Nervous System Midline Research Articles

Glial cell derived pathway directs regenerating optic nerve axons toward the CNS midline.

Several RGC intrinsic signaling pathways have been shown to enhance RGC survival and RGC axonal growth after optic nerve injury. Yet an unresolved challenge for regenerating RGC axons is to properly navigate the optic chiasm located at the Central Nervous System midline. Here, we use live-cell imaging in larval zebrafish to show that regrowing RGC axons initiate growth toward the midline and extend along a trajectory similar to their original projection. From a candidate genetic screen, we identify the glycosyltransferase Lh3 to be required during the process of regeneration to direct regrowing RGC axons toward the midline. Moreover, we find that mutants in collagen 18a1 (col18a1), a putative Lh3 substrate, display RGC axonal misguidance phenotypes similar to those we observe in lh3 mutants, suggesting that lh3 may act through col18a1 during regeneration. Finally, we show that transgenic lh3 expression in sox10+ presumptive olig2+ oligodendrocytes located near the optic chiasm restores directed axonal growth. Combined these data identify lh3 and col18a1 as part of a glial derived molecular pathway critical for guiding in vivo regenerating RGC axons towards and across the optic chiasm.

Midline Assembloids Reveal Regulators of Human Axon Guidance.

Organizers are specialized cell populations that orchestrate cell patterning and axon guidance in the developing nervous system. Although non-human models have led to fundamental discoveries about the organization of the nervous system midline by the floor plate, an experimental model of human floor plate would enable broader insights into regulation of human neurodevelopment and midline connectivity. Here, we have developed stem cell-derived organoids resembling human floor plate (hFpO) and assembled them with spinal cord organoids (hSpO) to generate midline assembloids (hMA). We demonstrate that hFpO promote Sonic hedgehog-dependent ventral patterning of human spinal progenitors and Netrin-dependent guidance of human commissural axons, paralleling non-human models. To investigate evolutionary-divergent midline regulators, we profiled the hFpO secretome and identified 27 evolutionarily divergent genes between human and mouse. Utilizing the hMA platform, we targeted these candidates in an arrayed CRISPR knockout screen and reveal that GALNT2 , a gene involved in O-linked glycosylation, impairs floor plate-mediated guidance of commissural axons in humans. This novel platform extends prior axon guidance discoveries into human-specific neurobiology with implications for mechanisms of nervous system evolution and neurodevelopmental disorders.

Identification of a novel downstream single-minded midline regulatory element in Drosophila melanogaster.

Development of the Drosophila melanogaster central nervous system midline depends on the gene single-minded ( sim ). Although sim regulation has been studied extensively, the fact that an enhancer mediating late embryonic sim transcription has not been identified suggests that additional regulatory sequences remain unknown. We tested several evolutionarily conserved sequences in the sim downstream region and isolated sim_3pB , whose midline activity in a reporter gene assay begins later than previously characterized sim enhancers. Its activity shares several key similarities with the Aedes aegypti sim _ 5P3 enhancer, though is sufficiently different to warrant further investigation into how sim_3pB functions in its native context.

Diagnostic and therapeutical approaches to H3K27M-altered diffuse midline glioma in children: a review

H3K27M-altered diffuse midline gliomas are the most devastating pediatric brain tumors. These tumors are characterized by lesion of central nervous system midline structures, diffuse infiltrative growth and fatal prognosis. The pathogenesis of H3K27M-altered diffuse midline glioma is based on unique epigenetic and genetic changes which are associated with histone 3 (H3) alterations. Clinical disease course usually is non-specific, that could hamper diagnosis establishment and defines high prevalence of disseminated tumor stages. Diagnostic approach includes neuroimaging, various laboratory and molecular methods, including high throughput sequencing, which allows finding potential targets for precise therapy. Despite the availability of anti-tumor technologies, including targeted therapy and immunotherapy, the standard of care for H3K27M-altered diffuse midline glioma is radiation therapy, which does not allow achieving long-term event-free survival. A dismal prognosis and absence of curative options for these tumors determine the necessity of new treatment methods search that could improve patients’ outcome. In this article we present current worldwide data of the diagnosis and treatment trends in H3K27M-altered diffuse midline glioma.

Analysis of Axon Guidance in the Drosophila Embryo.

