Shunsaku Homma

Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: Evidence for the role of cell death genes☆

Treatment of chick embryos in ovo for 10-12 hr with inhibitors of protein and RNA synthesis during the peak time of normal cell death (Embryonic Day 8) for motoneurons and dorsal root ganglion cells markedly reduces the number of degenerating neurons in these populations. The massive neuronal death induced by the early absence of the limbs was also blocked almost completely by these agents. Further, the death of neurons following peripheral axotomy at the end of the normal cell death period (Embryonic Day 10) was reduced significantly by treatment with inhibitors of biosynthetic reactions. These results indicate that, in vivo, naturally occurring neuronal death, neuronal death induced by the absence of peripheral targets, and axotomy-induced neuronal death later in development all require active gene expression and protein and RNA synthesis. Therefore, neuronal death in a variety of situations may reflect the expression of a developmental fate that can normally only be overridden or suppressed by specific environmental signals (e.g., neurotrophic molecules).

Peptide inhibitors of the ice protease family arrest programmed cell death of motoneurons in vivo and in vitro

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Expression pattern of LRR and Ig domain-containing protein (LRRIG protein) in the early mouse embryo.

The combination of leucine-rich repeat (LRR) and immunoglobulin-like (Ig) domains is found in the domain architecture of the Trk neurotrophin receptor protein. Recently dozens of such proteins simultaneously carrying LRR and Ig domains as the Trk receptors have been identified. Given the significant biological roles of Trk and such newly identified proteins, we have searched the public database for human proteins with LRR and Ig domains (collectively termed the leucine-rich repeat and Ig domain-containing protein, LRRIG protein, in this study), and have analyzed the mRNA expression pattern of mouse orthologs of obtained human LRRIG proteins at embryonic day 10. The list of the LRRIG proteins includes 36 human proteins: four LINGO, three NGL, five SALM, three NLRR, three Pal, two ISLR, three LRIG, two GPR, two Adlican, two Peroxidasin-like proteins, three Trk neurotrophin receptors, a yet unnamed protein AAI11068, and three AMIGO. Some molecules (LINGO2, LINGO4, NGL1, SALM1, SALM5, and TrkB) were expressed exclusively in neuronal tissues, whereas others (ISLR1, GPR124, and Adlican2) exhibited non-neuronal expression profiles. However, the majority of LRRIG protein family exhibited broad mRNA tissue-expression profiles.

Modulation of early but not later stages of programmed cell death in embryonic avian spinal cord by sonic hedgehog.

Sonic hedgehog (Shh) is a secreted glycoprotein expressed by the notochord and floor plate that is involved in the induction and specification of ventral phenotypes in the vertebrate neural tube. Recently, Shh has also been shown to promote the survival of cultured rat embryo ventral brain and spinal cord cells. We have examined whether Shh can promote the survival of chick embryo neurons in vivo or in vitro. In the chick, Shh is expressed in notochord, floor plate, and ventral neural tube/spinal cord at several stages at which programmed cell death (PCD) occurs. However, the administration of exogenous Shh to embryos in vivo or to motoneuron cultures at these stages failed to promote the survival of several different neuronal populations, including spinal motoneurons, spinal interneurons, sympathetic preganglionic neurons, sensory neurons, and neuronal precursor cells. Rather, at the earliest stage of PCD examined here (embryonic day 3) Shh selectively induced the death of ventral neuronal precursors and floor-plate cells, resulting in a net loss of cells in the neural tube. Altered concentrations of Shh induce aberrant phenotypes that are removed by PCD. Accordingly, normal PCD in the early neural tube may play a role in dorsal-ventral patterning.

Development of serotoninergic system in the brain and spinal cord of the chick.

