In the developing CNS, subtypes of neurons and glial cells are generated according to a schedule that is defined by cell-intrinsic mechanisms that function at the progenitor-cell level. However, no critical molecular switch for the temporal specification of CNS progenitor cells has been identified. We found that chicken ovalbumin upstream promoter-transcription factor I and II (Coup-tfI and Coup-tfII, also known as Nr2f1 and Nr2f2) are required for the temporal specification of neural stem/progenitor cells (NSPCs), including their acquisition of gliogenic competence, as demonstrated by their responsiveness to gliogenic cytokines. COUP-TFI and II are transiently co-expressed in the ventricular zone of the early embryonic CNS. The double knockdown of Coup-tfI/II in embryonic stem cell (ESC)-derived NSPCs and the developing mouse forebrain caused sustained neurogenesis and the prolonged generation of early-born neurons. These findings reveal a part of the timer mechanisms for generating diverse types of neurons and glial cells during CNS development.
Fibroblast growth factor (FGF) is among the most common growth factors used in cultures to maintain self-renewal and proliferative capabilities of a variety of stem cells, including neural stem cells (NSCs). However, the molecular mechanisms underlying the control by FGF have remained elusive. Studies on mutant mice of FGF receptor substrate 2α (FRS2α), a central mediator for FGF signaling, combined with FRS2α knockdown or gain-of-function experiments, allowed us to dissect the role of FGF signaling for the self-renewal and proliferation of NSCs and to provide novel molecular mechanisms for them. We identified Hes1 as a novel self-renewal target of FGF-signaling. Quantitatively different levels of Erk activation mediated by FRS2α may regulate self-renewal of NSCs and proliferation of neural stem/progenitor cells (NSPCs); low levels of Erk activation are sufficient for the former, however, higher levels are required for maximum activity of the latter. Thus, FRS2α fine-tunes the FGF-signaling to control qualitatively different biological activities, self-renewal at least partly through Hes1 versus proliferation of NSPCs. Stem Cells 2010; 28:1661–1673.
Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6-8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na(+) or Ca(2+) spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro.
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