Summary Crossovers (COs) between homologous chromosomes ensure their faithful segregation during meiosis. We identify C. elegans COSA-1 as a key component required to convert double-strand breaks (DSBs) into COs. COSA-1 localizes to foci during late meiotic prophase that correspond to the single CO site on each homolog pair. These foci represent sites of eventual concentration of other conserved CO proteins that initially exhibit broader distribution. Chromosomes gain and lose competence to load CO proteins at DSBs during meiotic progression, with competence to load COSA-1 requiring a prior licensing event. Our data further suggest a self-reinforcing mechanism maintaining CO designation. Modeling of a non-linear dose-response relationship between IR-induced DSBs and COSA-1 foci reveals efficient conversion of DSBs into COs when DSBs are limiting, and a robust capacity to limit the number of cytologically-differentiated CO sites when DSBs are in excess. COSA-1 foci serve as a biodosimeter for DSB levels and a unique live-cell readout of CO interference.

The raison d'etre of the germline is to produce oocytes and sperm that pass genetic material and cytoplasmic constituents to the next generation. To achieve this goal, many developmental processes must be executed and coordinated. ERK, the terminal MAP kinase of a number of signaling pathways, controls many aspects of development. Here we present a comprehensive analysis of MPK-1 ERK in Caenorhabditis elegans germline development. MPK-1 functions in four developmental switches: progression through pachytene, oocyte meiotic maturation/ovulation, male germ cell fate specification, and a nonessential function of promoting the proliferative fate. MPK-1 also regulates multiple aspects of cell biology during oogenesis, including membrane organization and morphogenesis: organization of pachytene cells on the surface of the gonadal tube, oocyte organization and differentiation, oocyte growth control, and oocyte nuclear migration. MPK-1 activation is temporally/spatially dynamic and most processes appear to be controlled through sustained activation. MPK-1 thus may act not only in the control of individual processes but also in the coordination of contemporaneous processes and the integration of sequential processes. Knowledge of the dynamic activation and diverse functions of MPK-1 provides the foundation for identification of upstream signaling cascades responsible for region-specific activation and the downstream substrates that mediate the various processes. I N the generation of oocytes and sperm, perhaps the most complex cells in animals, the germline lineage undergoes a multifaceted developmental process that begins in embryogenesis and continues into adulthood. While the details of the steps can differ between species due to differences in reproductive biology, a core set of events occurs in animals: germ cell fate specification, incorporation into the gonad, sexual fate specification, proliferative expansion, and gamete production. Two interconnected differentiation programs define gamete production: (1) meiosis where chromosomes pair, recombine, and then segregate to give a reassorted haploid content and (2) gametogenesis where biosynthetic and morphogenetic processes generate the large nutrient and developmental information-rich oocyte and the small motile sperm. In aggregate, these processes are essential to pass genetic material from generation to generation and to form the totipotent zygote necessary for the development of a new individual. Disruption of germline development can lead to sterility, germline tumors, and birth defects. Thus an important goal is to define the pathways and gene products that control and execute the various steps of germline development.The MAP kinase extracellular signal-regulated kinase (ERK) functions in many aspects of animal development and homeostasis (Marshall 1995;Rubin et al. 1997;Schlessinger 2000;Sundaram 2006). ERK is the terminal regulator of signaling cascades such as canonical receptor tyrosine kinase signaling, which contains core members, including the RA...

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.

SUMMARY High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach—profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes—pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

