For centuries analyses of tissues have depended on sectioning methods. Recent developments of tissue clearing techniques have now opened a segway from studying tissues in 2 dimensions to 3 ...dimensions. This particular advantage echoes heavily in the field of neuroscience, where in the last several years there has been an active shift towards understanding the complex orchestration of neural circuits. In the past five years, many tissue-clearing protocols have spawned. This is due to varying strength of each clearing protocol to specific applications. However, two main protocols have shown their applicability to a vast number of applications and thus are exponentially being used by a growing number of laboratories. In this review, we focus specifically on two major tissue-clearing method families, derived from the 3DISCO and the CLARITY clearing protocols. Moreover, we provide a "hands-on" description of each tissue clearing protocol and the steps to look out for when deciding to choose a specific tissue clearing protocol. Lastly, we provide perspectives for the development of tissue clearing protocols into the research community in the fields of embryology and cancer.
The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as
knockouts die at ...birth. To circumvent this drawback, we generated an eye-specific
mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of
, some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover,
-deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.
Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and ...the relative ease of producing them in large quantities, at low cost. Though they offer several advantages, they suffer from lower binding affinity and rapid clearance from circulation, which limits their therapeutic potential. However, these fragments can be genetically modified to enhance desirable properties, such as multivalency, high target retention and slower blood clearance, and as such, a variety of scFv formats have been generated. ScFvs can be administered by systemic injection for diagnostic and therapeutic purposes. They can be expressed in vivo through viral vectors in instances where large infection rates and sustenance of high levels of the antibody is required. ScFvs have found applications as tools for in vivo loss-of-function studies and inactivation of specific protein domains, diagnostic imaging, tumor therapy and treatment for neurodegenerative and infectious diseases. This review will focus on their in vivo applications.
Traumatic spinal cord injury (SCI) causes a cascade of degenerative events including cell death, axonal damage, and the upregulation of inhibitory molecules which prevent regeneration and limit ...recovery. Repulsive guidance molecule A (RGMa) is a potent neurite growth inhibitor in the central nervous system, exerting its repulsive activity by binding the Neogenin receptor. Here, we show for the first time that inhibitory RGMa is markedly upregulated in multiple cell types after clinically relevant impact-compression SCI in rats, and importantly, also in the injured human spinal cord. To neutralize inhibitory RGMa, clinically relevant human monoclonal antibodies were systemically administered after acute SCI, and were detected in serum, cerebrospinal fluid, and in the injured tissue. Rats treated with RGMa blocking antibodies showed significantly improved recovery of motor function and gait. Furthermore, RGMa blocking antibodies promoted neuronal survival, and enhanced the plasticity of descending serotonergic pathways and corticospinal tract axonal regeneration. RGMa antibody also attenuated neuropathic pain responses, which was associated with fewer activated microglia and reduced CGRP expression in the dorsal horn caudal to the lesion. These results show the therapeutic potential of the first human RGMa antibody for SCI and uncovers a new role for the RGMa/Neogenin pathway on neuropathic pain.
In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only ...the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.
Glycogen synthase kinase 3 (GSK3) proteins (GSK3α and GSK3β) are key mediators of signaling pathways, with crucial roles in coordinating fundamental biological processes during neural development. ...Here we show that the complete loss of GSK3 signaling in mouse retinal progenitors leads to microphthalmia with broad morphologic defects. A single wild-type allele of either
or
is able to rescue this phenotype. In this genetic context, all cell types are present in a functional retina. However, we unexpectedly detected a large number of cells in the inner nuclear layer expressing retinal ganglion cell (RGC)-specific markers (called displaced RGCs, dRGCs) when at least one allele of
is expressed. The excess of dRGCs leads to an increased number of axons projecting into the ipsilateral medial terminal nucleus, an area of the brain belonging to the non-image-forming visual circuit and poorly targeted by RGCs in wild-type retina. Transcriptome analysis and optomotor response assay suggest that at least a subset of dRGCs in
mutant mice are direction-selective RGCs. Our study thus uncovers a unique role of GSK3 in controlling the production of ganglion cells in the inner nuclear layer, which correspond to dRGCs, a rare and poorly characterized retinal cell type.
Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel ...and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.
