Constance L. Cepko *
Elizabeth Ryder1
Christopher Austin2
Jeffrey Golden3
Shawn Fields-Berry *
John Lin *
* Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA 02115
1 Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609
2 Merck Research Laboratories, West Point, PA 19486
3 Department of Pathology, Children's Hospital of Philadelphia, Philadelphia, PA 19104
ABSTRACT
Knowledge of the geneological relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Geneological relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. We describe the use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues. An overview of the relevant aspects of the retroviral life cycle, and the strategies and current methods in use in our laboratory are described.
Knowledge of the geneological relationships of cells during development can allow one to gain insight into 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, one can gain insight into whether the progenitor was committed to the production of one or multiple cell types. If multiple cell types are found in a clone, one can conclude that the progenitor that gave rise to these cells was not restricted to the production of only one cell type. Alternatively, if all of the cells that descend from a progenitor are the same type, the hypothesis is supported, but not proven, that the progenitor was committed to making only that cell type. In the latter case, a firm conclusion concerning the commitment of the progenitor can be reached only if the progenitor and/or progeny are exposed to a variety of environments. If only one cell type is produced despite variations in the environment, commitment of the progenitor to production of one cell type is supported. Analyses of clones generated after marking the progenitors of a tissue at various times in development can greatly aid in charting the stages of production of different cell types, allowing one to focus studies concerning cell fate decisions to particular times and places. In addition, analysis of the proliferation and migration patterns exhibited by clones can increase our understanding of the development of a particular area.
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 through the use of retrovirus vectors. The basis for this technique will be summarized, and the strategies and current methods in use in our laboratory will be detailed.
