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In response to a growing recognition over the past couple of decades that the field of toxicology needed to transition from a largely descriptive science to one based on the mechanisms of chemical effects in living systems, a partnership among several US government agencies (initially the Environmental Protection Agency, the National Toxicology Program and the NIH Chemical Genomics Center, later joined by the Food and Drug Administration) emerged in the early 21st century. This partnership, which was formally established in 2007 and came to be called Tox21, was based on the complementary expertise of the member agencies and was committed to advancing the science in a transparent manner, with open access to all methodologies and results, and with community involvement in science generation and analysis. The consortium represents a unique collaboration across the US federal government, and in its relatively short existence has had a significant impact on moving the field of toxicology to more mechanism-based in vitro approaches. This chapter reviews the history of the consortium, its major objectives and accomplishments, and identifies new strategic directions that are being formulated to address gaps in technology and knowledge.

Demonstration that cleavage of both APP and Notch are dependent on the product of the early onset Alzheimer's disease gene, presenilin-1 (PS1), has raised the possibility that Notch function may be altered in AD. This finding also suggests that Notch may be affected by APPgamma-secretase inhibitors under development for the treatment of Alzheimer's disease, as these target PS1. Data that address these questions have been lacking, due to inability to specifically modulate PS1 activity in a system directly relevant to the adult human brain. Using novel highly specific inhibitors of PS1/gamma-secretase, we demonstrate that modulation of PS1 activity in human CNS neurons not only affects Abeta generation, but also has unanticipated effects on Notch and its activity. We demonstrate that intracellular trafficking of Notch in human CNS neurons is altered by inhibition of PS1 and is accompanied by dramatic changes in neurite morphology, consistent with inhibition of Notch activity. These data, together with immunohistochemical evidence of elevation of Notch pathway expression in AD brain, suggest that Notch dysregulation may contribute to the neuritic dystrophy characteristically seen in Alzheimer's disease brain. In addition, they raise the possibility that inhibition of gamma-secretase/PS1 may have clinically beneficial effects on the neuritic pathology of AD, in addition to its expected effect to reduce amyloid burden.

Microtiter plate readers have evolved from photomultiplier and charged‐coupled device‐based readers, where a population‐averaged signal is detected from each well, to microscope‐based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536‐well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high‐throughput screening applications using laser‐scanning microplate cytometry, we used green fluorescent protein‐ and yellow fluorescent protein‐expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein‐coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput.

Publisher Summary Knowledge of the genealogical relationships of cells during development provides an insight concerning when and where developmental decisions are being made. Hypotheses can be ruled in or out concerning the commitment of cells to particular fates. For example, when analyzing the cell types that result from the marking of a single progenitor cell, insight can be gained as to whether the progenitor is committed to the production of one or multiple cell types. If multiple cell types are found in a clone, it may be concluded that the progenitor that gave rise to these cells is not restricted to the production of only one cell type. If all of the cells that descend from a progenitor are the same type, the hypothesis is supported that the progenitor is committed to making only that cell type. The complexity and inaccessibility of many types of embryos have made lineage analysis through direct approaches, such as time-lapse microscopy and injection of tracers, almost impossible. A genetic and clonal solution to lineage mapping is the use of retrovirus vectors. The chapter summarizes the basis of this technique, and discusses the current methods and strategies used in the laboratory.

Publisher Summary A genetic and clonal solution to lineage mapping is through the use of retrovirus vectors. The basis for this technique is summarized in this chapter, and the strategies and current methods in use in laboratory are described. A retrovirus vector is an infectious virus that transduces a nonviral gene into mitotic cells in vivo or in vitro . These vectors use the same efficient and precise integration machinery of naturally occurring retroviruses to produce a single copy of the viral genome stably integrated into the host chromosome. Those that are useful for lineage analysis have been modified so that their replication is incompetent, and thus cannot spread from one infected cell to another. They are, however, faithfully passed on to all daughter cells of the originally infected progenitor cell, making them ideal for lineage analysis. Retrovirus vector particles are essentially identical to naturally occurring retrovirus particles. They enter the host cell through interaction of a viral envelope glycoprotein (a product of the viral env gene) with a host cell receptor. The murine viruses have several classes of env glycoprotein that interact with different host cell receptors. The most useful class for lineage analysis of rodents is the ecotropic class.

Publisher Summary This chapter describes the lineage analysis using retrovirus vectors. The complexity and inaccessibility of many types of vertebrate embryos have made lineage analysis through direct approaches, such as time-lapse microscopy and injection of tracers, extremely difficult or impossible. A genetic and clonal solution to lineage mapping in some species is by infection with retrovirus vectors. The chapter summarizes the basis for this technique and details the strategies and current methods in use in the laboratory. A retrovirus vector is an infectious virus that transduces a nonviral gene into mitotic cells in vivo or in vitro. These vectors utilize the same efficient and precise integration machinery of naturally occurring retroviruses to produce a single copy of the viral genome stably integrated into the host chromosome. For lineage applications it is usually necessary to concentrate the virus to achieve sufficient titer. This is typically due to a limitation in the volume that can be injected at any one site. Viruses can be concentrated fairly easily by a relatively short centrifugation step. Virions also can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate is collected by centrifugation. The viral supernatant can be concentrated by centrifugation through a filter that allows only small molecules, to pass (for example, Centricon filters). Regardless of the protocols that are used, it must be kept in mind that retroviral particles are fragile, with short half-lives even under optimum conditions.