The perineuronal net forms the extracellular matrix of many neurons in the CNS, surrounding neuron cell bodies and proximal dendrites in a mesh-like structure with open "holes" at the sites of synaptic contacts. The perineuronal net is first detected late in development, approximately coincident with the transformation of the CNS from an environment conducive to neuronal growth and motility to one that is restrictive, suggesting a role for the perineuronal net in this developmental transition. Perineuronal nets show a great degree of molecular heterogeneity. Using monoclonal antibodies Cat-301, Cat-315, and Cat-316, we have shown previously that although all antibodies recognize chondroitin sulfate proteoglycans of similar sizes, each antibody recognizes perineuronal nets on distinct but overlapping sets of neurons in the adult cat CNS. An understanding of the heterogeneity demonstrated by these antibodies is critical to understanding the organization and function of perineuronal nets. Using aggrecan knock-out mice (cmd), we have now determined that all three antibodies recognize aggrecan. Chemical and enzymatic deglycosylation show that the differences revealed by the three antibodies arise from differential glycosylation of aggrecan. We further demonstrate that aggrecan mRNA is expressed relatively late in development and that neurons themselves are likely the predominant cellular sites of aggrecan expression. This work indicates that neurons can directly regulate the composition of their extracellular matrix by regulated synthesis and differential glycosylation of aggrecan in a cell type-specific manner. These results have important implications for the role of regulated microheterogeneity of glycosylation in the CNS.

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 ...

Rapid progress in the identification and characterization of axon guidance molecules and their receptors has left the field poised to explore the intracellular mechanisms by which signals are transduced into growth cone responses. The TUC (TOAD/Ulip/CRMP) family of proteins has emerged as a strong candidate for a role in growth cone signaling. The TUC family members reach their highest expression levels in all neurons during their peak periods of axonal growth and are strongly down-regulated afterward. When axonal regrowth in the adult is triggered by axotomy, TUC-4 is reexpressed during the period of regrowth. Mutations in unc-33, a homologous nematode gene, lead to severe axon guidance errors in all neurons. Furthermore, the TUC family is required for the growth cone-collapsing activity of collapsin-1. An important role for the TUC family is also suggested by its high degree of interspecies amino acid sequence identity, with the rat TUC-2 protein showing 98% identity with its chick ortholog and 89% identity with its Xenopus ortholog. Information gained from the study of the TUC family will be of key importance in understanding how growth cones find their targets.

We examined the expression of two members of theNotch family, Notch-1 and Notch-2, and one Notch ligand, Jagged-1, in hematopoietic cells. Both Notch-1 and Notch-2 were detected in murine marrow precursors (Lin−Sca-1+c-kit+). The Notch ligand, Jagged-1, was not detected in whole marrow or in precursors. However, Jagged-1 was seen in cultured primary murine fetal liver stroma, cultured primary murine bone marrow stroma, and in stromal cell lines. These results indicate a potential role for Notch-Notch ligand interactions in hematopoiesis. To further test this possibility, the effect of Jagged-1 on murine marrow precursor cells was assessed by coculturing sorted precursor cells (Lin−Sca-1+c-kit+) with a 3T3 cell layer that expressed human Jagged-1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged-1 (Jagged-1ext). We found that Jagged-1, presented both on the cell surface and on beads, promoted a twofold to threefold increase in the formation of primitive precursor cell populations. These results suggest a potential use for Notch ligands in expanding precursor cell populations in vitro.