Additional file 8 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 8: Supplementary Table 1. List of genes, ID number and their respective primer-Forward and primer-Reverse used for RT-qPCR analysis.

Adult Neurogenesis Is Sustained by Symmetric Self-Renewal and Differentiation

Somatic stem cells have been identified in multiple adult tissues. Whether self-renewal occurs symmetrically or asymmetrically is key to understanding long-term stem cell maintenance and generation of progeny for cell replacement. In the adult mouse brain, neural stem cells (NSCs) (B1 cells) are retained in the walls of the lateral ventricles (ventricular-subventricular zone [V-SVZ]). The mechanism of B1 cell retention into adulthood for lifelong neurogenesis is unknown. Using multiple clonal labeling techniques, we show that the vast majority of B1 cells divide symmetrically. Whereas 20%-30% symmetrically self-renew and can remain in the niche for several months before generating neurons, …

Axons take a dive

In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular-subventricular zone (V-SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the functio…

Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus(1-5). This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease(6-10). In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day(11), whereas other studies find many fewer putative new neurons(12-14). Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal …

Cellular Response to Spinal Cord Injury in Regenerative and Non-Regenerative Stages in Xenopus Laevis

Abstract Background The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. Methods We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with…

Annulate Lamellae in a Desmoplastic Small Round Cell Tumor

GFP immunogold staining, from light to electron microscopy, in mammalian cells.

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one …

Age-related changes in astrocytic and ependymal cells of the subventricular zone

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as ot…

Localization of GFP-Tagged Proteins at the Electron Microscope

Enhanced tissue penetration of antibodies through pressurized immunohistochemistry

ABSTRACTTo address the inefficiency of passive diffusion for antibody penetration in thick tissue samples, which limits clearing-technique applications, we developed a versatile and simple device to perform antibody incubation under increased barometric pressure. Pressurized immunohistochemistry greatly improves the uniformity, intensity, and depth of fluorescent immunostaining in thick human and mouse brain samples. Furthermore, pressurized immunohistochemistry substantially decreases the time required for classic staining of thin sections.SUBMISSION CATEGORYNew Results

Subventricular zone neural progenitors protect striatal neurons from glutamatergic excitotoxicity.

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-i…

Cellular and Molecular Mechanism of Spinal Cord Regeneration in the Frog Xenopus laevis

Does Adult Neurogenesis Persist in the Human Hippocampus?

Cellular composition and organization of the spinal cord central canal during metamorphosis of the frogXenopus laevis

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a…

Netrin-1 receptor antibodies in thymoma-associated neuromyotonia with myasthenia gravis.

Objective:To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications.Methods:Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers).Results:Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted i…

Axonal control of the adult neural stem cell niche.

SummaryThe ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain’s neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that acti…

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the …

The Adult Macaque Spinal Cord Central Canal Zone Contains Proliferative Cells And Closely Resembles The Human

The persistence of proliferative cells, which could correspond to progenitor populations or potential cells of origin for tumors, has been extensively studied in the adult mammalian forebrain, including human and nonhuman primates. Proliferating cells have been found along the entire ventricular system, including around the central canal, of rodents, but little is known about the primate spinal cord. Here we describe the central canal cellular composition of the Old World primate Macaca fascicularis via scanning and transmission electron microscopy and immunohistochemistry and identify central canal proliferating cells with Ki67 and newly generated cells with bromodeoxyuridine incorporation…

Unique Organization of the Nuclear Envelope in the Post-natal Quiescent Neural Stem Cells

Summary Neural stem cells (B1 astrocytes; NSCs) in the adult ventricular-subventricular-zone (V-SVZ) originate in the embryo. Surprisingly, recent work has shown that B1 cells remain largely quiescent. They are reactivated postnatally to function as primary progenitors for neurons destined for the olfactory bulb and some corpus callosum oligodendrocytes. The cellular and molecular properties of quiescent B1 cells remain unknown. Here we found that a subpopulation of B1 cells has a unique nuclear envelope invagination specialization similar to envelope-limited chromatin sheets (ELCS), reported in certain lymphocytes and some cancer cells. Using molecular markers, [3H]thymidine birth-dating, …

Vascular‐derived TGF‐β increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain

Neurogenesis decreases during aging and following cranial radiotherapy, causing a progressive cognitive decline that is currently untreatable. However, functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover, we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. I…

Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration.

Increased neurogenesis has been reported in neurodegenerative disease, but its significance is unclear. In a mouse model of prion disease, Gomez-Nicola et al. detect increased neurogenesis in the dentate gyrus that partially counteracts neuronal loss. Targeting neurogenesis may have therapeutic potential.

