[P50]: Chondroitin sulfate glycosaminoglycans are required for the development of telencephalic neural stem/progenitor cells

Abstract

Neural stem cells (NSCs) have the ability to self-renew and give rise to neurons, glia and oligodendrocytes; however, the signals that regulate NSCs are not well understood. In order to determine the role of ␤-catenin in neural stem and progenitor cells of the mouse embryonic forebrain, we used Emx1-Cre and Nestin-Cre to restrict inactivation of a floxed β-catenin allele to neural precursor cells in the cerebral cortex and neuroepithelium, respectively. Emx1-Cre-mediated knockout of β-catenin is compatible with viability; however, null mice possess a significantly smaller cerebral cortex, defects in cortical lamination and loss of the hippocampus. Nestin-Cre-mediated knockout of β-catenin also leads to a smaller cerebral cortex and perinatal death. To determine how the neural precursor cell population is affected by loss of β-catenin, we performed an in vitro clonal neurosphere assay on E14.5 striatal germinal zone cells obtained from Nestin-Cre/β-catenin-knockout embryos. Although β-catenin mutant cultures did not develop intact neurospheres, many individual cells were scattered throughout the cultures. This loss of cell adhesion seen in vitro may phenocopy the abnormal cortical morphogenesis that occurs in mutant mice in vivo. We also examined the self-renewal capacity of β-catenin mutant NSCs using a collagen-based, clonal colony forming assay. Although β-catenin mutant cells gave rise to smaller colonies than control cells, the total number of mutant colonies was significantly greater indicating the presence of more NSCs in the β-catenin mutant brain. These mutant colonies were passageable, demonstrating that ␤-catenin is not required for NSC self-renewal. Furthermore, β-catenin mutant colonies retained multipotentiality as demonstrated by their ability to differentiate into neurons, astrocytes and oligodendrocytes. Therefore, we hypothesize that ␤-catenin is required for proper adhesion of neural precursor cells via cadherins, and the proliferation of neural progenitor cells, but is not required for neural progenitor differentiation, and may act to inhibit the symmetric division of NSCs.The molecular basis of organized transition from proliferating neural stem cells to differentiating neurons is a central question in developmental neurobiology. FGF2 exhibits dual functions in maintaining neuroepithelial cells; it keeps the cells in a less differentiated and more proliferative state. It remains to be elucidated how FGF2 signaling links suppression of differentiation and promotion of proliferation. Here, we report that in cultured neuroepithelial cells, FGF2 signals through a PI 3 kinase (PI 3 K)-Akt pathway, leading to inhibition of kinase activity of glycogen synthase kinase 3␤ (GSK3␤), which is known as a major component of Wnt signaling pathway. Inactivation of GSK3␤ increases the nuclear ␤-catenin protein level and subsequent up-regulation of Cyclin D1 expression, promoting cell cycle progression of neuroepithelial cells. We further demonstrated that dominant active GSK3␤ inhibited secondary neurosphere f...

The Synergistic Effect of Cationic Moieties and GRGDSF‐Peptides in Hydrogels on Neural Stem Cell Behavior

“…To obtain sufficient amounts of NSCs for following cell experiments, fresh NSC containing tissue was dissected and cultivated as free floating neurospheres . To this end, neocortices of embryonic mouse (E12.5–E13.5) were dissected using microdissection forceps.…”

“…Thereafter collected protein probes were subjected to SDS‐PAGE procedure and subsequently transferred and visualized by immunoblotting as described previously . Thereby primary antibodies against 473HD and α‐Tubulin (Sigma‐Aldrich, St. Louis, USA) were diluted 1:200 and 1:5000, respectively. Appropriate secondary antibodies were applied in dilutions of 1:5000.…”

“…In this study following primary immunological reagents, with respective dilutions were used to identify specific epitopes. Monoclonal antibodies against O4 (1:30; mouse IgM; clone 81 to mark oligodendrocytes, nestin (1:500; mouse IgG1; Merck Millipore, Billerica; USA) for NPCs,] β‐III tubulin (1:300; mouse IgG2b; clone SDL3D10; Sigma‐Aldrich, St. Louis, USA) for neurons, a polyclonal antibody against Glial Fibrillary Acidic Protein (GFAP) (1:300; rabbit IgG; Dako GmbH, Hamburg, Germany) to mark astrocytes, as well as antibodies to identify cellular proliferation by detection of Ki67 (1:20; mouse IgG1, clone MM1; Novocastra; Leica Biosystems, New Castle, United Kingdom) and phosphorylated histone H3 (Ser10) (1:100; rabbit; IgG polyclonal; Merck Millipore, Darmstadt, Germany) epitopes, as well as 473HD to stain pericellular chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid structure (referred to as the DSD‐1 epitope) (DSD‐1 epitope) carrying proteoglycans (1:100) and biotinylated hyaluronic acid binding protein (HABP, 1:50, Merck Millipore, Darmstadt, Germany) to stain pericellular hyaluronic acid. Furthermore to label f‐actin epitopes of the cytoskeleton, an Atto 488‐dye coupled phalloidin solution was used (49409 Atto 488 Phalloidin; Sigma‐Aldrich, St. Louis, USA) in combination with antibodies against vinculin (1:100 mouse IgG1; clone hVIN‐1, Sigma‐Aldrich, St. Louis, USA) to stain focal adhesions.…”