AJs in the neuroepithelium of the developing mouse central nervous system. Thus a similar function earlier during development is conceivable but further experiments are needed to underpin this hypothesis. Our marker analysis revealed that PKCi-deficient embryos start to gastrulate and establish distinct anterior-posterior and dorsoventral body axes at around E7.5�CE7.75. However, axial elongation and mesoderm formation arrests around this stage and elaboration of all mesodermal tissues and organs fails. Absence of a functional cardiovascular system is the cause of the subsequent death. Given the fact that all embryonic tissue are affected our data suggest that PKCi is not regionally but cellularly BAY 73-4506 required. Applying the embryoid body formation assay we showed that mutant EBs failed to establish a single cavity instead they formed multiple small cavities. Nevertheless a proper formation of the basal lamina and the apical domain by various marker proteins could be detected. Particular the correct localization of Par-3 and Mupp1 implied an established apical pole which to some content did not surprise since studies performed in C. elegans showed that loss of PKC-3 did not affect Par-3 localization either. Interestingly EBs deficient for Cdc42 not only fail to form a single lumen cavity but also showed a complete loss of aPKC and Par-3 localization at the apical domain. One reason for this difference might be that Cdc42 is not able to distinguish between the two aPKC isoforms and we showed that PKCf is still present in PKCi deficient EBs. Thus we assume that depletion of Cdc42 resulted in a robust reduction of both aPKC activities causing a more severe phenotype. This finds support by the fact that overexpression of a dominant negative PKCf version in wt EBs mimics the Cdc42 phenotype. The formation of a single cavity has been described as a two-step process: first, multiple small lumen are formed in the periphery of the EB and within a second step smaller lumen are fused and remaining inner cells undergo apoptosis to clear the central lumen. Our data suggest that the loss of PKCi did not interfere with the first step but disrupted the second. Whether PKCi acts on the fusion of the small cavities or the subsequently induced apoptosis or both is not solved yet but from our data its clear that PKCi deficiency caused a decrease of activated caspase-3 and less apoptotic cells in EBs. An earlier study on the role of Par6 and aPKCs in Caco-2 cysts formation showed that down-regulation of either one of the proteins is causing multiple lumen formation as well. In this it was correlated to an Everolimus impaired spindle orientation during morphogenesis. These findings might represent an alternative explanation for the observed phenotype. However, we were not able to identify any indication for an impaired spindle formation, either in PKCi deficient embryos or in PKCi deficient EBs. Again, one likely reason for this difference might be that the knock-down of Par6 and both aPKCs is more dramatic than the loss of one single aPKC isoform. In this case the data would indicate compensatory functions in the context of spindle orientation in Caco-2 cysts. The immunohistochemical analysis of E7.5 embryo sections revealed by the E-cadherin staining that the general polarity of the embryonic ectoderm is preserved in mutants. But it became apparent that the fine structure of the apical side is changed when compared to the wt at higher magnification. As a possible explanation we found ZO-1, a tight junction protein, to be down-regulated and dislocated in epithelial cells deficient for PKCi. ZO-1 is believed to function as a junctional organizer by direct binding to 
tight junctional proteins and the actin cytoskeleton. Thus it could well be that the absence of ZO-1 in tight junction complexes could lead to a lack of proper connections of tight junctions to the actin cytoskeleton.