Fig. 2. Requirement of Sall4 for inner cell mass proliferation in blastocysts in vitro. (A) Normal development of wild-type (+/+), heterozygous (+/-) and Sall4-null (-/-) blastocysts (E 3.5). Sall4 is expressed in the inner cell mass and trophectoderm (left column). Oct3/4 (red) and Cdx2 (green) staining shows that commitment to the inner cell mass and trophectoderm occurs normally in the Sall4 homozygotes (compare middle and right columns). (B) Reduction of inner cell mass in Sall4-null blastocysts cultured in vitro. Uppermost row shows phase-contrast photo at 5 day of culture. Second row shows phase contrast at 3 day of culture. Lower two rows show immunostaining of Oct3/4 and Sall4. E, primitive endoderm; I, inner cell mass; T, trophectoderm. (C) RT-PCR analysis of markers in blastocysts cultured for 3 days. All the lineage markers are expressed. (D) Reduced proliferation of the inner cell mass of Sall4-null blastocysts cultured for 3 days. BrdU incorporation of the inner cell mass is reduced in Sall4-null blastocysts (arrow), compared with wild type (arrowhead). (E) Failure of growth of Sall4-null inner cell mass free from the trophectoderm.