Clonal analysis of the origin of primordial germ cells in the mouse.

Qualitative and quantitative clonal analysis has been used to answer three basic questions about the establishment of the germ cell lineage in the mouse. Where do primordial germ cells originate? What is the size of the founding population at the time of lineage restriction? When and where does lineage restriction occur? Single epiblast cells of 6.0 dpc and 6.5 dpc mouse embryos were injected with a short-term lineage label (lysinated rhodamine dextran, LRDX) and their descendants traced after 40 h embryo culture at neural plate and early somite stages, respectively. An objective matching technique was used to detect the lineage marker in primordial germ cells identified by their characteristic alkaline phosphatase staining. Precursors of the primordial germ cells were found in the proximal epiblast close to the extraembryonic ectoderm in both pregastrulation and early-streak stage embryos. They form part of the presumptive extraembryonic mesoderm and are not lineage restricted while in the epiblast. Quantitative analysis gives a best fit to a model of a founding population of 45 at the time of lineage restriction. The data indicate that the generation time lengthens at the time of allocation. Calculation of clonal histories gives a best fit of 16 h generation time after allocation compared with < 7 h before allocation, with lineage restriction occurring at the early midstreak stage, presumably in the region posterior to the streak in which primordial germ cells are first identifiable. Therefore primordial germ cells are probably allocated early during gastrulation in a group of > 40 cells already segregated in the extraembryonic mesoderm.