Role Of Close Homologue Of The L1(CHL1) In Proliferation And Differentiation Of Neural Stem/Progenitor Cells

During embryonic development, the brain is formed through a series of proliferation, migration and differentiation events deriving from neural progenitor cells (NPCs). Cell-cell adhesion may be involved in this process, because extracellular environment (or"niche") that stem cells inhabit can provide cells with information to their cellular neighborhood. Cell adhesion molecules (CAMs) regulate nervous system development, including proliferation and differentiation of NPCs. Close Homologue of L1 (CHL1) is an identified member of the L1 family of cell adhesion molecules.Expression of CHL1 appears to be restricted to the nervous system. In mice, CHL1 expression is first detectable in the brain at embryonic day 13, reaches peak levels from embryonicday 18 to postnatal day 7, and subsequently declines to lower levels in the adult brain. The role of CHL1 in the develpomen of NPCs remains unknown. Here, we have investigated the functions of CHL1 in the develpomen of NPCs.1. Expression of CHL1 in embryonic cortex and NPCsTo explore the role of CHL1 in embryonic cortex and NPCs, we performed a study of CHL1 protein expression in the saggital brain sections at E14.5. Immunofluorescence staining showed CHL1 mainly expressed along the the intermediate zone and cortical plate. CHL1 was also observed expressing in NPCs at the ventricular zone region and subventricular zone region. Then, we isolated and cultured NPCs derived from embryonic day 14.5 cortex. All nestin immunoreactive cells were CHL1 positive, and CHL1 was stably expressed during the proliferation of NPCs in vitro. Expression and localization of CHL1 in NPCs both in vitro and in vivo suggest that CHL1 have potential function on proliferation and differentiation of NPCs.2. Effect of CHL1 in the development of embryonic cortexWe then examined the development of cerebral cortex in CHL1-deficient mice. In our study, we found that CHL1-deficient mice had bigger brains than littermate wild-type during development. The incresed brain size was found in E14.5, E16.5 and P30. Nissl staining showed thicker cortices in CHL1-deficient mice at E14.5. The number of cells in cortical plate reduced, while the number of cells in intermediate zone and subpreplate increased.To assess proliferation of NPCs in vivo, the number of BrdU incorporation was detected after BrdU was injected ip at E14.5 and E16.5. No obvious differences were detected in the total number of BrdU-labeled cells between CHL1-deficient and control littermates. The immature neuronal marker,β-tubulin-Ⅲ(Tuj1) staining showed a remarkably incresed neuronal differentiation in CHL1-deficient mice compared with littermate control. Tuj1-positive cells increased in cotical plate, intermediate zone and ventricular zone. We next detected cell apoptosis in the cerebral cortex from CHL1-deficient and littermates control by using the TUNEL assay. A decreased number of apoptosis was observed in cerebral cortex from CHL1-deficient mice. Thus, these data showed that increased neurogenesis and decreased cell death in CHL1-deficient mice, which indicate CHL1 have potential function on the development of cerebral cortex.3. Role of CHL1 on proliferation and differentiation in NPCsWe further investigated the role of CHL1 deficiency on proliferation and differentiation of NPCs in vitro. NPCs were prepared from E14.5 littermates. Proliferation of NPCs was investigated by colony effiency, Brdu incorporation, FCAS and apoptosis analysis. CHL1-deficiency increased the formation of the primary cultured neuralspheres and the number of BrdU positive cells, while decreased the number of cell apoptosis. It indicates that CHL1-deficiency promoted proliferation of NPCs.The function of CHL1 on the differentiation of NPCs was also investigated. NPCs were differentiated for 5 days and stained with three major cell type markers: Tuj1 for neurons, GFAP for astrocytes and CNPase for oligodendrocytes. The percentage of Tuj1-positive cells increased compared with control. The percentage of oligodendrocytes did not change compared with control. Expression of Tuj1, GFAP and CNPase also was detected by Western blot analysis. The data showed that expression of Tuj1 in CHL1-deficient cells increased, while expression of GFAP reduced compared with control. These results suggested that CHL1-deficiency promoted the differentiation of NPCs into neurons.4. ERK1/2 MAPK is involved in proliferation and differentiation of CHL1-deficient NPCsERK1/2 MAPK is essential for the proliferation of NPCs derived from the embryonic cortex in vitro. Activated the phosphorylation of ERK1/2 but not JNK and p38 were detected in CHL1-deficient NPCs. ERK1/2 inhibitor, PD98059 blocked the increased number of neurospheres, and growth of CHL1-deficient NPCs. The function of CHL1-deficiency in NPCs could be abolished by PD98059. The phosphorylation of ERK1/2 was also activated on the differentiation of CHL1-deficient NPCs. Thus, it suggests ERK1/2 MAPK is essential for the proliferation of CHL1-deficient NPCs.In conclusion, here we first show that CHL1 plays a negative regulator in development of NPCs both in vivo and in vitro.We also provide evidence that ERK1/2 is involved in the function of CHL1-deficienct NPCs.