Overview

During animal development, the germ line serves the unique and critical role of producing gametes and offspring. To serve this role, germ cells require mechanisms to protect their “totipotency” and “immortality”. How do primordial germ cells acquire and preserve these and other germline traits? We are addressing this question in the model animal system, C. elegans, focusing on two regulatory systems: regulators of chromatin organization that are required for germline immortality, and unique cytoplasmic organelles that are required for proliferation and development of germ cells. In addition to germline development, we also study mitosis and patterning in early embryos.

C. elegans

C. elegans is a small (~1 mm) worm that lives in the soil and can be maintained in the lab by growth on bacteria. The adult worm contains only ~1000 somatic cells and ~1000 germ cells, organized into a relatively simple body plan. Sequencing of the worm genome has provided access to all of its ~20,000 genes, and we have both forward genetics and reverse genetic strategies such as RNAi to knock out selected genes and study the consequences. The germ line (highlighted in gray in the cartoon) is set apart from the soma by a series of asymmetric divisions of the early embryo. The primordial germ cells, Z2 and Z3, proliferate during the larval stages and in hermaphrodites generate sperm and then oocytes.

MES proteins and chromatin regulation

Through genetic screens we identified mes-2, mes-3, mes-4, and mes-6 as being essential for germline immortality: in the absence of a maternal supply of mes gene products, the nascent germ line dies. MES-2 and MES-6 are homologous to the Polycomb Group proteins E(Z) and ESC, which bind one another and function as chromatin regulators in insects and vertebrates. MES-2 and MES-6 form a complex with the novel protein MES-3. MES-4, a homolog of mammalian NSD1, appears to function independently of the other MES proteins. How do the MES proteins contribute to germline immortality? Studies from the Reinke and Kelly labs demonstrated a remarkable property of the C. elegans germ line: the X chromosomes are globally silenced during most stages of development. The MES proteins participate in that silencing, as evidenced by chromosome staining experiments and more recently by microarray analysis of gene expression patterns in gonads dissected from mes mutants. The MES-2/3/6 complex participates, probably directly, in X silencing by concentrating a repressive histone modification (methylation of Lys 27 on histone H3, abbreviated H3K27me) on the Xs. MES-4 participates in X silencing indirectly, by coating the autosomes and catalyzing a different histone modification (methylation of Lys36 on histone H3 or H3K36me). We think that MES-4 and/or its methyl mark repel a repressor and help focus repressor action on the Xs (see model). This system illustrates how chromatin modifiers can work in concert to achieve whole-chromosome regulation, and it focuses our attention on the X:autosome distinction. We are current addressing the following questions: To what chromosomal sites do the MES proteins bind? How are the MES proteins targeted to those sites? How do the methyl marks catalyzed by MES-2/3/6 and by MES-4 cause their respective effects on chromatin? Why do the primordial germ cells in mes mutant larvae die? It is an exciting time in both the chromatin field and the germline field – we are enjoying working at the intersection of the two areas.

P granules and control of RNA

"Germ granules" are distinctive organelles found in the germ cells of many species, including C. elegans. They have been invoked as "instructors" of germline development ever since their dramatic segregation to the germ line was first observed in fruit flies, frogs, and nematodes. We have demonstrated that the germ granules in C. elegans (a.k.a. P granules) are indeed required for fertility. The list of constitutive P-granule proteins currently includes PGL-1, PGL-2, PGL-3, GLH-1, GLH-2, GLH-3, GLH-4, and IFE-1. All 8 proteins are predicted to bind RNA and several have been shown to be required for germline development. The PGL proteins function redundantly, PGL-1 being the most critical – its loss leads to sterility but only at elevated temperature. Similarly, the GLH proteins function redundantly, GLH-1 being the most critical – like PGL-1, loss of GLH-1 leads to sterility but only at elevated temperature. Now that we have deletion alleles of all pgl and glh genes, we can finally eliminate the functions of both gene families and determine the effects on P-granule structure and on germline development. IFE-1 is one of the 5 nematode isoforms of eIF4E, the component of the translation initiation complex that binds to mRNA caps. IFE-1 is specifically required for spermatogenesis. The current working model is that germ granules control the trafficking, translation, and/or stability of mRNAs in the germline. Current projects in the lab are aimed at identifying additional P-granule components, defining the pathway of granule assembly, elucidating the roles of individual granule components, and using microarray analysis to investigate RNA regulation. Our studies will provide a better understanding of the composition and functions of these intriguing and still mysterious germline-specific organelles.

Control of mitosis in early development

We are collaborating with Bill Saxton's lab to investigate mitosis and patterning in the early embryo, with a focus on the roles of motor proteins (kinesins and dyneins) that carry cargoes along microtubules. By eliminating the functions of individual motors, we have identified several that are involved in mitotic spindle formation, chromosome segregation, and cytokinesis. Our ability to monitor microtubules, spindle poles, and chromosomes with GFP-tagged components enables us to track and quantify movements in living wild-type and motor-depleted embryos - the movies are very informative and cool to watch! In recent studies, we have shown that KLP-19 provides a polar ejection force on mitotic chromosomes and guarantees their faithful segregation at anaphase, and that dynein participates directly in rotation of the spindle onto the correct axis, chromosome congression at metaphase, and timely initiation of anaphase. Current efforts are focused on anaphase spindle pole separation and dissecting the pushing and pulling forces that operate. We are also interested in extending beyond motors and applying temperature-shift strategies to rapidly inactivate thermosensitive versions of proteins and study the effects in living cells, as we did for dynein.