(1) Development of serotonin positive cells and fibers was immunohistochemically studied by the use of an antibody against serotonin. (2) Serotoninergic neurons were first observed in the immature rohmbencephalon raphe nuclei on embryonic day (E)4, where two clusters of serotonin positive neurons were located: one observed at the rostral part of the rohmbencephalon corresponding to the dorsal raphe nuclei had many serotonin positive cells: the other located at the caudal part of the rohmbencephalon corresponding to the medullary raphe nuclei of the adult animals had only a small number of serotoninergic cells. (3) By E8 the number of serotonin positive cells in the brain stem increased, and virtually all the raphe nuclei found in an adult animal were located. (4) Serotonin positive fibers in the marginal layer reached up to the diencephalon and telencephalon on E6 and E8, respectively. (5) Serotonin positive cells were found beside the midline regions in the ventral part of the spinal cord of the embryonic as well as posthatching chick. (6) Because almost all the serotoninergic fibers in the spinal cord originated from the brain stem raphe nuclei, propriospinal serotonin positive cells were considered as phylogenetic vestiges. (7) Serotoninergic fibers were first found in the marginal layer of the cervical and lumbar spinal cord on E6 and E8, respectively. (8) There was a waiting period of a few days before they penetrated into the mantle layer. (9) Terminal arbolization of the serotoninergic fibers started from late embryonic periods (E16 less than), and was maximized within one week of hatching. (10) Thereafter the density of serotonin positive fibers decreased in all the regions of the spinal cord. (11) Developmental changes of the density of serotonin determined with a high performance liquid chromatography were the same as those determined through immunohistochemistry. Namely the density of serotonin increased linearly from E6 to hatching period, and reached the maximum value one week posthatching. (12( The density of the serotonin in the adult spinal cord was about half of the maximum value. (13) It is to say that the densities of serotonin and serotoninergic fibers transiently increased around one week posthatching. (14) Following the transient increase serotoninergic fibers were eliminated from the neuropil, the fibers were localized in the specific regions of the motor nucleus: motor neuron pools of extensor muscles of the hip joint in the lumbosacral spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential expression of the GDNF family receptors RET and GFRα1, 2, and 4 in subsets of motoneurons: A relationship between motoneuron birthdate and receptor expression

Previous studies have demonstrated the expression of specific members of the glial cell line‐derived neurotrophic factor (GDNF) receptor family α (GFRα) in subsets of motoneurons (MNs) in the developing mouse spinal cord. We examined the expression pattern of GFRα and RET in the avian lumbar spinal cord during the period of programmed cell death (PCD) of MNs by using double labeling in situ hybridization and immunohistochemistry. In the lateral motor column (LMC) of the lumbar spinal cord, a laminar organization of GFRα expression was observed: GFRα1‐positive MNs were located in the medial LMC; GFRα1‐, 2‐, and 4‐positive MNs were situated in the lateral LMC; and GFRα4‐positive MNs were located in the intermediate LMC. The species of GFRα receptor that was expressed in MNs was found to be related to their birthdates. The expression of subpopulation‐specific transcriptional factors was also used to define MNs that express a specific pattern of GFRα. This analysis suggests that motor pools as defined by these transcriptional factors have unique expression patterns of GFRα receptor. Early limb bud ablation did not affect the expression of GFRα in the spinal cord, indicating that regulation of receptor expression is independent of target‐derived signals. Finally, GDNF mRNA expression was found in the limb during the PCD period of MNs. In conclusion, these results indicate that time of withdrawal from the mitotic cycle may specify the expression pattern of GFRα in subsets of MNs and that GDNF may function as a target‐derived neurotrophic factor for specific subpopulations of MNs. J. Comp. Neurol. 456:245–259, 2003.

Distribution patterns of dendrites in motor neuron pools of lumbosacral spinal cord of the chicken.

SummaryThe morphology of dendritic trees (dendroarchitecture) of motor neurons innervating specific hindlimb muscles (motoneuron pools, MNP) was studied in the chick spinal cord. Motoneurons were labelled by intramuscular injections of horseradish peroxidase conjugated with cholera toxin subunit B. MNPs of posterior iliotibial and femorotibial muscles were located at the dorsolateral part of lateral motor column of lumbosacral segments (LS) 1–4 and 1–3, respectively. Although the dendritic profiles of femorotibialis motoneurons were fewer than those of posterior iliotibialis, these two MNPs had a similar distribution pattern of dendrites. Dendritic profiles were about equally distributed in the gray and white matter. Dendrites from the MNP of posterior iliotibialis radiated in all directions. A large number of dendrites penetrated into the white matter, and some even reached to the subpial regions of the lateral funiculus. One array of dendrites that projected dorsomedialwards extended to the base of the posterior horn. MNPs of both the iliofibularis (LS 4–7) and caudilioflexorius (LS 6–8) had dendritic trees with similar distribution patterns. There were two main arrays of dendritic extensions; one along the dorsal, and another along the ventral border of the lateral motor column. Dendrites from the iliofibularis and caudilioflexorius motoneurons were located more frequently in the white matter than in the gray matter. A large number of dendrites extended in all directions from the MNP of the adductor muscle, which was located in the medial region of lateral motor column of LS 1–2. The distribution of dendrites from a few other MNPs was also examined. From these observations, we conclude that there are major differences in the distribution of dendrites of MNPs innervating different chick hind limb muscles. We discuss the possibility that these differences may be associated with differences in the quantity or quality of afferent inputs received by motoneurons in the various MNPs.

