Showing papers by "Christopher A. Walsh published in 1995"

Cell lineage and patterns of migration in the developing cortex.

Abstract: Knowledge of cell lineage in the cortex is important for understanding normal development as well as brain malformations. We studied cell lineage in rats by injecting a library of up to 3400 retroviruses, distinguishable by PCR analysis and encoding alkaline phosphatase, at E14-19. Histological analysis at P15 revealed normal cell morphology and allowed identification of about 80% of all labelled cells. PCR amplification of DNA tags allowed clonal analysis. Cortical cells labelled at E15 formed clustered or widespread clones with equal frequency. Clustered clones contained one to four cells within about 1 mm that had similar morphology and laminar location. However, 48% of cortical clones contained multiple cell types with widely different locations (2.1-6.7 mm; mean, 3.8 mm). Widespread clones contained two to four 'subunits' (one to five neurons each), spaced at apparent intervals of 2-3 mm, with each subunit morphologically indistinguishable from a clustered clone. Distinct subunits in the same clone usually differed in laminar location suggesting sequential formation. Clones labelled at E17 contained fewer neurons and up to two subunits. Clustered clones seem to be produced by stationary progenitors, whereas progenitors of clusters may themselves be produced by migratory, multipotential cells.

The cell lineage of neuronal subtypes in the mammalian cerebral cortex - Discussion

Abstract: We have studied the lineage relationships of pyramidal and nonpyramidal neurons, the principal neuronal types in the cerebral cortex, using a recombinant retrovirus that carries the gene encoding Escherichia coli beta-galactosidase as a lineage marker. The phenotype of every cell of clones of beta-galactosidase-labelled neurons generated by intraventricular injection of recombinant retrovirus in rat embryos at different stages of cortical neurogenesis was identified using light and electron microscopy as well as immunohistochemistry for known markers of neuronal subtypes. We found that clonally related neurons in adult rats showed the same morphological and neurotransmitter phenotypes, suggesting that lineages of pyramidal and nonpyramidal neurons are specified as early as E14, the time of onset of neurogenesis. However, when we followed the development of cortical cell lineages, we noted that a significant number of neuronal clones showed a mixed pyramidal/nonpyramidal cell composition during the first three weeks of life. We suggest that the change in the composition of neuronal clones between the third week of postnatal life and adulthood may either be due to changes in the phenotype of some developing neurons or, more likely, to selective cell death.

Factors regulating the differentiation of neural precursors in the forebrain - Discussion

Abstract: Precursors from the neuroepithelium of the developing cortex and the adult subventricular zone can be cloned in vitro after stimulation with fibroblast growth factor 2 (FGF-2), and they have the potential to give rise to both neurons and glia. The generation of neurons from these clones can be stimulated by either a factor derived from an astrocyte precursor line, Ast-1, or FGF-1. We have shown that neuronal differentiation stimulated by FGF-1 can be inhibited by diacylglycerol lipase inhibitor and mimicked by arachidonic acid, suggesting that the neuronal differentiation is signalled through the phospholipase C gamma pathway. The sequential expression of FGF-2, followed by FGF within the developing forebrain neuroepithelium, fits with the different functions that the two FGFs play in precursor regulation. We have shown that the precursor response to FGF-1 is regulated by a heparan sulphate proteoglycan expressed within the developing neuroepithelium. Precursors restricted to the astrocyte cell lineage can be stimulated by epidermal growth factor or FGF-2F however, the differentiation into glial fibrillary acidic protein-positive astrocytes appears to require a cytokine acting through the leukaemia inhibitory factor-beta receptor.

The specificity of interactions between the cortex and the thalamus - Discussion

Abstract: The functioning of the adult mammalian cerebral cortex depends critically upon precise interconnections between specific thalamic nuclei and distinct cortical regions. Therefore, one central issue in understanding cortical development is determining the cellular and molecular strategies underlying the specification of thalamocortical projections. We address the role of axon-axon interactions and membrane-bound guidance molecules in the establishment of the development of layer-specific patterns of afferent and efferent cortical connections does not depend upon neuronal activity. We present evidence that activity conveyed by thalamic afferents is required for the elaboration of the columnar specificity of cortical circuits.

[18] Polymerase chain reaction-based techniques for utilizing retroviruses as cell lineage markers

Abstract: Publisher Summary This chapter describes polymerase chain reaction (PCR)-based techniques for utilizing retroviruses as cell lineage markers. “Lineage” refers to the patterns of cell division and differentiation that lead from single cells to complex tissues. To understand the molecular mechanisms that direct cellular development, precise information about intercellular interactions is indispensable. The advent of PCR allows the direct determination of retrovirally visualized clonal relationships without making assumptions about cell behavior. The chapter presents two techniques that broaden the applicability of retroviral vectors as lineage tracers. In the first method, the insertion site of retroviruses can be directly amplified and sequenced using a modification of PCR called “inverse PCR.” In the second method, retroviral vectors are modified to contain short DNA sequences that serve as DNA tags; these tags can then be amplified from single retrovirally labeled cells to allow determination of clonal relationships. Although the two techniques have been developed in the context of retrovirally mediated lineage analysis, both have broader applicability. The inverse PCR technique is a rapid and convenient method for amplifying and cloning unknown DNA sequences adjacent to known sequences. The technique starts with genomic DNA isolated from a large clone of cells carrying a single retroviral integration site.