During C. elegans development, two primordial germ cells (PGCs) must acquire germ fate and proliferate into hundreds of germ cells to form a complete and fertile adult germline. How PGCs acquire and maintain germ cell fate is poorly understood. Two important contributors are the chromatin regulators MES-4, a histone methyltransferase, and MRG-1, a chromodomain-containing protein. Without maternal delivery of these proteins to embryos, germ cells in early larvae fail to proliferate and die, resulting in sterile adults. We hypothesize that MES-4 and MRG-1 transmit the memory of a germline gene expression program from parent germ cells to progeny germ cells to ensure that PGCs express the appropriate genes for their survival and proliferation. Our working model is that 1) during embryogenesis, MES-4 maintains a memory of which genes were previously expressed in the parental germline by maintaining H3K36me3 on nucleosomes that package those genes, and 2) MRG-1 effects the memory by binding to H3K36me3 generated by MES-4 and activating an appropriate germline transcription program in PGCs. To test our model, I am determining whether PGCs from L1s that lack MES-4 or MRG-1 fail to launch an appropriate germline gene expression program. Due to the previous limitation in gathering enough PGCs for mRNA profiling, I first profiled mRNAs from mutant adult germlines that inherited maternally provided MES-4 and MRG-1, but cannot express one of the gene products (M+Z-).
mes-4 M+Z- and
mrg-1 M+Z- mutant adult germlines misexpress a similar set of genes that are normally repressed in germ cells: somatic genes and genes on the X chromosome. Thus, MES-4 and MRG-1 are required for a proper germline gene expression program and they regulate similar genes in adult germlines. We predict that PGCs in L1 progeny of these mutant adults, which completely lack MES-4 or MRG-1 (M-Z-), fail to acquire germ cell fate because they fail to launch a proper germline transcription program and more severely misexpress somatic genes and genes on the X chromosome. To test this prediction, I developed a method to isolate PGC pairs from wild-type and mutant L1 larvae to profile their mRNAs. I showed that this approach can successfully profile the mRNA population from single sister pairs of wild-type PGCs. This dissection approach offers advantages over FACS sorting large populations of PGCs: I can analyze mutant PGCs instead of RNAi PGCs, PGCs are of a uniform age, and PGCs spend minimal time in buffer before RNA preparation. I am currently analyzing the transcriptome of PGCs isolated from
mes-4 and
mrg-1 M-Z- mutant larvae, to learn how maternal MES-4 and MRG-1 ensure PGC survival and proliferation.