Immunocytochemical detection of Reissner's fiber-like glycoproteins in the subcommissural organ and the floor plate of wildtype and cyclops mutant zebrafish larvae

The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glycoproteins that polymerize in the form of the Reissner's fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish larvae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ventricle, over the SCO surface, thin immunoreactive fibers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pontine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an immunoreactive SCO and fibrous material in the ventricle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some specimens in which the caudal FP persisted and was immunoreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glycoproteins but for their polymerization into an RF in the brain ventricles.     

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.     

The role of floor plate contact in the elaboration of contralateral commissural projections within the embryonic mouse spinal cord

In vertebrate embryos, commissural axons extend toward and across the floor plate (FP), an intermediate target at the ventral midline (VM) of the spinal cord. After decussating, many commissural axons turn into the longitudinal plane and elaborate diverse projections. FP contact is thought to alter the responsiveness of these axons so that they can exit the FP and adopt new trajectories. However, a requirement for the FP in shaping contralateral commissural projections has not been established in higher vertebrates. Here we further analyze to what extent FP contact is necessary for the elaboration of decussated commissural projections both in cultured, FP-excised spinal cord preparations and in gli2-deficient mice, which lack a FP. In FP-lacking spinal cords, we observe a large number of appropriately projecting contralateral commissural projections in vivo and in vitro. Surprisingly, even though gli2 mutants lack a FP, slit1-3 mRNA and their receptors (Robo1/2) are expressed in a wild-type-like manner. In addition, blocking Robo-Slit interactions in FP-lacking spinal cord explants prevents commissural axons from leaving the VM and turning longitudinally. Thus, compared to FP contact, Slit-Robo interactions are more critical for driving commissural axons out of the VM and facilitating the elaboration of a subset of contralateral commissural projections.     

Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.     

Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene

The processes that specify early regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The mouse open brain (opb) gene plays an essential role in dorsal neural patterning: in the caudal spinal cord of opb mutants, dorsal cell types are absent and markers of ventral fates, including Shh, expand into dorsal regions. Analysis of the opb mutant phenotype and of opb/opb <--> wild-type chimeric embryos reveals that early in neural development, the wild-type opb gene (opb(+)) is required cell autonomously for the expression of Pax7 in dorsal cells and Pax6 in lateral cells. Thus the opb(+) gene product acts intracellularly in the reception or interpretation of signals that determine cell types in the dorsal 80% of the neural tube. At later stages, the lack of opb(+) causes a non-cell-autonomous expansion of ventral cell types into dorsal regions of the neural tube, revealing that opb(+) controls the production of a diffusible molecule that defines the domain of Shh expression. The data indicate that opb(+) could act as either a novel component of a dorsalizing pathway or a novel intracellular negative regulator of the Shh signal transduction pathway.     

Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord

The three vertebrate Gli proteins play a central role in mediating Hedgehog (Hh)-dependent cell fate specification in the developing spinal cord; however, their individual contributions to this process have not been fully characterized. In this paper, we have addressed this issue by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. We show that Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. We also show that motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, we provide evidence that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation.     

Ectopic Wnt/beta-catenin signaling induces neurogenesis in the spinal cord and hindbrain floor plate

The most ventral structure of the developing neural tube, the floor plate (FP), differs in neurogenic capacity along the neuraxis. The FP is largely non-neurogenic at the hindbrain and spinal cord levels, but generates large numbers of dopamine (mDA) neurons at the midbrain levels. Wnt1, and other Wnts are expressed in the ventral midbrain, and Wnt/beta catenin signaling can at least in part account for the difference in neurogenic capacity of the FP between midbrain and hindbrain levels. To further develop the hypothesis that canonical Wnt signaling promotes mDA specification and FP neurogenesis, we have generated a model wherein beta-catenin is conditionally stabilized throughout the FP. Here, we unambiguously show by fate mapping FP cells in this mutant, that the hindbrain and spinal cord FP are rendered highly neurogenic, producing large numbers of neurons. We reveal that a neurogenic hindbrain FP results in the altered settling pattern of neighboring precerebellar neuronal clusters. Moreover, in this mutant, mDA progenitor markers are induced throughout the rostrocaudal axis of the hindbrain FP, although TH+ mDA neurons are produced only in the rostral aspect of rhombomere (r)1. This is, at least in part, due to depressed Lmx1b levels by Wnt/beta catenin signaling; indeed, when Lmx1b levels are restored in this mutant, mDA are observed not only in rostral r1, but also at more caudal axial levels in the hindbrain, but not in the spinal cord. Taken together, these data elucidate both patterning and neurogenic functions of Wnt/beta catenin signaling in the FP, and thereby add to our understanding of the molecular logic of mDA specification and neurogenesis.     

Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

Dynactin is a complex motor protein involved in the retrograde axonal transport disturbances of which may lead to amyotrophic lateral sclerosis (ALS). Mice with hSOD1G93A mutation develop ALS-like symptoms and are used as a model for the disease studies. Similar symptoms demonstrate Cra1 mice, with Dync1h1 mutation. Dynactin heavy (DCTN1) and light (DCTN3) subunits were studied in the CNS of humans with sporadic ALS (SALS), mice with hSOD1G93A (SOD1/+), Dync1h1 (Cra1/+), and double (Cra1/SOD1) mutation at presymptomatic and symptomatic stages. In SALS subjects, in contrast to control cases, expression of DCTN1-mRNA but not DCTN3-mRNA in the motor cortex was higher than in the sensory cortex. However, the mean levels of DCTN1-mRNA and protein were lower in both SALS cortexes and in the spinal cord than in control structures. DCTN3 was unchanged in brain cortexes but decreased in the spinal cord on both mRNA and protein levels. In all SALS tissues immunohistochemical analyses revealed degeneration and loss of neuronal cells, and poor expression of dynactin subunits. In SOD1/+ mice both subunits expression was significantly lower in the frontal cortex, spinal cord and hippocampus than in wild-type controls, especially at presymptomatic stage. Fewer changes occurred in Cra1/SOD1 and Cra1/+ mice.It can be concluded that in sporadic and SOD1-related ALS the impairment of axonal retrograde transport may be due to dynactin subunits deficiency and subsequent disturbances of the whole dynein/dynactin complex structure and function. The Dync1h1 mutation itself has slight negative effect on dynactin expression and it alleviates the changes caused by SOD1G93A mutation.     

Localization of the netrin guidance receptor, DCC, in the developing peripheral and enteric nervous systems

Over recent years the secreted guidance cue, netrin-1, and its receptor, DCC, have been shown to be an essential guidance system driving axon pathfinding within the developing vertebrate central nervous system (CNS). Mice lacking DCC exhibit severe defects in commissural axon extension towards the floor plate demonstrating that the DCC-netrin guidance system is largely responsible for directing axonal projections toward the ventral midline in the developing spinal cord (Fazeli et al., Nature 386 (1997) 796). In addition, these mutants lack several major commissures within the forebrain, including the corpus callosum and the hippocampal commissure. In contrast to the CNS, the role of the DCC guidance receptor in the development of the mammalian peripheral and enteric nervous systems (PNS and ENS) has not been investigated. Here we demonstrate using immunohistochemical analysis that the DCC receptor is present in the developing mouse PNS where it is found on spinal, segmental, and sciatic nerves, and in developing sensory ganglia and their associated axonal projections. In addition, DCC is present in the ENS throughout the early developmental phase.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.     