The Molecular mechanisms required for the proper wiring of both the Central Nervous System (CNS) as well as the Peripheral Nervous System (PNS) share similar features. However, once the CNS or PNS undergo a lesion or begin degenerating, both systems respond in distinctive ways. Here, we discuss recent advances and current challenges in the fields of neural development as well as regeneration.
Glycogen Synthase Kinase 3 (GSK) proteins (GSK3α and GSK3β) are key mediators of signaling pathways, with crucial roles in coordinating fundamental biological processes during neural development. ...Here we show that the complete loss of GSK3 signaling in mouse retinal progenitors leads to microphthalmia with broad morphological defects. A single wild-type allele of either Gsk3α or Gsk3β is able to rescue this phenotype. In this genetic context, all cell types are present with a functional retina. However, we unexpectedly detect a large number of cells in the inner nuclear layer expressing retinal ganglion cell (RGC)-specific markers (called displaced RGCs, dRGCs) when at least one allele of Gsk3α is expressed. Excess dRGCs lead to increased number of axons projecting into the ipsilateral medial terminal nucleus, an area of the brain belonging to the non-image-forming visual circuit and poorly targeted by RGCs in wild-type retina. Transcriptome analysis and optomotor response assay suggest that at least a subset of dRGCs in Gsk3 mutant mice are direction-selective RGCs. Our study thus uncovers a unique role of GSK3 in controlling the production of ganglion cells in the inner nuclear layer, which correspond to dRGCs, a rare and poorly characterized retinal cell type.Significance StatementGlycogen Synthase Kinase 3 (GSK) proteins (Gsk3α or Gsk3β) are key mediators of signaling pathways, especially in the central nervous system but poorly described in the retina. Here we show that the complete loss of GSK3 in mouse retinal progenitors leads to microphthalmia. However, when only one allele of Gsk3α or Gsk3β are present, all cell types are present with a functional retina. More importantly, we unexpectedly uncover a unique role of GSK3 in controlling the genesis of retinal ganglion cells in the inner nuclear layer which could correspond to a rare and poorly characterized retinal cell type. Therefore, our mouse models potentially offer a unique and powerful model system to study the visual function of dRGCs in mammals.
The Repulsive Guidance Molecule a (RGMa) has recently been implicated in the development of Multiple sclerosis (MS). We report for the first time that the third member of the RGM family, RGMc, is ...downregulated in the sera of mice induced with Experimental Autoimmune Encephalomyelitis (EAE), a well-characterized animal model of MS. Treatment with soluble RGMc (sRGMc) significantly delayed the onset of EAE and reduced the clinical severity of disease. Furthermore, RGMc knockout animals developed a stronger disease progression than wild-type controls. In vitro binding studies outlined a mechanism by which soluble RGMa and RGMc competed for interaction with their shared receptor, Neogenin. Finally, we demonstrated that administration of soluble RGMc halted leukocyte infiltration by interfering with the RGMa-mediated RhoA permeabilisation of the Blood-Brain Barrier (BBB). Overall, we show that RGMc treatment alleviated the clinical severity of EAE by enhancing BBB integrity and preventing leukocyte infiltration.
Chronic GvHD-related keratoconjunctivitis sicca (cGvHD-related KCS) can significantly alter the quality of life of patients after allogeneic hematopoietic stem cell transplantation. The aim of this ...work was to assess the efficacy and tolerability of scleral lenses to treat severe cGvHD-related KCS. In this retrospective, multicenter study, we included 60 consecutive patients diagnosed with cGvHD-related KCS and fitted with scleral lenses. Patients were evaluated at baseline and at 2 months with the following tests: the Ocular Surface Disease Index (OSDI) to assess quality of life, the Oxford score to grade corneal damage and the logarithm of minimal angle of resolution (Log MAR) scale to determine visual acuity. We observed improvement in quality of life in 58 patients (97%). All parameters improved at 2 months. We observed significant differences at 2 months compared with baseline for the mean OSDI (86 versus 30, respectively, P<0.001), the mean Oxford score (3.2 versus 1.3, respectively, P<0.001) as well as visual acuity (Log MAR of 0.33 versus 0.10, respectively, P<0.001). Treatment with scleral lenses was discontinued in only 5 patients (8%) with a median follow-up of 20.5 months (range: 2-125 months). Scleral lenses were very efficient and well tolerated in patients with severe cGvHD-related KCS.
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