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In JoVE (2)
- Dechiffrera axonal vägar genetiskt definierade grupper av nervceller i Chick Neural Tube Använda I ovo Elektroporation
- Elektroporering av hindbrain att spåra axonal Trajectories och Synaptic Mål i Chick Embryo
Other Publications (13)
- Development (Cambridge, England)
- Journal of Neuroimmunology
- The International Journal of Biochemistry & Cell Biology
- Journal of Neurochemistry
- The Journal of Cell Biology
- Molecular Cancer Research : MCR
- Neural Development
- PLoS Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Molecular and Cellular Neurosciences
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- PloS One
- Development (Cambridge, England)
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Articles by Avihu Klar in JoVE
JoVE General
Dechiffrera axonal vägar genetiskt definierade grupper av nervceller i Chick Neural Tube Använda I ovo Elektroporation
Denna video visar hur du visualisera axonal vägar genetiskt definierade grupper av nervceller i embryonala ungen ryggmärgen att utnyttja
JoVE Neuroscience
Elektroporering av hindbrain att spåra axonal Trajectories och Synaptic Mål i Chick Embryo
1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, 2Department of Medical Neurobiology, The Hebrew University of Jerusalem
Hur neuronala nätverk är etablerade i den embryonala hjärnan är en grundläggande fråga i utvecklingspsykologi neurobiologi. Här har vi kombinerat en elektro teknik med nya genetiska verktyg, såsom Cre / Lox-plasmider och piggyBac-medierad DNA införlivande systemet i aviär hindbrain att märka rygg interneurons och spåra sina axonal prognoser och synaptiska mål på olika utvecklingsstadier.
Other articles by Avihu Klar on PubMed
Irx4-mediated Regulation of Slit1 Expression Contributes to the Definition of Early Axonal Paths Inside the Retina
Although multiple axon guidance cues have been discovered in recent years, little is known about the mechanism by which the spatiotemporal expression patterns of the axon guidance cues are regulated in vertebrates. We report that a homeobox gene Irx4 is expressed in a pattern similar to that of Slit1 in the chicken retina. Overexpression of Irx4 led to specific downregulation of Slit1 expression, whereas inhibition of Irx4 activity by a dominant negative mutant led to induction of Slit1 expression, indicating that Irx4 is a crucial regulator of Slit1 expression in the retina. In addition, by examining axonal behavior in the retinas with overexpression of Irx4 and using several in vivo assays to test the effect of Slit1, we found that Slit1 acts positively to guide the retinal axons inside the optic fiber layer (OFL). We further show that the regulation of Slit1 expression by Irx4 is important for providing intermediate targets for retinal axons during their growth within the retina.
Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) Activity is Regulated by a GM-CSF Binding Molecule in Wallerian Degeneration Following Injury to Peripheral Nerve Axons
The hematopoietic factor and inflammatory cytokine GM-CSF is involved in PNS and CNS injury and disease, and in macrophage and microglia function regulation. We presently document that injury to PNS axons induces in vivo production of GM-CSF-inhibitor and GM-CSF-augmenter activities. GM-CSF-inhibitor activity was detected in extract and conditioned medium (CM) of injured PNS but not in extract of intact PNS, and was removed from CM by GM-CSF affinity chromatography, suggesting it is carried by a secreted GM-CSF binding molecule. CM further displayed GM-CSF-augmenter activity along with GM-CSF-inhibitor activity but at contrasting concentrations; augmentation at lowest and inhibition at highest. GM-CSF activity is thus regulated during Wallerian degeneration (WD); augmenter activity characterizes the onset and inhibitor activity the later stages of WD.
The Neuronal Class 2 TSR Proteins F-spondin and Mindin: a Small Family with Divergent Biological Activities
F-spondin and Mindin are members of a subgroup of the thrombospondin type 1 (TSR) class molecules, defined by two domains of homology, the FS1/FS2 and TSR domains. The TSRs of F-spondin proteins are typical of class 2 TSRs. F-spondin and Mindin are evolutionarily conserved proteins. The embryonic expression of the vertebrate genes is enriched in the nervous system, mainly at the floor plate and the hippocampus. Similar to thrombospondin, F-spondin and Mindin are extracellular matrix attached molecules that promote neurite outgrowth and inhibit angiogenesis. Analysis of gain and loss of function experiments reveal that F-spondin is required for accurate pathfinding of embryonic axons. F-spondin plays a dual role in patterning axonal trajectories: it promotes the outgrowth of commissural and inhibits the outgrowth of motor axons. Macrophages of Mindin-deficient mice exhibit defective responses to a broad spectrum of microbial stimuli. This may implicate Mindin and F-spondin in inflammatory processes in the nervous system.