The reovirus sigma1s protein is a determinant of hematogenous but not neural virus dissemination in mice

Nonstructural protein σ1s is a critical determinant of hematogenous dissemination by type 1 reoviruses, which reach the central nervous system (CNS) by a strictly blood-borne route. However, it is not known whether σ1s contributes to neuropathogenesis of type 3 reoviruses, which disseminate by both vascular and neural pathways. Using isogenic type 3 viruses that vary only in σ1s expression, we observed that mice survived at a higher frequency following hind-limb inoculation with σ1s-null virus than when inoculated with wild-type virus. This finding suggests that σ1s is essential for reovirus virulence when inoculated at a site that requires systemic spread to cause disease. Wild-type and σ1s-null viruses produced comparable titers in the spinal cord, suggesting that σ1s is dispensable for invasion of the CNS. Although the two viruses ultimately achieved similar peak titers in the brain, loads of wild-type virus were substantially greater than those of the σ1s-null mutant at early times after inoculation. In contrast, wild-type virus produced substantially higher titers than the σ1s-null virus in peripheral organs to which reovirus spreads via the blood, including the heart, intestine, liver, and spleen. Concordantly, viral titers in the blood were higher following infection with wild-type virus than following infection with the σ1s-null mutant. These results suggest that differences in viral brain titers at early time points postinfection are due to limited virus delivery to the brain by hematogenous pathways. Transection of the sciatic nerve prior to hind-limb inoculation diminished viral spread to the spinal cord. However, wild-type virus retained the capacity to disseminate to the brain following sciatic nerve transection, indicating that wild-type reovirus can spread to the brain by the blood. Together, these results indicate that σ1s is not required for reovirus spread by neural mechanisms. Instead, σ1s mediates hematogenous dissemination within the infected host, which is required for full reovirus neurovirulence.     

Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways

The floor plate is a morphologically distinct structure of epithelial cells situated along the midline of the ventral spinal cord in vertebrates. It is a source of guidance molecules directing the growth of axons along and across the midline of the neural tube. In the zebrafish, the floor plate is about three cells wide and composed of cuboidal cells. Two cell populations can be distinguished by the expression patterns of several marker genes, including sonic hedgehog (shh) and the fork head-domain gene fkd4: a single row of medial floor plate (MFP) cells, expressing both shh and fkd4, is flanked by rows of lateral floor plate (LFP) cells that express fkd4 but not shh. Systematic mutant searches in zebrafish embryos have identified a number of genes, mutations in which visibly reduce the floor plate. In these mutants either the MFP or the LFP cells are absent, as revealed by the analysis of the shh and fkd4 expression patterns. MFP cells are absent, but LFP cells are present, in mutants of cyclops, one-eyed pinhead, and schmalspur, whose development of midline structures is affected. LFP cells are absent, but MFP cells are present, in mutants of four genes, sonic you, you, you-too, and chameleon, collectively called the you-type genes. This group of mutants also shows defects in patterning of the paraxial mesoderm, causing U- instead of V-shaped somites. One of the you-type genes, sonic you, was recently shown to encode the zebrafish Shh protein, suggesting that the you-type genes encode components of the Shh signaling pathway. It has been shown previously that in the zebrafish shh is required for the induction of LFP cells, but not for the development of MFP cells. This conclusion is supported by the finding that injection of shh RNA causes an increase in the number of LFP, but not MFP cells. Embryos mutant for iguana, detour, and umleitung share the lack of LFP cells with you-type mutants while somite patterning is not severely affected. In mutants that fail to develop a notochord, MFP cells may be present, but are always surrounded by LFP cells. These data indicate that shh, expressed in the notochord and/or the MFP cells, induces the formation of LFP cells. In embryos doubly mutant for cyclops (cyc) and sonic you (syu) both LFP and MFP cells are deleted. The number of primary motor neurons is strongly reduced in cyc;syu double mutants, while almost normal in single mutants, suggesting that the two different pathways have overlapping functions in the induction of primary motor neurons.     

Melanophore differentiation in the periodic albino mutant of Xenopus laevis

That embryonic ventral truck tissue might play a role in expression of the periodic albino mutant phenotype (ap/ap) in Xenopus laevis was suggested from the experiments of MacMillan (1980). In contrast, the present experiments, involving the culture of isolated regions of Xenopus embryos, have demonstrated that both mutant and wild-type melanoblasts differentiate independently of a ventral trunk factor. A similar conclusion, that mutant melanoblasts differentiate independently of a ventral trunk factor, is derived from observations on neural crest cultures, wherein melanization of neural crest cells in both wild-type and mutant cultures occurred in a manner consistent with their genotype.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.