ramus fibers may be caused by Sema3A‐induced axon repulsion derived from the myotome. Next, by performing culture experiments, we confirmed that E12.5 mouse axons responded to Sema3A‐induced repulsion. Together, our results imply that Sema3A may play a key role in the proper development of the dorsal ramus projection.