The establishment of neural connectivity is a major part of neural development. The central nervous system (CNS) midline is the most characterized axon guidance choice point, and work in Drosophila has played a pivotal role in understanding the molecular mechanisms responsible. Axons respond to attractive cues such as Netrin via the Frazzled receptor, and repulsive cues such as Slit via Robo receptors. Both signals are expressed at the CNS midline, affect pioneer axons, and have dramatic effects on the axon scaffold as a whole. Here, we focus on previous research analyzing classic mutants in the Slit/Robo pathway, which can readily be detected with a dissecting microscope. We also discuss analyzing these mutants in a teaching lab situation. The combination of sophisticated genetics and reliable axonal markers in Drosophila allows phenotypic analysis to be performed at the single-cell level. The elaborate architecture of neurons is very sensitive to disruption by genetic mutations, allowing the effects of novel mutations to be easily detected and assessed.

Diffuse Midline Glioma, H3K27M-mutant Subtype, Confused for Viral Encephalitis: A Case Report

The diffuse midline glioma, H3K27M-mutant subtype, occurs mainly in children as a result of mutations in the histone H3 (H3F3A) and HIST1H3B (K27M) genes and is characterized by diffuse tumor growth in central nervous system (CNS) midline structures. Due to its nonspecific clinical manifestations, viral encephalitis is often confused with other central nervous system diseases. In this case, we reported a young male patient who was admitted to the hospital chiefly complaining of “diplopia for more than two months and aggravated walking instability for more than 10 days”. After admission, relevant patient blood and cerebrospinal fluid (CSF) tests were completed, and no obvious abnormalities were found. Chest CT suggested pulmonary infection; magnetic resonance imaging (MRI) and contrast-enhanced CT, PET-CT and other imaging examinations of the head all indicated a high possibility of viral encephalitis. Symptoms of fever were improved in the patient after treatment with antiviral therapy and anti-infection therapy. However, symptoms of neurological function loss, such as double vision and adverse right limb movement, persisted. Finally, stereotactic biopsies of deep brain lesions were carried out and sent to the pathology department, which led to a diagnosis of diffuse midline glioma, H3K27M-mutant subtype (WHO IV). His family chose to perform conservative treatment in another hospital, and the patient died four months later. To conclude, when clinical symptoms of suspected viral encephalitis appear in the course of diffuse midline glioma, it can result in confusion regarding clinical diagnosis and treatment. Clinicians should ensure proper early recognition and identification of the disease.

Floor-plate-derived netrin-1 is dispensable for commissural axon guidance.

Netrin-1 is an evolutionarily conserved secreted extracellular matrix protein discovered using genetic and biochemical screens for its role in axon guidance at the central nervous system (CNS) midline1,2. Netrin-1 is expressed by cells localized at CNS midline, such as the floor plate in vertebrate embryos1,3. Growth cone turning assays and 3D gel diffusion assays showed that netrin-1 can attract commissural axons2,4–6. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in absence of netrin-13,7–9. Together these data support a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here, we selectively ablated netrin-1 expression in floor plate cells using a Netrin-1 conditional mouse line. We found that hindbrain and spinal cord commissural axons develop normally in absence of floor plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone with the potential to release it at the pial surface where it binds to commissural axons. Importantly, netrin-1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Netrin-1 knockout mice. These results show that the classical textbook view that attraction of commissural axons is mediated by a gradient of floor plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.

The use of modern ultrasound tridimensional techniques for the evaluation of fetal cerebral midline structures- a practical approach.

Fetal central nervous system midline structures represent an essential landmark for the confirmation of normality or for the identification of severe pathology. The ultrasound examination of the fetal brain using modern 3D techniques allows the creation of high sensitivity reconstructions. The facility of 3D volume acquisition permits the identification of corpus callosum, median septum, cavum septi pellucid and cerebellar vermis even in difficult cases. The examination should rely on both static (3D) and dynamic acquisition (4D). The use of a practical ultrasound protocol in clinical settings ensures the visualization of the midline cerebral structures in the vast majorities of fetuses. In selected cases MRI can be performed.

The Drosophila FHOD1-like formin Knittrig acts through Rok to promote stress fiber formation and directed macrophage migration during the cellular immune response

A tight spatiotemporal control of actin polymerization is important for many cellular processes that shape cells into a multicellular organism. The formation of unbranched F-actin is induced by several members of the formin family. Drosophila encodes six formin genes, representing six of the seven known mammalian subclasses. Knittrig, the Drosophila homolog of mammalian FHOD1, is specifically expressed in the developing central nervous system midline glia, the trachea, the wing and in macrophages. knittrig mutants exhibit mild tracheal defects but survive until late pupal stages and mainly die as pharate adult flies. knittrig mutant macrophages are smaller and show reduced cell spreading and cell migration in in vivo wounding experiments. Rescue experiments further demonstrate a cell-autonomous function of Knittrig in regulating actin dynamics and cell migration. Knittrig localizes at the rear of migrating macrophages in vivo, suggesting a cellular requirement of Knittrig in the retraction of the trailing edge. Supporting this notion, we found that Knittrig is a target of the Rho-dependent kinase Rok. Co-expression with Rok or expression of an activated form of Knittrig induces actin stress fibers in macrophages and in epithelial tissues. Thus, we propose a model in which Rok-induced phosphorylation of residues within the basic region mediates the activation of Knittrig in controlling macrophage migration.

