During animal development, the germline 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 specific traits? We are addressing this question in the model animal system, C. elegans, focusing on two germline-specific 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.
C. elegans is a small (~1 mm) worm that dwells 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. The worm genome is relatively small (100 Mbp) and has been cloned and completely sequenced. Thus, we have access to all of the worm's ~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.
Through genetic screens
we previously identified MES-2, MES-3, MES-4, and MES-6 as being essential
for germline immortality: in the absence of a maternal supply of these
proteins, the nascent germline dies. MES-2 and MES-6 are homologous
to the Polycomb Group proteins Enhancer of zester [E(Z)] and Extra
sex combs [ESC], which bind one another and function as chromatin
repressors in insects and vertebrates. MES-2 and MES-6 form a complex
with MES-3, which has no recognizable homologs in other species. MES-4
appears to function independently of the other MES proteins. Furthermore,
it has a distinct and remarkable distribution: MES-4 binds along the
lengths of the 5 autosomes but is absent from most of the X chromosome.
It was recently shown that the X chromosomes are maintained in a silenced
chromatin state throughout most of C. elegans germline development.
The MES system is involved in regulating X-chromosome silencing, as
loss of the MES-2/3/6 complex causes MES-4 to spread to the X chromosomes
and causes the XÕs to acquire marks of active chromatin. Thus, the
MES proteins participate in controlling global patterns of chromatin
organization and presumably patterns of gene expression in the germline.
"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 germline 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, the 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, and elucidating the roles of individual granule components. One new and already-successful approach is to use worms that express a GFP-tagged version of PGL-1 to screen for mutants that lack normal P granules (i.e., lack granular GFP::PGL-1). Our studies will provide a better understanding of the composition and functions of these intriguing and still mysterious germline-specific organelles.
In a collaboration with Bill Saxton's lab, we are investigating the roles in early embryos of the ~20 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! Among our recent advances are elucidation of some roles of the kinesin KLP-19 and cytoplasmic dynein. KLP-19 provides a polar ejection force on mitotic chromosomes and guarantees their faithful segregation at anaphase. We hypothesize that KLP-19's function is to maintain constant tension on microtubule-kinetochore attachments, which helps orient kinetochores toward the spindle poles and minimizes aberrant kinetochore attachments. Our dynein studies have made use of an allelic series of dominant, temperature-sensitive mutant alleles of dynein heavy chain (DHC-1). Rapid inactivation of DHC-1 at different times in the first cell cycle has revealed direct roles of dynein in rotation of the spindle onto the correct axis, chromosome congression to a well-ordered metaphase plate, and timely initiation of anaphase. Our current motor efforts are focused on anaphase spindle pole separation and dissecting the pushing and pulling forces that operate.