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...

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...

Kinase Suppressor of Ras (KSR) is a conserved protein that positively regulates Ras signaling and may function as a scaffold for Raf, MEK, and ERK. However, the precise role of KSR is not well understood, and some observations have suggested that KSR might act in a parallel pathway. In C. elegans, ksr-1 is only required for a specific Ras-mediated process (sex myoblast migration) and is a nonessential positive regulator of other Ras-mediated developmental events. We report the existence of a second C. elegans ksr gene, ksr-2, which is required for Ras-mediated signaling during germline meiotic progression and functions redundantly with ksr-1 during development of the excretory system, hermaphrodite vulva, and male spicules. Thus, while the ksr-1 and ksr-2 genes are individually required only for specific Ras-dependent processes, together these two genes appear necessary for most aspects of Ras-mediated signaling in C. elegans. The finding that ksr-2; ksr-1 double mutants have strong ras-like phenotypes and severely reduced or absent levels of diphosphorylated MPK-1 ERK strongly supports models where KSR acts to promote the activation or maintenance of the Raf/MEK/ERK kinase cascade.

Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth.

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.