Late proliferation of retinal Müller cell progenitors facilitates preferential targeting with retroviral vectors in vitro.

During vertebrate neural retina development, the relationship between mitotic activity in progenitor cells and the acquisition of a mature cell phenotype remains an area of controversy. The Müller glial cell has long been recognized as one of the last cell types of the retina to mature, which occurs under the influence of cell-cell interactions. In this report we examine the acquisition of the Müller cell phenotype in relation to mitotic activity. Using immunohistochemical markers, we demonstrate that a gene product characteristic of mature Müller cells, the 2M6 antigen, is expressed in mitotically active cells, even after all the major retina architectural features have been laid down. Furthermore, we show that retroviral infection, a process that requires mitotically active cells, preferentially targets Müller cell progenitors when late embryonic retina is infected in vitro. The two lines of evidence are consistent with a model for Müller cell differentiation that includes a mitotically active progenitor that has already begun to express specific differentiation gene products.

Characterization, cloning and expression of the 67-kDA annexin from chicken growth plate cartilage matrix vesicles.

Analysis of a 67 kDa lipid-dependent Ca(2+)-binding protein from avian matrix vesicles revealed amino acid sequences homologous to mammalian annexin VI. PCR methods were used to identify a clone from an avian cDNA library that contained a full length copy of the 67-kDa annexin cDNA. This was restriction mapped, subcloned and sequenced. The cDNA sequence of the open reading frame showed 70 percent identity to that of murine annexin VI; the predicted amino acid sequence, 78 percent identity. There was no homology in the 5'- and 3'-untranslated regions. A plasmid was constructed that overexpresses the intact chicken 67-kDa matrix vesicle annexin in E. coli DH5 alpha in high yield; the physicochemical properties and the amino terminal sequence of the expressed protein exactly matched those of the native protein.

Establishment of the primary structure of the major lipid-dependent Ca2+ binding proteins of chicken growth plate cartilage matrix vesicles: identity with anchorin CII (annexin V) and annexin II.

Electron microscopic studies of calcifying vertebrate tissues reveal the locus of de novo mineral formation within matrix vesicles (MV). The direct involvement of MV in the initiation of mineral formation is supported by the fact that MV isolated from avian growth plate cartilage rapidly accumulate large amounts of Ca2+ and P(i) and induce mineral formation. Exploration of the constituents of MV has revealed two major protein components, a 33 and a 36 kD protein, the former of which binds to cartilage-specific collagens. These annexin-like proteins bind to acidic phospholipids in the presence of submicromolar levels of Ca2+. Antibodies raised against both the purified 33 and the 36 kD MV annexin do not cross-react with the other, indicating that they are distinct proteins. Reported here are studies elucidating the primary structure of both MV proteins using both conventional protein and molecular biologic methods. These studies establish that the 33 kD protein is nearly identical to anchorin CII (annexin V) and that the 36 kD protein is identical to avian annexin II. Immunolocalization studies show that hypertrophic chondrocytes at the calcification front of avian growth plate contain the highest level of these annexins. Further, immunogold labeling indicates that the annexins are localized within MV isolated from the growth plate. Recent studies indicate that annexin V is a new type of ion-selective Ca2+ channel protein that possesses selective collagen binding properties. Since MV are tightly associated with the collagen- and proteoglycan-rich matrix, it is tempting to speculate that this MV protein may be a component of stretch-activated ion channels that enhance Ca2+ uptake during mechanical stress.