F-spondin Promotes Nerve Precursor Differentiation
Cells in the developing nervous system secrete a large number of proteins that regulate the migration and differentiation of their neighbors. It is shown here that a clonal central nervous system cell line secretes a protein that causes both a rat hippocampal progenitor cell line and primary cortical neural cells to differentiate into cells with the morphological and biochemical features of neurons. This protein was identified as F-spondin. Analysis of F-spondin isoforms secreted from transfected cells shows that the core protein without the thrombospondin type 1 repeats is sufficient to promote neuronal differentiation when adsorbed to a surface. F-spondin can also inhibit neurite outgrowth while allowing the expression of nerve-specific proteins when present in a soluble form at high concentrations. Therefore, F-spondin can alter cell differentiation in multiple ways, depending upon its concentration and distribution between substrate-attached and soluble forms.
Proteolysis and Membrane Capture of F-spondin Generates Combinatorial Guidance Cues from a Single Molecule
The formation of neuronal networks is governed by a limited number of guidance molecules, yet it is immensely complex. The complexity of guidance cues is augmented by posttranslational modification of guidance molecules and their receptors. We report here that cleavage of the floor plate guidance molecule F-spondin generates two functionally opposing fragments: a short-range repellent protein deposited in the membrane of floor plate cells and an adhesive protein that accumulates at the basement membrane. Their coordinated activity, acting respectively as a short-range repellant and a permissive short-range attractant, constricts commissural axons to the basement membrane beneath the floor plate cells. We further demonstrate that the repulsive activity of the inhibitory fragment of F-spondin requires its presentation by the lipoprotein receptor-related protein (LRP) receptors apolipoprotein E receptor 2, LRP2/megalin, and LRP4, which are expressed in the floor plate. Thus, proteolysis and membrane interaction coordinate combinatorial guidance signaling originating from a single guidance cue.
A Key Role for Cyclic AMP-responsive Element Binding Protein in Hypoxia-mediated Activation of the Angiogenesis Factor CCN1 (CYR61) in Tumor Cells
Hypoxia is a prominent feature of solid tumors known to contribute to malignant progression and therapeutic resistance. Cancer cells adapt to hypoxia using various pathways, allowing tumors to thrive in a low oxygen state. Induction of new blood vessel formation via the secretion of proangiogenic factors is one of the main adaptive responses engaged by tumor cells under hypoxic conditions. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays a pivotal role in mediating such responses. In addition, several other transcription factors have also been implicated in hypoxic gene regulation, either independently or in cooperation with HIF-1. In this work, we show that the expression of the angiogenesis-related, immediate early gene CCN1 (formerly known as CYR61), considered to be involved in tumor growth and invasiveness, is enhanced upon hypoxia stress primarily in a protein kinase A and cyclic AMP-responsive element binding protein (CREB) and CRE-dependent manner in various cell lines. The hypoxia-mediated activation of the CCN1 promoter is independent of HIF-1 and HIF-2, as shown by small interfering RNA knockdown. We identify the cis element in the mouse CCN1 promoter responsible for CREB binding to be one of two partial CRE sites present in the promoter. Moreover, we report for the first time that CREB-mediated CCN1 transcription is enhanced in hypoxic regions of tumors in vivo. Identifying and characterizing the molecular mechanisms that govern the response of tumors to hypoxia may be instrumental to identify the tumors that will respond favorably to inhibition of angiogenesis and thus lead to the development of treatments that could complement hypoxia-inducing treatment modalities.
Transcriptional Control of Axonal Guidance and Sorting in Dorsal Interneurons by the Lim-HD Proteins Lhx9 and Lhx1
Lim-HD proteins control crucial aspects of neuronal differentiation, including subtype identity and axonal guidance. The Lim-HD proteins Lhx2/9 and Lhx1/5 are expressed in the dorsal spinal interneuron populations dI1 and dI2, respectively. While they are not required for cell fate acquisition, their role in patterning the axonal trajectory of dI1 and dI2 neurons remains incompletely understood.
Foxp1 and Lhx1 Coordinate Motor Neuron Migration with Axon Trajectory Choice by Gating Reelin Signalling
Topographic neuronal maps arise as a consequence of axon trajectory choice correlated with the localisation of neuronal soma, but the identity of the pathways coordinating these processes is unknown. We addressed this question in the context of the myotopic map formed by limb muscles innervated by spinal lateral motor column (LMC) motor axons where the Eph receptor signals specifying growth cone trajectory are restricted by Foxp1 and Lhx1 transcription factors. We show that the localisation of LMC neuron cell bodies can be dissociated from axon trajectory choice by either the loss or gain of function of the Reelin signalling pathway. The response of LMC motor neurons to Reelin is gated by Foxp1- and Lhx1-mediated regulation of expression of the critical Reelin signalling intermediate Dab1. Together, these observations point to identical transcription factors that control motor axon guidance and soma migration and reveal the molecular hierarchy of myotopic organisation.
Motor and Dorsal Root Ganglion Axons Serve As Choice Points for the Ipsilateral Turning of DI3 Axons
The axons of the spinal intersegmental interneurons are projected longitudinally along various funiculi arrayed along the dorsal-ventral axis of the spinal cord. The roof plate and the floor plate have a profound role in patterning their initial axonal trajectory. However, other positional cues may guide the final architecture of interneuron tracks in the spinal cord. To gain more insight into the organization of specific axonal tracks in the spinal cord, we focused on the trajectory pattern of a genetically defined neuronal population, dI3 neurons, in the chick spinal cord. Exploitation of newly characterized enhancer elements allowed specific labeling of dI3 neurons and axons. dI3 axons are projected ipsilaterally along two longitudinal fascicules at the ventral lateral funiculus (VLF) and the dorsal funiculus (DF). dI3 axons change their trajectory plane from the transverse to the longitudinal axis at two novel checkpoints. The axons that elongate at the DF turn at the dorsal root entry zone, along the axons of the dorsal root ganglion (DRG) neurons, and the axons that elongate at the VLF turn along the axons of motor neurons. Loss and gain of function of the Lim-HD protein Isl1 demonstrate that Isl1 is not required for dI3 cell fate. However, Isl1 is sufficient to impose ipsilateral turning along the motor axons when expressed ectopically in the commissural dI1 neurons. The axonal patterning of dI3 neurons, revealed in this study, highlights the role of established axonal cues-the DRG and motor axons-as intermediate guidepost cues for dI3 axons.
Foxa2 Regulates the Expression of Nato3 in the Floor Plate by a Novel Evolutionarily Conserved Promoter
The development of the neural tube into a complex central nervous system involves morphological, cellular and molecular changes, all of which are tightly regulated. The floor plate (FP) is a critical organizing center located at the ventral-most midline of the neural tube. FP cells regulate dorsoventral patterning, differentiation and axon guidance by secreting morphogens. Here we show that the bHLH transcription factor Nato3 (Ferd3l) is specifically expressed in the spinal FP of chick and mouse embryos. Using in ovo electroporation to understand the regulation of the FP-specific expression of Nato3, we have identified an evolutionarily conserved 204 bp genomic region, which is necessary and sufficient to drive expression to the chick FP. This promoter contains two Foxa2-binding sites, which are highly conserved among distant phyla. The two sites can bind Foxa2 in vitro, and are necessary for the expression in the FP in vivo. Gain and loss of Foxa2 function in vivo further emphasize its role in Nato3 promoter activity. Thus, our data suggest that Nato3 is a direct target of Foxa2, a transcription activator and effector of Sonic hedgehog, the hallmark regulator of FP induction and spinal cord development. The identification of the FP-specific promoter is an important step towards a better understanding of the molecular mechanisms through which Nato3 transcription is regulated and for uncovering its function during nervous system development. Moreover, the promoter provides us with a powerful tool for conditional genetic manipulations in the FP.
