β-Catenin has been widely implicated in the regulation of mammalian development and cellular homeostasis. However, the mechanisms by which Wnt/β-catenin signaling components regulate physiological events during brain development remain undetermined. Inactivation of glycogen synthase kinase (GSK)-3β leads to β-catenin accumulation in the nucleus, where it couples with T-cell factor (TCF), an association that is disrupted by ICAT (inhibitor of β-catenin and T cell factor). In this study, we sought to determine whether regulation of ICAT by members of the microRNA-29 family plays a role during neurogenesis and whether deregulation of ICAT results in defective neurogenesis due to impaired β-catenin-mediated signaling. We found that miR-29b, but not miR-29a or 29c, is significantly upregulated in three-dimensionally cultured neural stem cells (NSCs), whereas ICAT is reduced as aged. Treatment with a miR-29b reduced the reporter activity of a luciferase-ICAT 3'-UTR construct whereas a control (scrambled) miRNA oligonucleotide did not, indicating that miR-29b directly targets the 3'-UTR of ICAT. We also found that treatment with miR-29b diminished NSC self-renewal and proliferation, and controlled their fate, directing their differentiation along certain cell lineages. Furthermore, our in vivo results showed that inhibition of miR-29b by in utero electroporation induced a profound defect in corticogenesis during mouse development. Taken together, our results demonstrate that miR-29b plays a pivotal role in fetal mouse neurogenesis by regulating ICAT-mediated Wnt/β-catenin signaling.

The A9 dopaminergic (DA) neuronal group projecting to the dorsal striatum is the most vulnerable in Parkinson's disease (PD). We genetically engineered mouse embryonic stem (ES) cells to express the transcription factors Nurr1 or Pitx3. After in vitro differentiation of Pitx3-expressing ES cells, the proportion of DA neurons expressing aldehyde dehydrogenase 2 (AHD2) increased, while the total number of DA neurons remained the same. The highest levels of AHD2 expression were observed in mouse A9 DA neurons projecting to the dorsal striatum. Furthermore, real-time PCR analyses of in vitro differentiated Pitx3-expressing ES cells revealed that genes highly expressed in A9 DA neurons were up-regulated. When transplanted into the mouse striatum, Pitx3-expressing cells generated an increased proportion of AHD2-expressing DA neurons. Contrastingly, in Nurr1-expressing ES cells, increases of all midbrain DA markers were observed, resulting in a higher total number of DA neurons in vitro and in vivo, whereas the proportion of AHD2-expressing DA neurons was not changed. Our data, using gain-of-function analysis of ES cells, suggest that Pitx3 may be important for specification and/or maintenance of A9-like neuronal properties, while Nurr1 influences overall midbrain DA specification. These findings may be important for modifying ES cells to generate an optimal cell source for transplantation therapy of PD.

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.