Abstract
Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs.
Highlights
During mammalian central nervous system (CNS) development, neural stem/neural progenitor cells (NSPCs) generate neural and glial lineages by mitotic cell division
We used adherent fibroblast growth factor 2 (FGF2)- and EGFexpanded NSPCs derived from the telencephalon of E11.5 embryos or the adult mouse subventricular zone (SVZ) as the tester cDNA pool, and each sister culture under a differentiating condition for 3 days for the driver cDNA pool
Among 2000 candidate cDNAs that were enriched in subtracted libraries, 120 clones were verified to be differentially expressed in the cDNA pool of NSPCs
Summary
During mammalian central nervous system (CNS) development, neural stem/neural progenitor cells (NSPCs) generate neural and glial lineages by mitotic cell division. At the early embryonic stage, neuroepithelial cells spanning the neural tube serve as primary NSPCs. As the neuroepithelium thickens, neuroepithelial cells differentiate into radial glial cells (apical progenitors), and shift their mode of proliferation from symmetric to asymmetric cell division [1,2,3]. These cells undergo cell division at the ventricular zone (VZ), and display a defined apico-basal polarity with a radially oriented fiber (radial process) extending from the VZ to the pial surface of the cortical wall [4]. Another type of neural progenitor cell, called intermediate progenitors or basal progenitors, originate from asymmetric divisions of radial glial cells. In the adult rodent SVZ, slowly dividing glial fibrillary acidic protein (GFAP)-positive cells are thought to be neural stem cells (NSCs; type-B cells) that give rise to rapidly proliferating progenitors (type-C cells) [2,6]
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