A clonal analysis of spinal cord development in the zebrafish

 We describe the results of a clonal analysis of spinal cord development in the zebrafish. The data were obtained from embryos in which fluorescent lineage tracer was injected into single cells in the neural plate at the two-somite stage. Injected animals were allowed to survive until either 4 days or 2 weeks postfertilization. In other experiments, bromodeoxy uridine (BrdU) was injected intraperitoneally at 30 h postfertilization (hpf) after lineage tracer injection in the neural plate at the two-somite stage, and the embryos fixed at 38 hpf. We restricted our experiments to the thoracic region of the spinal cord. Our experiments were aimed at answering questions regarding the proliferative abilities of neuroepithelial cells during embryonic development (as deduced from the size of the clones), the modes of cell division (as deduced from the uptake of BrdU into clone cells), positional differences in the proliferation of cells within the neural plate itself, the cellular composition of the clones, and cell dispersion (deduced from the regional distribution of clone cells). Received: 30 December 1994 / Accepted: 9 March 1997     

Alarm substance from adult zebrafish alters early embryonic development in offspring

Alarm substances elicit behavioural responses in a wide range of animals but effects on early embryonic development are virtually unknown. Here we investigated whether skin injury-induced alarm substances caused physiological responses in embryos produced by two Danio species (Danio rerio and Danio albolineatus). Both species showed more rapid physiological development in the presence of alarm substance, although there were subtle differences between them: D. rerio had advanced muscle contraction and heart function, whereas D. albolineatus had advanced heart function only. Hence, alarm cues from injured or dying fish may be of benefit to their offspring, inducing physiological responses and potentially increasing their inclusive fitness.     

Changes within maturing neurons limit axonal regeneration in the developing spinal cord

Embryonic birds and mammals display a remarkable ability to regenerate axons after spinal injury, but then lose this ability during a discrete developmental transition. To explain this transition, previous research has emphasized the emergence of myelin and other inhibitory factors in the environment of the spinal cord. However, research in other CNS tracts suggests an important role for neuron-intrinsic limitations to axon regeneration. Here we re-examine this issue quantitatively in the hindbrain-spinal projection of the embryonic chick. Using heterochronic cocultures we show that maturation of the spinal cord environment causes a 55% reduction in axon regeneration, while maturation of hindbrain neurons causes a 90% reduction. We further show that young neurons transplanted in vivo into older spinal cord can regenerate axons into myelinated white matter, while older axons regenerate poorly and have reduced growth cone motility on a variety of growth-permissive ligands in vitro, including laminin, L1, and N-cadherin. Finally, we use video analysis of living growth cones to directly document an age-dependent decline in the motility of brainstem axons. These data show that developmental changes in both the spinal cord environment and in brainstem neurons can reduce regeneration, but that the effect of the environment is only partial, while changes in neurons by themselves cause a nearly complete reduction in regeneration. We conclude that maturational events within neurons are a primary cause for the failure of axon regeneration in the spinal cord.     

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development

Background  

Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.     

First transplantation of embryonic serotonergic neurons in primate spinal cord

Three adult monkeys (Macaca fascicularis) underwent a total section of the spinal cord at thoracic level (T6). 1 week later, two of them received at T8 level an injection of a cellular suspension prepared from the raphe region of a foetal macaque 39 days old. The third animal received one injection of buffer solution. 1 month later, the animals were sacrificed, and their spinal cord was processed for the immunocytochemical detection of serotonin with light and electron microscopy. Serotonergic neurons had survived after transplantation, and had grown axons and dendrites. Afferent and efferent synapses to serotonergic neurons were readily identified.     

The development of a population of spinal cord neurons and their axonal projections revealed by GABA immunocytochemistry in frog embryos

The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77–86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

gsh1 demarcates hypothalamus and intermediate spinal cord in zebrafish

We have cloned and characterized the expression of zebrafish genetic screen homeobox 1 (gsh1). Early expression is confined to hindbrain rhombomeres; by mid-somitogenesis gsh1 is expressed in precise domains within the mesencephalon and diencephalon, as well as in intermediate spinal cord. Double-label experiments revealed that the diencephalic domain is coincident with hypothalamus and that spinal cord expression is in a region that generates interneurons. These data suggest gsh1 may play a role in patterning cell types generated in these domains.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

The JIP1 scaffold protein regulates axonal development in cortical neurons

The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling.JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1-4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification,it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.     

Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo

In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.     

Synaptic localization and axonal targeting of agrin secreted by ventral spinal cord neurons in culture

Agrin secreted by motor neurons is a critical signal for postsynaptic differentiation at the developing neuromuscular junction. We used cultures of chick ventral spinal cord neurons with rat myotubes and immunofluorescence with species-specific antibodies to determine the distribution of agrin secreted by neurons and compare it to the distribution of agrin secreted by myotubes. In addition, we determined the distribution of agrin secreted by isolated chick ventral spinal cord neurons and rat motor neurons grown on a substrate that binds agrin. In cocultures, neuronal agrin was concentrated along axons at sites of axon-induced acetylcholine receptor (AChR) aggregation and was found at every such synaptic site, consistent with its role in synaptogenesis. Smaller amounts of agrin were found on dendrites and cell bodies and rarely were associated with AChR aggregation. Muscle agrin, recognized by an antibody against rat agrin, was found at nonsynaptic sites of AChR aggregation but was not detected at synaptic sites, in contrast to neuronal agrin. In cultures of isolated chick neurons or rat motor neurons, agrin was deposited relatively uniformly around axons and dendrites during the first 2-3 days in culture. In older cultures, agrin immunoreactivity was markedly more intense around axons than dendrites, indicating that motor neurons possess an intrinsic, developmentally regulated program to target agrin secretion to axons.     

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine “irregular” patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.     

Neurovascular development in the embryonic zebrafish hindbrain

The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos.