Spinal Cord Of Zebrafish Embryos Research Articles

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord.

Subtypes of hypoxia-responsive cells differentiate into neurons in spinal cord of zebrafish embryos after hypoxic stress.

Neuron stem/progenitor cells (NSPCs) of zebrafish central nervous system (CNS) are known to thrive during oxygen recovery after hypoxia, but not all cell types have been fully characterised due to their heterogeneities. In addition, an in vivo model system is not available that can help us to identify what type-specific cell populations that are involved in neural regeneration and to track their cell fate after regeneration. To solve these issues, we employed a zebrafish transgenic line, huORFZ, which harbours an inhibitory upstream open reading frame of human chop mRNA fused downstream with GFP reporter and driven by cytomegalovirus promoter. When huORFZ embryos were exposure to hypoxic stress, followed by oxygen recovery, GFP was exclusively expressed in some particular cells of CNS. Unlike GFP-negative cells, all GFP-expressing cells were not apoptotic, indicating that cell populations that are able to survive after hypoxia can be identified through this approach. When GFP-expressing cells of spinal cord were studied, we found mostly NSPCs and radial glia cells (RGs), along with a few oligodendrocyte progenitor cells and oligodendrocytes, all termed as hypoxia-responsive recovering cells (HrRCs). After hypoxic stress, these GFP-positive HrRCs did not undergo apoptosis, but GFP-negative neurons did. Prolonged recovery time after hypoxia was correlated with higher proportions of GFP(+)-NSPCs and GFP(+)-RGs, in contrast to lower proportions of proliferating/differentiating GFP(-)-NSPCs and GFP(-)-RGs. Among HrRCs subtypes, only GFP(+)-NSPCs and GFP(+)-RGs proliferated, migrated and differentiated into functional neurons during oxygen recovery. When some HrRCs were ablated in the spinal cord of hypoxia-exposed huORFZ embryos, swimming performance was impaired, suggesting that HrRCs are involved in neuronal regeneration. We demonstrated type-specific cell populations able to respond sensitively to hypoxic stress in the spinal cord of zebrafish embryos and that these type-specific populations play a role in neural regeneration. Among heterogeneous cell types that exist in the spinal cord of zebrafish embryos after hypoxia, the particular cells that are resistant to hypoxia and also involved in neuronal regeneration can be clearly identified and dynamically traced using a transgenic model fish.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

Indian Hedgehog b Function Is Required for the Specification of Oligodendrocyte Progenitor Cells in the Zebrafish CNS

A subset of ventral spinal cord precursors, known as pMN precursor cells, initially generate motor neurons and then oligodendrocyte progenitor cells (OPCs), which migrate and differentiate as myelinating oligodendrocytes in the developing neural tube. The switch between motor neuron and oligodendrocyte production by the pMN neural precursors is an important step in building a functional nervous system. However, the precise mechanism that orchestrates the sequential generation of motor neurons and oligodendrocytes within the common population of pMN precursors is still unclear. The current study demonstrates that Indian Hedgehog b (Ihhb), previously known as Echidna Hedgehog, begins to be expressed in the floor plate cells of the ventral spinal cord at the time of OPC specification in zebrafish embryos. Ihhb loss-of-function analysis revealed that Ihhb function is required for OPC specification from pMN precursors by negatively regulating the proliferation of neural precursors. Finally, results showed that Sonic Hedgehog (Shh) could not replace Ihhb function in OPC specification, suggesting that Ihhb and Shh play separate roles in OPC specification. Altogether, data from the present study suggested a novel mechanism, mediated by Ihhb, for the sequential generation of motor neurons and oligodendrocytes from pMN precursors in the ventral spinal cord of zebrafish embryos.

Tcf3 function is required for the inhibition of oligodendroglial fate specification in the spinal cord of zebrafish embryos.

The generation of various subtypes of neurons and glial cells at the right time and place is crucial for the proper development of the vertebrate CNS. Although the mechanisms and factors for the regulation of neuronal diversity in the CNS have been well studied, the mechanisms regulating the sequential production of neuronal and glial cells from neural precursors remain poorly understood. This study shows that Tcf3, a member of the Lef/Tcf family of proteins, is required to inhibit the premature oligodendroglial fate specification of spinal cord precursors using the transgenic zebrafish, which expresses a dominant repressor form of Tcf3 under the control of a heat-shock inducible promoter. In addition, the data revealed that Tcf3 function in oligodendroglial fate specification is mediated independently of canonical Wnt signaling. Altogether, these results show a novel function for Tcf3 in regulating the timing of oligodendroglial fate specification in the spinal cord.

Modulation of Glial and Neuronal Migration by Lipocalin-2 in Zebrafish

BackgroundGlial cells are involved in immune and inflammatory responses in the central nervous system (CNS). Glial cells such as microglia and astrocytes also provide structural and functional support for neurons. Migration and morphological changes of CNS cells are associated with their physiological as well as pathological functions. The secreted protein lipocalin-2 (LCN2) has been previously implicated in regulation of diverse cellular processes of glia and neurons, including cell migration and morphology.MethodsHere, we employed a zebrafish model to analyze the role of LCN2 in CNS cell migration and morphology in vivo. In the first part of this study, we examined the indirect effect of LCN2 on cell migration and morphology of microglia, astrocytes, and neurons cultured in vitro.ResultsConditioned media collected from LCN2-treated astrocytes augmented migration of glia and neurons in the Boyden chamber assay. The conditioned media also increased the number of neuronal processes. Next, in order to further understand the role of LCN2 in the CNS in vivo, LCN2 was ectopically expressed in the zebrafish spinal cord. Expression of exogenous LCN2 modulated neuronal cell migration in the spinal cord of zebrafish embryos, supporting the role of LCN2 as a cell migration regulator in the CNS.ConclusionThus, LCN2 proteins secreted under diverse conditions may play an important role in CNS immune and inflammatory responses by controlling cell migration and morphology.

Zebrafish Motor Neuron Subtypes Differ Electrically Prior to Axonal Outgrowth

Different muscle targets and transcription factor expression patterns reveal the presence of motor neuron subtypes. However, it is not known whether these subtypes also differ with respect to electrical membrane properties. To address this question, we studied primary motor neurons (PMNs) in the spinal cord of zebrafish embryos. PMN genesis occurs during gastrulation and gives rise to a heterogeneous set of motor neurons that differ with respect to transcription factor expression, muscle targets, and soma location within each spinal cord segment. The unique subtype-specific soma locations and axonal trajectories of two PMNs-MiP (middle) and CaP (caudal)-allowed their identification in situ as early as 17 h postfertilization (hpf), prior to axon genesis. Between 17 and 48 hpf, CaPs and MiPs displayed subtype-specific electrical membrane properties. Voltage-dependent inward and outward currents differed significantly between MiPs and CaPs. Moreover, by 48 hpf, CaPs and MiPs displayed subtype-specific firing behaviors. Our results demonstrate that motor neurons that differ with respect to muscle targets and transcription factor expression acquire subtype-specific electrical membrane properties. Moreover, the differences are evident prior to axon genesis and persist to the latest stage studied, 2 days postfertilization.

Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos

Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch-evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA-type glutamate receptor activation. Transection experiments suggest that the circuits required for touch-evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA-type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation.

Notch-regulated perineurium development from zebrafish spinal cord

In the developing central nervous system, nerve fascicles are surrounded by a protective sheath known as the perineurium. Perineurium is composed of perineurial cells that have both epithelial and myofibroblastoid properties, including tight and gap junctions and contractility. However, the molecular mechanism that governs perineurial development remains unclear. Here we focused on fabp7a, a brain lipid binding protein that is reportedly crucial for the proper differentiation of radial glial progenitor cells. Our study reveals that fabp7a is expressed in the lateral floor plate cells, and later perineurial glia in the spinal cord of zebrafish embryos. Furthermore, Notch signaling regulated perineurial glia development by maintaining fabp7a-expressing cells, demonstrating a novel mechanism for perineurial development.

Notch‐regulated oligodendrocyte specification from radial glia in the spinal cord of zebrafish embryos

During vertebrate neural development, many dividing neuroepithelial precursors adopt features of radial glia, which are now known to also serve as neural precursors. In mammals, most radial glia do not persist past early postnatal stages, whereas zebrafish maintain large numbers of radial glia into adulthood. The mechanisms that maintain and specify radial glia for different fates are still poorly understood. We investigated formation of radial glia in the spinal cord of zebrafish and the role of Notch signaling in their maintenance and specification. We found that spinal cord precursors begin to express gfap+, a marker of radial glia, during neurogenesis and that gfap cells give rise to both neurons and oligodendrocytes. We also determined that Notch signaling is continuously required during embryogenesis to maintain radial glia, limit motor neuron formation and permit oligodendrocyte development, but that radial glia seem to be refractory to changes in Notch activity in postembryonic animals.

Axonal trajectories and distribution of GABAergic spinal neurons in wildtype and mutant zebrafish lacking floor plate cells.

The role of the midline floor plate cells in the neuronal differentiation of the spinal cord was examined by comparing putative GABAergic neurons in wildtype zebrafish embryos with those in cyc-1 mutant embryos. The mutation produces a pleiotropic recessive lethal phenotype and is severe in rostral brain regions, but its direct effect in the caudal hindbrain and the spinal cord is apparently restricted to the depletion of the midline floor plate cells. In wildtype embryos, an antibody against the neurotransmitter GABA labeled the cell bodies, axons, and growth cones of three classes of previously identified neurons; dorsal longitudinal neurons (DoLA), commissural secondary ascending neurons (CoSA), and ventral longitudinal neurons (VeLD). A novel ventral cell type, Kolmer-Agduhr (KA) neurons, was also labeled. In the cyc-1 mutant, abnormalities were observed in some, but not all, of the GABAreactive CoSA, VeLD, and KA axons, while the axonal trajectories of DoLA neurons were not affected. Furthermore, the number of KA cells was reduced in the mutant while the numbers of the other GABAreactive cells were unperturbed. These observations corroborate our earlier hypothesis that the floor plate cells are one of several guidance cues that direct axonal outgrowth near the ventral midline of the spinal cord. They also suggest that the floor plate cells may play a role in the cellular differentiation of the spinal cord of zebrafish embryos.

Growth cone guidance by floor plate cells in the spinal cord of zebrafish embryos

The spinal cord of early zebrafish embryos contains a small number of neuronal classes whose growth cones all follow stereotyped, cell-specific pathways to their targets. Two classes of spinal neurons make cell-specific turns at or near the ventral midline of the spinal cord, which is occupied by a single row of midline floor plate cells. We tested whether these cells guide the growth cones by examining embryos missing the midline floor plate cells due either to laser ablation of the cells or to a mutation (cyc-1). In these embryos the growth cones followed both normal and aberrant pathways once near the ventral midline. This suggests that normally the midline floor plate cells do provide guidance cues, but that these cues are not obligatory.

Pathfinding by identified growth cones in the spinal cord of zebrafish embryos

The spinal cord of early (18-20 hr) zebrafish embryos consists of a small number of neurons per hemisegment. The earliest neurons are identified and project growth cones that follow stereotyped, cell-specific pathways to reach their termination sites. We have studied the pathways taken by 4 of the early neurons in order to delineate the cells and structures their growth cones encounter during pathfinding. These neurons are 3 classes of commissural neurons (CoPA, CoSA, and CoB), which have contralateral longitudinal axons, and the VeLD neuron, which has an ipsilateral longitudinal axon. These growth cones encounter a defined set of cells and structures. Commissural growth cones appear to bypass the longitudinal axons of several identified neurons, including those from contralateral commissural neurons they encounter immediately following projection from the cell bodies. In contrast, these growth cones appear to extend in association with the longitudinal axons of commissural cells after crossing the ventral midline. Another set of cells of interest are the floor plate cells, a row of cells that constitute the ventral floor of the cord. At the floor plate growth cones exhibit cell-specific behaviors which may be influenced by the floor plate. (1) The floor plate may attract specific growth cones. The CoPA, CoSA, CoB, and VeLD growth cones all extend to the floor plate while other identified growth cones do not. (2) The floor plate may mediate cell-specific turns and induce some growth cones to cross the midline while inhibiting others from doing so. The commissural growth cones extend directly under the floor plate to cross the midline and turn anterior (CoPA and CoSA) or bifurcate (CoB); the VeLD growth cone turns away from the midline and extends posteriorly. (3) The floor plate may mediate changes in the substrate affinities of growth cones. Commissural growth cones bypass longitudinal pathways before they have encountered the floor plate, but not after. The description of pathfinding by these growth cones suggests that some elements in their environment are ignored while others are not. Most interestingly, a single structure (the floor plate) may mediate multiple, cell-specific effects on spinal growth cones.

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.