The Drosophila FHOD1-like formin Knittrig acts through Rok to promote stress fiber formation and directed macrophage migration during the cellular immune response

A tight spatiotemporal control of actin polymerization is important for many cellular processes that shape cells into a multicellular organism. The formation of unbranched F-actin is induced by several members of the formin family. Drosophila encodes six formin genes, representing six of the seven known mammalian subclasses. Knittrig, the Drosophila homolog of mammalian FHOD1, is specifically expressed in the developing central nervous system midline glia, the trachea, the wing and in macrophages. knittrig mutants exhibit mild tracheal defects but survive until late pupal stages and mainly die as pharate adult flies. knittrig mutant macrophages are smaller and show reduced cell spreading and cell migration in in vivo wounding experiments. Rescue experiments further demonstrate a cell-autonomous function of Knittrig in regulating actin dynamics and cell migration. Knittrig localizes at the rear of migrating macrophages in vivo, suggesting a cellular requirement of Knittrig in the retraction of the trailing edge. Supporting this notion, we found that Knittrig is a target of the Rho-dependent kinase Rok. Co-expression with Rok or expression of an activated form of Knittrig induces actin stress fibers in macrophages and in epithelial tissues. Thus, we propose a model in which Rok-induced phosphorylation of residues within the basic region mediates the activation of Knittrig in controlling macrophage migration.

A Comparison of Midline and Tracheal Gene Regulation during Drosophila Development

Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, we identified and analyzed novel enhancers that drive midline and/or tracheal expression and compared them to previously characterized enhancers. In addition, we tested expression of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.

Preparing for Your Future as You Grow

Commissural axons cross the nervous system midline and connect to targets in the contralateral hemisphere. In this issue of Neuron, Michalski etal. (2013) demonstrate that synapses formed by ipsilaterally misrouted commissural axons exhibit defects in synapse maturation. Thus, midline crossing primes axons for subsequent synaptogenesis.

Localized Netrins Act as Positional Cues to Control Layer-Specific Targeting of Photoreceptor Axons in Drosophila

SummaryA shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.

Mastermind Mutations Generate a Unique Constellation of Midline Cells within the Drosophila CNS

BackgroundThe Notch pathway functions repeatedly during the development of the central nervous system in metazoan organisms to control cell fate and regulate cell proliferation and asymmetric cell divisions. Within the Drosophila midline cell lineage, which bisects the two symmetrical halves of the central nervous system, Notch is required for initial cell specification and subsequent differentiation of many midline lineages.Methodology/Principal FindingsHere, we provide the first description of the role of the Notch co-factor, mastermind, in the central nervous system midline of Drosophila. Overall, zygotic mastermind mutations cause an increase in midline cell number and decrease in midline cell diversity. Compared to mutations in other components of the Notch signaling pathway, such as Notch itself and Delta, zygotic mutations in mastermind cause the production of a unique constellation of midline cell types. The major difference is that midline glia form normally in zygotic mastermind mutants, but not in Notch and Delta mutants. Moreover, during late embryogenesis, extra anterior midline glia survive in zygotic mastermind mutants compared to wild type embryos.Conclusions/SignificanceThis is an example of a mutation in a signaling pathway cofactor producing a distinct central nervous system phenotype compared to mutations in major components of the pathway.

Avoiding DEET through Insect Gustatory Receptors

DEET is the most widely used insect repellent worldwide. In Drosophila olfactory receptor neurons (ORNs), DEET is detected through a mechanism employing the olfactory receptor, OR83b. However, it is controversial as to whether ORNs respond directly to DEET or whether DEET blocks the response to attractive odors. Here, we showed that DEET suppressed feeding behavior in Drosophila, and this effect was mediated by gustatory receptor neurons (GRNs). DEET was potent in suppressing feeding as <0.1% DEET elicited aversive behavior. Inhibition of feeding required multiple gustatory receptors (GRs) expressed in inhibitory GRNs. DEET stimulated action potentials in GRNs that respond to aversive compounds, and this response was lost in the Gr32a, Gr33a, and Gr66a mutants. Since 0.02% DEET elicited action potentials, we conclude that DEET directly activates of GRNs. We suggest that the effectiveness of DEET in pest control owes to its dual action in inducing avoidance simultaneously via GRNs and ORNs.