Axonal Patterns and Targets of DA1 Interneurons in the Chick Hindbrain
Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is formed along the dorsal-most region of the caudal rhombic lip, gives rise to the cochlear and precerebellar nuclei. These centers project sensory inputs toward upper-brain regions. The fundamental role of dA1 interneurons in the assembly and function of these brainstem nuclei is well characterized. However, the precise en route axonal patterns and synaptic targets of dA1 interneurons are not clear as of yet. Novel genetic tools were used to label dA1 neurons and trace their axonal trajectories and synaptic connections at various stages of chick embryos. Using dA1-specific enhancers, two contralateral ascending axonal projection patterns were identified; one derived from rhombomeres 6-7 that elongated in the dorsal funiculus, while the other originated from rhombomeres 2-5 and extended in the lateral funiculus. Targets of dA1 axons were followed at later stages using PiggyBac-mediated DNA transposition. dA1 axons were found to project and form synapses in the auditory nuclei and cerebellum. Investigation of mechanisms that regulate the patterns of dA1 axons revealed a fundamental role of Lim-homeodomain (HD) proteins. Switch in the expression of the specific dA1 Lim-HD proteins Lhx2/9 into Lhx1, which is typically expressed in dB1 interneurons, modified dA1 axonal patterns to project along the routes of dB1 subgroup. Together, the results of this research provided new tools and knowledge to the assembly of trajectories and connectivity of hindbrain dA1 interneurons and of molecular mechanisms that control these patterns.
Distinct Cis Regulatory Elements Govern the Expression of TAG1 in Embryonic Sensory Ganglia and Spinal Cord
Cell fate commitment of spinal progenitor neurons is initiated by long-range, midline-derived, morphogens that regulate an array of transcription factors that, in turn, act sequentially or in parallel to control neuronal differentiation. Included among these are transcription factors that regulate the expression of receptors for guidance cues, thereby determining axonal trajectories. The Ig/FNIII superfamily molecules TAG1/Axonin1/CNTN2 (TAG1) and Neurofascin (Nfasc) are co-expressed in numerous neuronal cell types in the CNS and PNS - for example motor, DRG and interneurons - both promote neurite outgrowth and both are required for the architecture and function of nodes of Ranvier. The genes encoding TAG1 and Nfasc are adjacent in the genome, an arrangement which is evolutionarily conserved. To study the transcriptional network that governs TAG1 and Nfasc expression in spinal motor and commissural neurons, we set out to identify cis elements that regulate their expression. Two evolutionarily conserved DNA modules, one located between the Nfasc and TAG1 genes and the second directly 5' to the first exon and encompassing the first intron of TAG1, were identified that direct complementary expression to the CNS and PNS, respectively, of the embryonic hindbrain and spinal cord. Sequential deletions and point mutations of the CNS enhancer element revealed a 130bp element containing three conserved E-boxes required for motor neuron expression. In combination, these two elements appear to recapitulate a major part of the pattern of TAG1 expression in the embryonic nervous system.
A Dynamic Code of Dorsal Neural Tube Genes Regulates the Segregation Between Neurogenic and Melanogenic Neural Crest Cells
Understanding when and how multipotent progenitors segregate into diverse fates is a key question during embryonic development. The neural crest (NC) is an exemplary model system with which to investigate the dynamics of progenitor cell specification, as it generates a multitude of derivatives. Based on 'in ovo' lineage analysis, we previously suggested an early fate restriction of premigratory trunk NC to generate neural versus melanogenic fates, yet the timing of fate segregation and the underlying mechanisms remained unknown. Analysis of progenitors expressing a Foxd3 reporter reveals that prospective melanoblasts downregulate Foxd3 and have already segregated from neural lineages before emigration. When this downregulation is prevented, late-emigrating avian precursors fail to upregulate the melanogenic markers Mitf and MC/1 and the guidance receptor Ednrb2, generating instead glial cells that express P0 and Fabp. In this context, Foxd3 lies downstream of Snail2 and Sox9, constituting a minimal network upstream of Mitf and Ednrb2 to link melanogenic specification with migration. Consistent with the gain-of-function data in avians, loss of Foxd3 function in mouse NC results in ectopic melanogenesis in the dorsal tube and sensory ganglia. Altogether, Foxd3 is part of a dynamically expressed gene network that is necessary and sufficient to regulate fate decisions in premigratory NC. Their timely downregulation in the dorsal neural tube is thus necessary for the switch between neural and melanocytic phases of NC development.