Differential innervation of specific motor neuron pools by serotoninergic fibers in the chick spinal cord

A new technique in which cholera toxin subunit B conjugated to horseradish peroxidase is injected in chick muscles followed by perfusion with Zambonis fixative for serotonin immunocytochemistry allows one to visualize immunoreactive fibers and retrogradely labelled motoneurons in alternate sections. Using these procedures, we have found that there is a differential innervation by serotoninergic fibers of motoneuron pools that project to specific muscles or muscle groups. Dense clusters of serotonin-positive fibers were located in the motoneuron pools of extensors of the hip joint consisting of the lateral iliotibialis, ischioflexorius, iliofibularis, accessorius and caudilioflexorius muscles.

The terminal distribution pattern of spinocerebellar fibers

SummaryThe terminal fields of spinocerebellar fibers from different levels (cervical, thoracic and lumbar) of the spinal cord in the chick were determined by using wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), an anterograde labelling technique. More terminals were found in the anterior lobe than in the posterior lobe. Following injections in the lumbar spinal cord, mossy fiber terminals were found mostly in the anterior lobe (lobules I–V). The highest density of labelled terminals was found in lobule I, and labelled terminals were found in all parts of the lobule. However in lobules II–IV, labelled fibers were mediolaterally arranged in three longitudinal strips on both sides of the midline, and formed a distinct zonal distribution pattern. Labelled terminals were distributed evenly from the apical to basal regions of lobules I–V. A large number of mossy fiber terminals originating from the thoracic spinal cord were located in the anterior lobe and lobule VI; some labelled terminals were found in lobule IX. Three less distinct longitudinal strips, compared to those following WGA-HRP injections in the lumbar spinal cord, were recognized on each side of the midline. Labelled mossy terminals were observed in lobules II–IX following WGA-HRP injections in the cervical spinal cord. In transverse sections two longitudinal strips were found at the apical parts of lobules II and III, whereas four thin longitudinal strips were located in lobules IV and V.The present study showed that the terminal fields of spinocerebellar fibers from each level of the spinal cord have different distribution patterns.

IMMUNOHISTOCHEMICAL STUDY OF TYROSINE-HYDROXYLASE-POSITIVE CELLS AND FIBERS IN THE CHICKEN SPINAL CORD

Tyrosine hydroxylase (TH)-positive cells and fibers were examined by immunohistochemistry in the chick spinal cord. TH-positive cells, which were located in laminae I, V and X, were most frequently found in the rostral part of the cervical spinal cord, with fewer cells being found in more caudal levels of the spinal cord. TH-positive cells located in lamina X, which were bipolar in shape, were mainly found in regions lateral as well as just ventral to the central canal. They had processes reaching to the central canal. The terminals of these cerebrospinal-fluid-contacting cells were oval in shape, and were most frequently found at the ventral wall of the central canal. There were dense clusters of TH-positive fibers in lamina X. A meshwork-like structure of TH-positive fibers was found over the lateral wall of the central canal. A high density of TH-positive fibers was also found in the medial part of laminae V-VII. In lamina IX, small numbers of TH-positive fibers were observed in the lateral motor column of the brachial spinal cord, and in the medial and lateral motor columns of the lumbosacral spinal cord. However, within the medial motor column of the brachial spinal cord TH-positive fibers were densely distributed around somal as well as dendritic profiles. Similar to our previous observations on serotoninergic fibers. TH-positive fibers were also differentially distributed in the ventral horn of the chicken spinal cord: a high density of TH-positive fibers was localized to specific regions of the spinal motor nucleus.