Search PubMedSearch

Biomedical subjects

R J Greenspan

Publications and source records attributed to R J Greenspan.

8 recordsLinked to original sources

Expression pattern of Motch, a mouse homolog of Drosophila Notch, suggests an important role in early postimplantation mouse development.

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell-cell interactions and cell fate decisions in the Drosophila embryo. To determine if a gene homologous to Drosophila Notch plays a role in early mouse development, we screened a mouse embryo cDNA library with probes from the Xenopus Notch homolog, Xotch. A partial cDNA clone encoding the mouse Notch homolog, which we have termed Motch, was used to analyze expression of the Motch gene. Motch transcripts were detected in a wide variety of adult tissues, which included derivatives of all three germ layers. Differentiation of P19 embryonal carcinoma cells into neuronal cell types resulted in increased expression of Motch RNA. In the postimplantation mouse embryo Motch transcripts were first detected in mesoderm at 7.5 days post coitum (dpc). By 8.5 dpc, transcript levels were highest in presomitic mesoderm, mesenchyme and endothelial cells, while much lower levels were detected in neuroepithelium. In contrast, at 9.5 dpc, neuroepithelium was a major site of Motch expression. Transcripts were also abundant in cell types derived from neural crest. These data suggest that the Motch gene plays multiple roles in patterning and differentiation of the early postimplantation mouse embryo.

Amino Acid Sequence

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10.

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.

Animals

The Notch locus of Drosophila is required in epidermal cells for epidermal development.

The Notch locus of Drosophila plays an important role in cell fate decisions within the neurogenic ectoderm, a role thought to involve interactions at the cell surface. We have assayed the requirement for Notch gene expression in epidermal cells by two kinds of genetic mosaics. First, with gynandromorphs, we removed the wild-type gene long before the critical developmental events to produce large mutant clones. The genotype of cells in large clones was scored by means of an antibody to the Notch protein. Second, using mitotic recombination, we removed the gene at successively later times after completion of the mitotically active early cleavage stages, to produce small clones. These clones were detected by means of a linked mutation of cuticle pattern, armadillo. The results of both experiments demonstrate a requirement for Notch expression by epidermal cells, and thus argue against the model that the Notch product acts as a signal required only in the neuroblast to influence neighboring epidermal cells. The mitotic recombination experiment revealed that Notch product is required by epidermal cells subsequent to neuroblast delamination. This result implies that the Notch gene functions to maintain the determined state of epidermal cells, possibly by mediating cell surface interactions within the epidermis.

Animals

The Notch gene, adhesion, and developmental fate in the Drosophila embryo.

The Notch locus in Drosophila encodes a transmembrane protein required for the determination of cell fate in ectodermal cells. When these cells are faced with a choice of two possible fates, Notch enables some of them to advance from the fate that represents a "default" state. The decision appears to involve both an interaction between cells and the presence of the Notch product on the surface of those advancing from the default state. The timing of gene action suggests that Notch plays a role in the stabilization of the choice of cell fate and that the Notch-mediated interaction occurs between presumptive epidermal cells. Several properties of the Notch product are characteristic of an adhesion molecule, and thus cell adhesion may play a role in the determination of cell fate.

Animals

Conversion of placental alkaline phosphatase from a phosphatidylinositol-glycan-anchored protein to an integral transmembrane protein.

Placental alkaline phosphatase (PLAP) is normally anchored to the plasma membrane of cells by a phosphatidylinositol-glycan anchor after removal of a carboxyl-terminal peptide from the nascent enzyme. To investigate the signals required for this processing we constructed a chimeric cDNA. The latter was designed to code for a truncated precursor form of PLAP, containing the phosphatidylinositol-glycan attachment site but incapable of any form of membrane attachment, fused to a carboxyl-terminal peptide of vesicular stomatis virus glycoprotein. Expression of the PLAP-vesicular stomatis virus glycoprotein chimeric cDNA in transfected COS cells produced an enzymatically active protein that was attached to the plasma membrane, with the PLAP domain on the outer surface. Assays for the presence of phosphatidylinositol-glycan attachment proved negative, whereas an antibody assay confirmed the presence of the vesicular stomatis virus glycoprotein carboxyl-terminal peptide, leading to the conclusion that the truncated PLAP is attached to the cells by the membrane-spanning domain of the vesicular stomatis virus glycoprotein. In light of previous findings on carboxyl-terminal requirements of PLAP these studies suggest that an essential signal for correct sorting between transmembrane insertion and phosphatidylinositol-glycan attachment resides in the cytoplasmic domain.

Alkaline Phosphatase

Genetic evidence for the role of Thy-1 in neurite outgrowth in the mouse.

Non-neuronal accessory cells of mouse dorsal root ganglion cultures secrete a complex that stimulates neurite outgrowth in neonatal sympathetic ganglion neurons. A monoclonal antibody that binds to these two cell types also immunoprecipitates neurite outgrowth complex from mouse conditioned medium and binds to mouse brain tissue. Genetic analysis of the component recognized by the antibody revealed that it maps to the Thy-1 locus on mouse chromosome 9. Further studies of cell-type specificity, sedimentation analysis and antibody competition, confirmed that it is indistinguishable from the product of the Thy-1 locus. The finding of an association between Thy-1 and neurite outgrowth complex in the mouse argues for a role of the Thy-1 locus in the interactions of neurons with their surroundings.

Alleles