Expression of Bmp4 in the lateral plate mesoderm, the extraembryonic mesoderm and the extraembryonic ectoderm was found in mutant embryos as in the wt. Tbx20 was expressed in the allantois, a derivative of the extraembryonic mesoderm, and in the linear heart tube, which derives from the cardiac subregion of the anterior lateral plate mesoderm in the wt. Again, both expression domains were established at opposite poles of the mutant embryos but neither domain was appropriately developed into an allantois. Finally, Gja, that is expressed in the cardiac endoderm and dorsal aorta in wt embryos was restricted to cell clusters at the anterior and posterior pole of the mutant embryos but clearly lacked an organization into vessel like structures. Together, this analysis suggests that PKCi-deficient embryos exhibit a normal anterior-posterior polarization of their main body axis. Mesoderm formation is initiated but comes to a premature halt, mesoderm differentiation into axial, paraxial, lateral and extraembryonic subtypes occurs but subsequent formation of tissues and organs completely fails. As a consequence PKCi-deficient embryos die due to lack of a cardiovascular system. ESCs, when cultured as aggregates, form spherical structures which are defined as embryoid bodies. These structures are thought to recapitulate early steps of the pre-implantation development including endoderm formation, basement membrane assembly, epiblast polarization and subsequent cavity formation. When cultured in suspension for 5 days, EBs form epithelial cysts consisting of an outer endoderm and an inner columnar epiblast epithelium, separated by a BM. As PKCi mutants displayed an abnormal amniotic cavity at E7.5 we decided to use the embryoid body formation assay to analyze cavity formation in more detail. When wt and PKCi deficient ESCs were subjected to this assay, we detected obvious differences among the two genotypes. Wt EBs displayed the expected appearance in a phase contrast representation, PKCiD/D EBs showed a disorganized structure. Most of the wt EBs formed a single large cavity enclosed by CEE after 5 days, whereas the mutant EBs did not form a single cavity at all or formed multiple small cavities adjacent to the polarized CEE, although the endoderm differentiated normally. When cultured for additional 2 days, most mutant EBs formed small cavities but failed to complete cavitation. Since caspase-dependent NSC 136476 structure apoptosis has been described as a key mechanism involved in the early steps of cavity formation we immunostained 5-day EBs for cleaved caspase-3. Wt EBs displayed massive central apoptosis as evidenced by cleaved caspase-3 staining. By contrast, only scattered WY 14643 50892-23-4 apoptotic cells were detected in PKCi deficient EBs. DAPI staining of condensed and fragmented nuclei, another hallmark of apoptosis, also showed reduced apoptosis at the center of mutant EBs. To further analyze the apoptosis during EB cavitation, we cultured EBs of both genotypes for 2 to 5 days and performed immunoblotting for cleaved caspase-3. Indeed, PKCi deficient EBs expressed less apoptotic activity at all time points tested than the corresponding wt extracts. These results demonstrate that PKCi deficient EBs fail to form a single cavity possibly due to reduced central apoptosis. Gene targeting experiments have shown that laminin-mediated BM formation is essential for primitive ectoderm epithelialization and cavitation in EBs. The laminin a-1 chain is a key component of the embryonic BM expressed early on during development. In the wt EB the laminin a-1 staining formed a continuous thin layer between endoderm and epiblast cells. We did not observe obvious changes in BM assembly in mutant EBs. These findings were confirmed by using perlecan as an alternative marker for the BM. However, ectopic BM formation was detected at the center of,20% mutant EBs.