We used a recombinant retrovirus to study cell lineage in the chicken optic tectum. The virus inserts the Escherichia coli lacZ (.3-galactosidase) gene into the genome of an infected cell; a histochemical stain marks the progeny of infected cells with a blue precipitate. We had previously shown that individual clones frequently contain diverse neuronal types. Now we asked whether individual clones contain glia as well as neurons. To this end, we constructed a virus in which lacZ is fused to a nuclear localization signal sequence from the simian virus 40 large tumor antigen. Cells infected with this virus are marked with blue nuclei instead of blue somata. In embryos injected with a mixture of the two retroviruses, individual clusters contained cells with only one label type (nuclear or cytoplasmic), thus verifying that clusters of cells were clones. Furthermore, it was possible to immunostain the somata of cells that had blue nuclei, whereas the blue cytoplasmic precipitate hampered immunostaining. Together, these methods allowed us to show that some clones contained neurons (neurofilament-positive) and two types of glia (glutamine synthetase-positive and glial fibrillary acidic proteinpositive). This result demonstrates the existence of a common progenitor for neurons and glia in optic tectum.A few years ago, we (1) and Price et al. (2) form coherent radial arrays (4), many putative glia were displaced tangentially. It was therefore unclear whether the glia had migrated tangentially from their neuronal relatives or arisen from separate, nearby progenitors. Thus, our tentative conclusion, that some tectal progenitors give rise to both neurons and glia, remained unverified because of ambiguities in the identification of glial cells and of clonal boundaries.To circumvent both of these problems, we constructed a retroviral vector that targets LacZ to the nucleus. This was done by replacing the lacZ gene in the virus with a construct in which lacZ is fused to a short "signal" sequence from the nuclear large tumor antigen of the simian virus 40 virus (5-7); in fact, Kalderon et al. (5) originally used this fusion to show that the sequence in question codes for a nuclear localization signal (5). As expected, cells infected with the nuclear signal-lacZ (nslacZ) virus had blue nuclei instead of blue somata after staining. We were then able to use the nslacZ virus in two types of double-staining protocols. (i) Embryos were infected with a mixture of lacZ and nslacZ viruses to determine whether individual clusters of cells presumed to represent clones indeed contained cells with only one type of label, nuclear or cytoplasmic. (ii) We combined immunofluorescent visualization of cytoplasmic antigens with f8-galactosidase histochemistry to determine the phenotype of cells that had blue nuclei. Together, these methods allowed us to demonstrate unambiguously that some clones of tectal cells contain both neurons and glia. METHODSViruses. The recombinant retroviral vectors used in this study (Fig. 1) were constructed ...

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.

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The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.