In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

In the nervous system, cilia dysfunction perturbs the circulation of the cerebrospinal fluid, thus affecting neurogenesis and brain homeostasis. A role for planar cell polarity (PCP) signaling in the orientation of cilia (rotational polarity) and ciliogenesis is established. However, whether and how PCP regulates cilia positioning in the apical domain (translational polarity) in radial progenitors and ependymal cells remain unclear. By analysis of a large panel of mutant mice, we show that two PCP signals are operating in ciliated cells. The first signal, controlled by cadherin, EGF-like, laminin G-like, seven-pass, G-type receptor (Celsr) 2, Celsr3, Frizzled3 (Fzd3) and Van Gogh like2 (Vangl2) organizes multicilia in individual cells (single-cell polarity), whereas the second signal, governed by Celsr1, Fzd3, and Vangl2, coordinates polarity between cells in both radial progenitors and ependymal cells (tissue polarity). Loss of either of these signals is associated with specific defects in the cytoskeleton. Our data reveal unreported functions of PCP and provide an integrated view of planar polarization of the brain ciliated cells.

Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140) in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general.

After their generation and specification in periventricular regions, neuronal precursors maintain an immature and migratory state until their arrival in the respective target structures. Only here are terminal differentiation and synaptic integration induced. Although the molecular control of neuronal specification has started to be elucidated, little is known about the factors that control the latest maturation steps. We aimed at identifying factors that induce terminal differentiation during postnatal and adult neurogenesis, thereby focusing on the generation of periglomerular interneurons in the olfactory bulb. We isolated neuronal precursors and mature neurons from the periglomerular neuron lineage and analyzed their gene expression by microarray. We found that expression of the bHLH transcription factor NeuroD1 strikingly coincides with terminal differentiation. Using brain electroporation, we show that overexpression of NeuroD1 in the periventricular region in vivo leads to the rapid appearance of cells with morphological and molecular characteristics of mature neurons in the subventricular zone and rostral migratory stream. Conversely, shRNA-induced knockdown of NeuroD1 inhibits terminal neuronal differentiation. Thus, expression of a single transcription factor is sufficient to induce neuronal differentiation of neural progenitors in regions that normally do not show addition of new neurons. These results suggest a considerable potential of NeuroD1 for use in cell-therapeutic approaches in the nervous system. adult neurogenesis | bHLH transcription factor | interneurons | postnatal electroporation

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2 cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.