Chimerism analysis reveals 2 distinct roles of Csk. (A-B) X-gal–stained yolk sac membranes at E9.5. Panel A is from a Csk+/+, Flt-1+/– embryo, and panel B is from a chimeric embryo produced by aggregation between R1 ES cells and Csk–/–, Flt-1+/– morula. The yolk sac in panel A contains vitelline vessels (arrowhead) and capillary sprouts. The blood vessels in panel B are similar to the capillary-like vessels in panel A but drastically improved over those shown in Figure 1D, indicating that sprouting of small blood vessels is essentially normal. However, –/– endothelial cells do not form vitelline vessels in the chimera. (C-D) These panels are portions of body trunks to show intersomitic vessels (arrowheads). Because the genotypes of morulas were unknown at the time chimeric aggregations were set up, they were determined retrospectively after chimeric embryos were dissected, using a previously established protocol.17 For a chimeric embryo, the trophoblasts in its placenta could be derived only from morula embryos but not from R1 ES cells. Thus, during dissection, placental tissue containing trophoblasts were carefully removed and cultured for 2 weeks. Subsequently, DNA samples were extracted and used for genotyping analysis by polymerase chain reaction (PCR). Bars are 100 μm.
