The EMS blastomere divides to produces MS, a progenitor of mesoderm, and E, the endoderm precursor. The generation of unequal daughters by this asymmetric division requires both the Wnt and MAP kinase pathways, which are activated in the endoderm lineage by inductive signals from the neighboring P 2 blastomere. As a result of these signals, the levels of the POP-1 transcription factor are reduced in the nucleus of E compared to MS, and POP-1 is converted from a repressor to an activator of the endoderm-determining
end-1 and -3 genes (Kasmir and Rothman, IWM,1999). We are investigating the regulation of
end-1 in an effort to learn more about the mechanisms that regulate developmentally asymmetric cell divisions. Using affinity purification methods and mass spectrometry we identified several factors that bind to key regulatory sites in the
end-1 promoter (Witze and Rothman, WCWM, 2000). One such factor, PLP-1 ( p ur alpha l ike p rotein), is similar to the mammalian transcription factor pur alpha. RNAi of the
plp-1 gene results in impenetrant phenotypes suggesting a role in the asymmetric division of EMS: in mutant embryos we periodically observe derepression of endoderm in the MS lineage, reminiscent of the
pop-1(-) phenotype. However, unlike
pop-1 mutants, we also observe a gutless phenotype. While the latter phenotype is quite impenetrant, we find that it is greatly enhanced by mutations in the Wnt pathway: for example,
mom-2 ;
plp-1(RNAi) doubles are nearly fully penetrant for the absence of gut. Interestingly, this synergy is not seen with mutations in the MAP kinase pathway, suggesting that PLP-1 may act in the MAPK pathway. Such a possibility is supported by our finding that early embryonic extracts can phosphorylate recombinant PLP-1 protein and that phosphatase treatment of extracts containing PLP-1 eliminates its in vitro binding to the
end-1 promoter. We found that PLP-1 immunoreactive protein is present throughout all nuclei of early embryos up to the 100 cell stage and also associates with P granules. However, it distributes asymmetrically between nuclei of dividing cells: staining is more intense in the future posterior nucleus during telophase in EMS and in many other dividing cells, including the AB granddaughters (which undergo a POP-1-dependent developmentally asymmetric cell division) and even in the zygote, P0. Early in the subsequent cell cycles, staining once again equalizes in the two sister cells. This transient asymmetry in PLP-1 levels may reflect a role in specifying differences in sister cells: for example,
plp-1 mutant embryos do not express the AB-specific component of
lag-2 expression, consistent with a possible conversion in fate of the anterior AB great-granddaughters to that of their posterior sisters. These results lead us to propose that PLP-1 may collaborate with POP-1 to establish differences between daughter cells recursively throughout C. elegans development.