We are investigating how genes control germ cell development in Caenorhabditis elegans. C. elegans is an important metazoan model organism for developmental biology because of its tractable genetics, transparency (allowing observation of development in living animals using Nomarski microscopy as well as the dynamics of gene product expression and subcellular localization using GFP tagged molecules), and the large body of information already known about its development and genome.

The germ line is a unique immortal tissue of metazoans that is used in sexual reproduction for the continued propagation of the species. We are studying a number of processes that are critical for germ cell development:
the decision of stem cells to proliferate or enter meiotic prophase,
the control of progression through meiotic prophase,
the regulation of meiotic maturation and ovulation, and
the determination of sexual identity.

Three general strategies are employed in the analysis. 1) Genes that control germ cell development are identified by mutations that disrupt one or more germline developmental processes. For example, mutations which result in the failure of germ cells to enter meiotic prophase can have a tumorous phenotype, while mutations that disrupt sex determination can result in the inappropriate specification of sexual fate (i.e., a male with oocytes). Further genetic, cellular and developmental studies are used to deduce the wild-type process(es) that a gene controls, and to order gene functions in genetic pathways. 2) Molecular analysis of specific genes that control germline development is used to translate the genetic pathways into molecular pathways of gene function. The sequences of gene products that we have analyzed to date indicate that the molecules and the molecular pathways have been conserved during evolution. 3) The role of the somatic gonad in controlling germline development is investigated by eliminating specific somatic gonad cells, either by laser ablation or by mutations that alter their cell fate, and analyzing the effect of the change on germline development.

The distal portion of the C. elegans germ line consists of a population of mitotically active stem cells whose proliferation is regulated by signaling from the somatically-derived distal tip cell (DTC). The DTC promotes germ cell proliferation and/or inhibits entry of germ cells into the meiotic pathway - in the absence of the inductive signal from the DTC, all germ cells enter meiosis. The signaling molecule produced by the DTC is LAG-2, a protein related to the Drosophila Delta and Serrate proteins. The germline receptor for LAG-2 is GLP-1, a member of the Notch family of receptors.

We are interested in how the decision to proliferate versus enter the meitic developmental pathway is made. We have isolated a gain-of-function, oncogenic allele of glp-1 which prevents germ cells from entering meiosis and causes germline tumor formation. We have also isolated enhancer mutations of a weakly tumorous glp-1 allele. These teg genes [for tumorous enhancer of glp-1(oz112oz120)] may define negative regulators of germ cell proliferation or positive regulators of entry into the meiotic development pathway.

In addition, we have isolated a gain-of-function allele of the let-42 gene which, like the glp-1(gf) mutation, has a tumorous germline phenotype and may prevent germ cells from entering the meiotic pathway. LET-42 is an evolutionarily conserved protein of unknown biochemical function.

Additionally, we have found that loss-of-function alleles of gld-1, which encodes a putative cytoplasmic RNA binding protein, acts as part of a redundant control system to negatively regulate stem cell proliferation.

Finally, we have found that the ablation of certain somatic gonad cells can lead to ectopic germ cell proliferation.

Having entered the meiotic cell cycle, a germ cell progresses through the stages of meiotic prophase: leptotene, zygotene, pachytene, diplotene and diakinesis. We are interested in understanding how the transitions between different stages of meiotic prophase are controlled. To this end, we have isolated mutations in a number of genes that disrupt various aspects of meiotic prophase progression.

Mutations in the gld-1 gene affect only female germ cell development. Severe loss-of-function gld-1 alleles cause female germ cells to exit the meiotic cell cycle at the pachytene stage to resume proliferation. A germline tumor results. Partial loss-of-function alleles of gld-1 cause female germ cells to arrest in pachytene.

Loss-of-function mutations in several genes encoding components of the MAP kinase signaling cascade mpk-1 (MAP kinase), mek-2 (MEK), lin-45 (Raf) and let-60 (Ras), cause both male and female germ cells to arrest at the pachytene stage. Mutations in the six currently identified pex (pachytene exit) genes, whose products are yet unknown, produce a similar meiotic prophase progression defect.

In C. elegans, germ cells undergoing spermatogenesis proceed directly from meiotic prophase through the meiosis I and meiosis II divisions to produce four haploid spermatids. Germ cells undergoing oogenesis, in contrast, experience considerable growth during diplotene/diakinesis and, in females, arrest at diakinesis for long periods until mating with a male occurs. The transition from diakinesis of meiotic prophase to metaphase of meiosis I is called meiotic maturation.

An oocyte begins meiotic maturation just prior to its ovulation into the spermatheca. At this time the nuclear envelope breaks down, the nucleus migrates posteriorly, and oocyte undergoes cortical rearrangement. These changes in the oocyte are accompanied by increased contractile activity of the myoepithelial somatic sheath cells that surround the proximal end of the germ line. The oocyte is then ovulated by dilation of the distal spermatheca. Once the oocyte is ovulated into the spermatheca, it is fertilized and completes the MI and MII divisions in the uterus.

We have uncovered five cell-cell interactions that regulate maturation and ovulation. 1) Sperm induce oocyte maturation; this process is temporally and spatially distinct from fertilization. 2) The somatic sheath cells that surround the oocytes promote maturation. 3) Sperm promote sheath contraction. 4) The maturing oocyte modulates sheath contractions at ovulation. 5) The maturing oocyte induces distal spermathecal dilation.

We have used time-lapse video microscopy to characterize the time course of oocyte meiotic prophase progression and ovulation in wildtype animals. We further used this technique to analyze the interaction between the germ line and somatic gonad during this process, as well as to characterize mutations that disrupt oocyte meiotic maturation and ovulation.

We have isolated a collection of mutations that disrupt oocyte meiotic maturation and ovulation. One group of genes, the Emo (endomitotic oocyte) loci, cause oocytes to undergo chromosome replication without karyokinesis. Detailed characterization of these mutations reveals that their oocyte endomitosis phenotype is a secondary consequence of a defect in ovulation. Two genes that show an Emo phenotype are let-23, which encodes the Caenorhabditis EGF receptor homolog, and lin-3, which encodes an EGF-like molecule. lin-3 is required in the oocyte and let-23 is needed in the soma to regulate ovulation.

We are currently screening for mutations that specifically affect female meiotic cell cyle progression.

The C. elegans hermaphrodite produces both sperm and oocytes from a common pool of cells. The first 40 or so germ cells that enter meiosis in the hermaphrodite adopt the male sexual identity and form sperm, while the remaining germ cells develop into oocytes.

To investigate the molecular basis of germline sex determination, we have identified mutations that transform all germ cells into sperm or oocytes. We have cloned two genes that act to promote adoption of the male germ cell fate, gld-1 and fog-2. GLD-1 is a cytoplasmic protein containing a KH type RNA binding domain. FOG-2 is a member of a novel protein family.

In addition, we have found that the ablation of certain somatic gonad cells in the hermaphrodite - either by mutation or by laser killing - can cause all germ cells to adopt a female sexual identity.