Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells

Anne-Marie Marzesco 1 Peggy Janich 1 Michaela Wilsch-Bruninger 1 Vronique Dubreuil 1 Katja Langenfeld 1 Denis Corbeil 0 1 Wieland B. Huttner ) 1 0 Medical Clinic and Polyclinic I, Dresden University of Technology , Fetscherstrasse 74, 01307 Dresden , Germany 1 Max-Planck-Institute of Molecular Cell Biology and Genetics , Pfotenhauerstrasse 108, 01307 Dresden , Germany - Apical plasma membrane constituents of mammalian neural stem/progenitor cells have recently been implicated in maintaining their stem/progenitor cell state. Here, we report that in the developing embryonic mouse brain, the fluid in the lumen of the neural tube contains membrane ce particles carrying the stem cell marker prominin-1 n (CD133), a pentaspan membrane protein found on ice membrane protrusions of the apical surface of S neuroepithelial cells. Two size classes of prominin-1ll containing membrane particles were observed in the eC ventricular fluid: 600-nm particles, referred to as P2 f particles, and 50-80-nm vesicles, referred to as P4 particles. lo The P2 and P4 particles appeared in the ventricular fluid na at the very onset and during the early phase of ru neurogenesis, respectively. Concomitant with their Jo appearance, the nature of the prominin-1-containing apical plasma membrane protrusions of neuroepithelial cells changed, in that microvilli were lost and large pleiomorphic protuberances appeared. P4 particles were found in various body fluids of adult humans, including saliva, Introduction The proliferation versus differentiation of stem/progenitor cells is a fundamental issue of biology and medicine. Yet, the underlying cell biological mechanisms are poorly understood. A key aspect in this context is cell polarity, which has moved into the focus especially in the case of mammalian neuroepithelial cells (Chenn et al., 1998; Huttner and Brand, 1997; Wodarz and Huttner, 2003), the somatic stem/progenitor cells from which directly or indirectly all neurons of the central nervous system derive (Alvarez-Buylla et al., 2001; Fishell and Kriegstein, 2003; Kriegstein and Gtz, 2003). As is the case for other polarized epithelial cells, a characteristic feature of neuroepithelial cells are junctional complexes at the apical-most end of the lateral plasma membrane (Aaku-Saraste et al., 1996; Chenn et al., 1998; Ho et al., 2000; Manabe et al., 2002) and an apical plasma membrane facing a lumen (Aaku-Saraste et al., 1997; Huttner and Brand, 1997). Focusing on the latter membrane, our group recently reported that the switch of mouse neuroepithelial cells seminal fluid and urine, and were released by the epithelial model cell line Caco-2 upon differentiation. Importantly, P4 particles were distinct from exosomes. Our results demonstrate the widespread occurrence of a novel class of extracellular membrane particles containing proteins characteristic of stem cells, and raise the possibility that the release of the corresponding membrane subdomains from the apical surface of neural progenitors and other epithelial cells may have a role in tissue development and maintenance. Moreover, the presence of prominin-1containing membrane particles in human body fluids may provide the basis for a protein-based diagnosis of certain diseases. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/118/13/2849/DC1 from symmetric, proliferative to asymmetric, neurongenerating cell divisions is associated with a halving of the size of the apical plasma membrane (Kosodo et al., 2004). Moreover, as hypothesized previously (Huttner and Brand, 1997), apical plasma membrane is inherited by both daughter cells upon symmetric, proliferative division of neuroepithelial cells but by only one daughter cell upon asymmetric, neurongenerating division (Kosodo et al., 2004). These studies suggest a critical role of apical plasma membrane constituents in maintaining neuroepithelial cells in a stem/progenitor cell state (Huttner and Brand, 1997; Kosodo et al., 2004; Wodarz and Huttner, 2003). A notable constituent of the apical plasma membrane of neuroepithelial cells is prominin-1 (CD133), a pentaspan membrane glycoprotein expressed on the surface of many somatic stem cells (Alessandri et al., 2004; Corbeil et al., 2001b; Richardson et al., 2004; Weigmann et al., 1997; Yin et al., 1997). Remarkably, prominin-1 is specifically associated with plasma membrane protrusions, irrespective of the cell type (Corbeil et al., 2001b; Corbeil et al., 2000; Giebel et al., 2004; Maw et al., 2000; Rper et al., 2000; Weigmann et al., 1997). Here, we report that, during the early stages of neurogenesis, neuroepithelial cells reduce the extent, and reorganize the nature, of their apical plasma membrane protrusions, and prominin-1-containing membrane particles appear in the lumen of the neural tube. Furthermore, the release of prominin-1containing membrane particles by epithelial cells appears to be a widespread phenomenon. Materials and Methods Immunofluorescence microscopy E7-E12.5 NMRI mouse embryos were fixed by immersion for 24 hours at 4C in 4% paraformaldehyde, 150 mM sodium phosphate buffer, pH 7.4. (In some experiments, 1% rather than 4% paraformaldehyde was used, without any obvious difference in the results obtained.) The fixed embryos were infiltrated overnight at 4C with 30% sucrose in phosphate buffer, embedded in TissueTek and frozen on dry-ice. Cryosections (20 m) were collected onto HistoBond microscope slides (Paul Marienfeld GmbH, LaudaKnigshofen, Germany). The sections, dried overnight at 4C, were hydrated with PBS and permeabilised for 15 minutes with 0.3% Triton X-100 in PBS. Residual aldehyde was quenched for 15 minutes with 50 mM NH4Cl in PBS. Sections were blocked for 1 ce hour with buffer A (1% BSA, 5% fetal calf serum in PBS) and n incubated overnight at 4C in buffer A containing primary antibody ice against mouse prominin-1 [rat mAb 13A4 (Weigmann et al., 1997) S at 8 g/ml and rabbit antiserum E3 (Maw et al., 2000) at 1:300 ll dilution]. Sections were extensively rinsed in buffer A, incubated in e buffer A containing secondary antibody (affinity-purified goat antifC rat or anti-rabbit IgG conjugated either to Cy2 or Cy3), rinsed o several times with buffer A, with PBS and once with distilled water, la and mounted in Moviol 4.88. rn Stained sections were observed using either an Olympus u epifluorescence microscope with a 100 oil immersion objective Jo connected to a CCD camera, or a Zeiss or Leica confocal laser scanning microscope. The confocal microscope settings were such that 5- m-thick optical sections were obtained in the middle of the cryosection or at the level of the glass slide, as shown schematically in Fig. 1e. The images shown were prepared from the digital data files using Adobe Photoshop and Illustrator. For the quantification of the ring-like particles in the neural tube lumen, the area analysed in the optical sections (4-14 sections per developmental stage and br (...truncated)