Navigating Intermediate Targets: The Nervous System Midline

In a bilaterally symmetric animal, the midline plays a key role in directing axon growth during wiring of the nervous system. Midline cells provide a variety of guidance cues for growing axons, to which different types of axons respond in different ways and at different times. For some axons, the midline is an intermediate target. They first seek it out, but then move on towards their final targets on the opposite side. For others, the midline is a repulsive barrier that keeps them on their own side of the midline. And for many of these axons the midline provides signals that guide them along specific lateral pathways or up and down the longitudinal axis.

CNS midline cells influence the division and survival of lateral glia in the Drosophila nervous system

Central nervous system (CNS) midline cells are essential for identity determination and differentiation of neurons in the Drosophila nervous system. It is not clear, however, whether CNS midline cells are also involved in the development of lateral glial cells. The roles of CNS midline cells in lateral glia development were elucidated using general markers for lateral glia, such as glial cell missing and reverse polarity, and specific enhancer trap lines labeling the longitudinal, A, B, medial cell body, peripheral, and exit glia. We found that CNS midline cells were necessary for the proper expression of glial cell missing, reverse polarity, and other lateral glia markers only during the later stages of development, suggesting that they are not required for initial identity determination. Instead, CNS midline cells appear to be necessary for proper division and survival of lateral glia. CNS midline cells were also required for proper positioning of three exit glia at the junction of segmental and intersegmental nerves, as well as some peripheral glia along motor and sensory axon pathways. This study demonstrated that CNS midline cells are extrinsically required for the proper division, migration, and survival of various classes of lateral glia from the ventral neuroectoderm.

Robo4 Signaling in Endothelial Cells Implies Attraction Guidance Mechanisms

Roundabouts (robo) are cell-surface receptors that mediate repulsive signaling mechanisms at the central nervous system midline. However, robos may also mediate attraction mechanisms in the context of vascular development. Here, we have performed structure-function analysis of roundabout4 (Robo4), the predominant robo expressed in embryonic zebrafish vasculature and found by gain of function approaches in vitro that Robo4 activates Cdc42 and Rac1 Rho GTPases in endothelial cells. Indeed, complementary robo4 gene knockdown approaches in zebrafish embryos show lower amounts of active Cdc42 and Rac1 and angioblasts isolated from these knockdown embryos search actively for directionality and guidance cues. Furthermore, Robo4-expressing endothelial cells show morphology and phenotype, characteristic of Rho GTPase activation. Taken together, this study suggests that Robo4 mediates attraction-signaling mechanisms through Rho GTPases in vertebrate vascular guidance.

The Divergent Robo Family Protein Rig-1/Robo3 Is a Negative Regulator of Slit Responsiveness Required for Midline Crossing by Commissural Axons

Commissural axons in vertebrates and insects are initially attracted to the nervous system midline, but once they reach this intermediate target they undergo a dramatic switch, becoming responsive to repellent Slit proteins at the midline, which expel them onto the next leg of their trajectory. We have unexpectedly implicated a divergent member of the Robo family, Rig-1 (or Robo3), in preventing premature Slit sensitivity in mammals. Expression of Rig-1 protein by commissural axons is inversely correlated with Slit sensitivity. Removal of Rig-1 results in a total failure of commissural axons to cross. Genetic and in vitro analyses indicate that Rig-1 functions to repress Slit responsiveness similarly to Commissureless (Comm) in Drosophila. Unlike Comm, however, Rig-1 does not produce its effect by downregulating Robo receptors on precrossing commissural axon membranes. These results identify a mechanism for regulating Slit repulsion that helps choreograph the precise switch from attraction to repulsion at a key intermediate axonal target.

SOX3 is required during the formation of the hypothalamo-pituitary axis.

The pituitary develops from the interaction of the infundibulum, a region of the ventral diencephalon, and Rathke's pouch, a derivative of oral ectoderm. Postnatally, its secretory functions are controlled by hypothalamic neurons, which also derive from the ventral diencephalon. In humans, mutations affecting the X-linked transcription factor SOX3 are associated with hypopituitarism and mental retardation, but nothing is known of their etiology. We find that deletion of Sox3 in mice leads to defects of pituitary function and of specific central nervous system (CNS) midline structures. Cells in the ventral diencephalon, where Sox3 is usually highly expressed, have altered properties in mutant embryos, leading to abnormal development of Rathke's pouch, which does not express the gene. Pituitary and hypothalamic defects persist postnatally, and SOX3 may also function in a subset of hypothalamic neurons. This study shows how sensitive the pituitary is to subtle developmental defects and how one gene can act at several levels in the hypothalamic-pituitary axis.