The generation of oligodendroglial cells is preserved in the rostral migratory stream during aging

The subventricular zone (SVZ) is the largest source of newly generated cells in the adult mammalian brain. SVZ-derived neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into mature neurons. Additionally, a small proportion of SVZ-derived cells contribute to the generation of myelinating oligodendrocytes. The production of new cells in the SVZ decreases during aging, affecting the incorporation of new neurons into the OB. However, the age-related changes that occur across the RMS are not fully understood. In this study we evaluate how aging affects the cellular organization of migrating neuroblast chains, the proliferation, and th…

The adult spinal cord harbors a population of GFAP-positive progenitors with limited self-renewal potential

Adult neural stem cells (aNSCs) of the forebrain are GFAP-expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP-CreERT2) to conditionally label GFAP-expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self-renewal and multipotential properties in vitro. We demonstrate that a population of GFAP+ cells that share the morphology and the antigenic pr…

Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

The ventricular-subventricular zone (V-SVZ), on the walls of the lateral ventricles, harbors the largest neurogenic niche in the adult mouse brain. Previous work has shown that neural stem/progenitor cells (NSPCs) in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. The molecular signatures that underlie this regional heterogeneity remain largely unknown. Here, we present a single-cell RNA-sequencing dataset of the adult mouse V-SVZ revealing two populations of NSPCs that reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. These regional differences in gene expression were further validated using a single-nucl…

Additional file 6 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 6: Figure S6. Analysis of EdU+ cells in the intestine. (A-B) Click-iT staining of EdU+ (red) of the intestine in (A) sham control animals (2 dps), and at (B) 2 dpt. Nuclei were stained with Hoechst (blue). (C) Graph of EdU+ cells per mm3 in the intestine. n = 3.

Additional file 4 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 4: Figure S4. Transgenic line Xla.Tg(Dre.gfap:EGFP)Larra. (A-C) Three different animals’ electroporated in the spinal cord with the CAG promoter driving the expression of EGFP in central canal cells. (D-F) Three different animals electroporated in the spinal cord with the zGFAP::EGFP construct driving specific expression in radial glial like cells in contact with the central canal. (G-J) Animals at different developmental stages of the transgenic line Xla.Tg(Dre.gfap:EGFP)Larra showing expression of EGFP in the neural tube at (G-G’) NF stage 23; (H-H′) NF stage 27; (I-I′) NF stage 31 and in the CNS at (J-J’) NF stage 41. (K-M) Double staining against (K) EGFP and (L) Sox2 in…

Additional file 7 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 7: Figure S7. Transgenic line Xla.Tg(Dre.gfap:mCherry-Nitroreductase) allows selective cell ablation. (A) Diagram of injection and electroporation of the spinal cord at NF stage 50, indicating volume, concentration and parameters of electroporation. (B) Scheme of electroporation of the Dre.gfap:mCherry-Nitroreductase construct and treatment with vehicle or metronidazol (MTZ) at NF stage 50. (C-R) mCherry (red) expression in the spinal cord of animal electroporated at (C-D; I-J) 2 days post electroporation (dpe), before treatment; (E-F; K-L) 4 dpe and 2 days post treatment (dtt); (G-H; M-N) 7 dpe and 5 dtt, and (O- R) at 8 dpe and 6 dtt co-stained with Hoechst (blue). (S) The…

Additional file 5 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 5: Figure S5. RNAseq of EGFP+ and EGFP− cells isolated from the transgenic line Xla.Tg(Dre.gfap:EGFP)Larra. (A) Flow chart of RNAseq bioinformatics analysis from EGFP+ and EGFP− cells. (B) Graph of the Log2 fold change of the differential gene expression between EGFP+ cells versus EGFP− cells after FACS and RNAseq. EGFP expression in EGFP+ cells (green) is highlighted.

Additional file 2 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 2: Figure S2. In vivo time-lapse imaging of cells being extruded into the central canal. (A) Rostral stump of the transected spinal cord from a zGFAP::EGFP transgenic animal at R-stage 2 dpt. A time-lapses during 7 h for EGFP and transmitted light (T-PMT) z-stack were capture at the following time points: (B-B′) 0 min; 60 min (C-C′); 120 min (D-D′); 180 min (E-E’); 240 min (F-F′); 300 min (G-G’); 360 min (H-H′). White and purple arrows point to extrusion events from the cells lining the central canal.

Additional file 1 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 1: Figure S1. Cellular response to spinal cord injury in R- and NR-stages. (A) Centriolar satellite ultrastructure (arrowheads) in cells surrounding the rostral stump. (B) Radial projection of cells lining the central canal (yellow shadow). (C) Neutrophil in the injury site at 2 dpt in animals at NF stage 50. (C-E) Cells lining a rosette structure at 6 dpt are characterized by a (D) basal collagen lamina (blue shadow), (E) interdigitations and adherent junctions (arrowheads), and (F) intermediate filaments (arrowheads). Graphs of the number of red blood cells/μm2 × 105 at (G) 2 and 6 dpt in NF stage 50, and (H) at 2 and 6 dpt in NF stage 66. Graphs of the number of macrophag…

Additional file 3 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Additional file 3: Figure S3. Quantification of Vimentin Western Blot and Collagen AFOG staining. Western blot replicates for Vimentin and GAPDH in uninjured animals (ui), and after 2, 6, 10, 20 dpt in (A, B) R-Stage and (C, D) NR-Stage. Graphs of the adjusted relative density bands of Vimentin to the GAPDH control and normalized to the uninjured sample (ui) in (E) R-stage and (F) NR-stage at 2, 6, 10 and 20 days post transection (dpt) spinal cord samples. (G) Graph of the adjusted collagen staining area relative to the uninjured (ui) animals at 6, 10 dpt of R-stage and 10, 20 dpt of NR-stage. Red line defined no changes of Vimentin levels or Collagen staining. t-Test: * p