RAS-extracellular signal regulated kinase (ERK) signaling governs multiple aspects of cell fate specification, cellular transitions, and growth by regulating downstream substrates through phosphorylation. Understanding how perturbations to the ERK signaling pathway lead to developmental disorders and cancer hinges critically on identification of the substrates. Yet, only a limited number of substrates have been identified that function in vivo to execute ERK-regulated processes. The Caenorhabditis elegans germ line utilizes the well-conserved RAS-ERK signaling pathway in multiple different contexts. Here, we present an integrated functional genomic approach that identified 30 ERK substrates, each of which functions to regulate one or more of seven distinct biological processes during C. elegans germ-line development. Our results provide evidence for three themes that underlie the robustness and specificity of biological outcomes controlled by ERK signaling in C. elegans that are likely relevant to ERK signaling in other organisms: (i) multiple diverse ERK substrates function to control each individual biological process; (ii) different combinations of substrates function to control distinct biological processes; and (iii) regulatory feedback loops between ERK and its substrates help reinforce or attenuate ERK activation. Substrates identified here have conserved orthologs in humans, suggesting that insights from these studies will contribute to our understanding of human diseases involving deregulated ERK activity.functional genomics ͉ signaling ͉ MPK-1 ͉ RNAi screen T he RTK-RAS-ERK pathway relays extracellular signals via a conserved kinase cascade that results in phosphorylation and activation of ERK, the terminal member of this pathway (1, 2). Active ERK in turn phosphorylates substrates to execute many cellular and developmental processes (2) (Fig. 1A). Comprehensive insight into mechanisms underlying ERK-dependent control of biological processes depends on identification of ERK substrates. Although bona fide ERK substrates have been identified in cultured cells (3-5) and potential substrates documented from proteomic studies (6, 7), the function of most of these substrates in vivo has not been defined. Additionally, currently identified substrates do not account for most ERK-dependent events in mammals, making it likely that more substrates remain to be identified. Forward genetic studies in Caenorhabditis elegans and Drosophila have identified a few ERK substrates that act in defined biological contexts (8-10); however, the mutant phenotypes of these genes account for some but not all ERK-regulated processes. To obtain molecular insight into how ERK signaling controls multiple biological processes in vivo through substrates, we devised an integrated bioinformatic, genetic, and biochemical approach by using C. elegans germ-line development as the model system.In C. elegans RAS and ERK are encoded by let-60 and mpk-1, respectively, and loss-of-function or null mutations in these core components abrogate pat...

Signaling pathways and small RNAs direct diverse cellular events, but few examples are known of defined signaling pathways directly regulating small RNA biogenesis. We show that ERK phosphorylates Dicer on two conserved residues in its RNAse IIIb and dsRNA-binding domain, and phosphorylation of these residues is necessary and sufficient to trigger Dicer’s nuclear translocation in worms, mice, and human cells. Phosphorylation of Dicer on either site inhibits Dicer function in the female germ line and dampens small RNA repertoire. Our data demonstrate that ERK phosphorylates and inhibits Dicer during meiosis I for oogenesis to proceed normally in C. elegans and that this inhibition is released before fertilization for embryogenesis to proceed normally. The conserved Dicer residues, their phosphorylation by ERK, and the consequences of the resulting modifications implicate an ERK-Dicer nexus as a fundamental component of the oocyte-to-embryo transition and an underlying mechanism coupling extracellular cues to small RNA production.

In 1893 August Weismann proposed that information about the environment could not pass from somatic cells to germ cells1, a hypothesis now known as the Weismann barrier. However, recent studies have indicated that parental exposure to environmental stress can modify progeny physiology2–7 and that parental stress can contribute to progeny disorders8. The mechanisms regulating these phenomena are poorly understood. We report that the nematode C. elegans can protect itself from osmotic stress by entering a state of arrested development and can protect its progeny from osmotic stress by increasing the expression of the glycerol biosynthetic enzyme GPDH-2 in progeny. Both of these protective mechanisms are regulated by insulin-like signalling: insulin-like signalling to the intestine regulates developmental arrest, while insulin-like signalling to the maternal germline regulates glycerol metabolism in progeny. Thus, there is a heritable link between insulin-like signalling to the maternal germline and progeny metabolism and gene expression. We speculate that analogous modulation of insulin-like signalling to the germline is responsible for effects of the maternal environment on human diseases that involve insulin signalling, such as obesity and type-2 diabetes8.

Tissues that generate specialized cell-types in a production line must coordinate developmental mechanisms with physiological demand, although how this occurs is largely unknown. In the C. elegans hermaphrodite, the developmental sex-determination cascade specifies gamete sex in the distal germline, while physiological sperm signaling activates MPK-1/ERK in the proximal germline to control plasma membrane biogenesis/organization during oogenesis. We discovered repeated utilization of a self-contained negative regulatory module, consisting of NOS-3 translational repressor, FEM-CUL-2 (E3 ubiquitin ligase) and TRA-1 (Gli transcriptional repressor), which acts both in sex-determination and in physiological demand control of oogenesis, coordinating these processes. In the distal germline, where MPK-1 is not activated, TRA-1 represses the male fate as NOS-3 functions in translational repression leading to inactivation of the FEM-CUL-2 ubiquitin ligase. In the proximal germline, sperm-dependent physiological MPK-1 activation results in phosphorylation-based inactivation of NOS-3, FEM-CUL-2 mediated degradation of TRA-1 and the promotion of membrane organization during oogenesis.