Ronald E. Ellis

Complementary Signaling Pathways Regulate the Unfolded Protein Response and Are Required for C. elegans Development

The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). We have used C. elegans as a genetic model system to dissect UPR signaling in a multicellular organism. C. elegans requires ire-1-mediated splicing of xbp-1 mRNA for UPR gene transcription and survival upon ER stress. In addition, ire-1/xbp-1 acts with pek-1, a protein kinase that mediates translation attenuation, in complementary pathways that are essential for worm development and survival. We propose that UPR transcriptional activation by ire-1 as well as translational attenuation by pek-1 maintain ER homeostasis. The results demonstrate that the UPR and ER homeostasis are essential for metazoan development.

Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females.

In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage–induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem.

Turning Clustering Loops: Sex Determination in Caenorhabditis elegans

The nematode Caenorhabditis elegans has two sexes: males and hermaphrodites. Hermaphrodites are essentially female animals that produce sperm and oocytes. In the past few years tremendous progress has been made towards understanding how sexual identity is controlled in the worm. These analyses have revealed that the regulatory pathway controlling sexual development is far from linear and that it contains a number of loops and branches that play crucial roles in regulating sexual development. This review summarizes our current understanding of the mechanisms that regulate sexual cell fate in C. elegans.

Mutations in Two Independent Pathways Are Sufficient to Create Hermaphroditic Nematodes

Life Histories to Suit Nematode worms can profoundly manipulate their life histories in several ways. For example, Caenorhabditis elegans has two genders: males and hermaphrodites. Some clues for the evolution of this peculiar mating system have been revealed by Baldi et al. (p. 1002), who turned females of a related species, Caenorhabditis remanei, into hermaphrodites by modifying a gene involved in making sperm and another gene required for activating the spermatids. In most animals, the germ line is fully established during adulthood and a reproductive period is determined, at least in part, by aging of the germ line and the viability of oocytes. The reproductive longevity of hermaphrodite C. elegans can be increased at least 15-fold by starvation. Angelo and Van Gilst (p. 954, published online 27 August; see the Perspective by Ogawa and Sommer) found that in starved worms, the germline component of the reproductive system is actively killed, with the exception of a small set of preserved stem cells. When the worms are able to feed again, these cells regenerate into an entirely new and functional germ line. But this is not all. Kim et al. (p. 994, published online 1 October; see the Perspective by Ogawa and Sommer) show that subsets of the complex mixture of structurally related molecules in dauer pheromone act via distinct G protein–coupled receptors either to initiate longterm effects on development and physiology by modulating the neuroendocrine axis, or to trigger short-term acute effects on behavior by altering neuronal responses. Female nematode worms can be turned into hermaphrodites through the modification of two genes. Although the nematode Caenorhabditis elegans produces self-fertile hermaphrodites, it descended from a male/female species, so hermaphroditism provides a model for the origin of novel traits. In the related species C. remanei, which has only male and female sexes, lowering the activity of tra-2 by RNA interference created XX animals that made spermatids as well as oocytes, but their spermatids could not activate without the addition of male seminal fluid. However, by lowering the expression of both tra-2 and swm-1, a gene that regulates sperm activation in C. elegans, we produced XX animals with active sperm that were self-fertile. Thus, the evolution of hermaphroditism in Caenorhabditis probably required two steps: a mutation in the sex-determination pathway that caused XX spermatogenesis and a mutation that allowed these spermatids to self-activate.

The regulation of spermatogenesis and sperm function in nematodes

In the nematode C. elegans, both males and self-fertile hermaphrodites produce sperm. As a result, researchers have been able to use a broad range of genetic and genomic techniques to dissect all aspects of sperm development and function. Their results show that the early stages of spermatogenesis are controlled by transcriptional and translational processes, but later stages are dominated by protein kinases and phosphatases. Once spermatids are produced, they participate in many interactions with other cells - signals from the somatic gonad determine when sperm activate and begin to crawl, signals from the female reproductive tissues guide the sperm, and signals from sperm stimulate oocytes to mature and be ovulated. The sperm also show strong competitive interactions with other sperm and oocytes. Some of the molecules that mediate these processes have conserved functions in animal sperm, others are conserved proteins that have been adapted for new roles in nematode sperm, and some are novel proteins that provide insights into evolutionary change. The advent of new techniques should keep this system on the cutting edge of research in cellular and reproductive biology.

A cGMP-dependent protein kinase is implicated in wild-type motility in C. elegans

In mammals, cyclic GMP and cGMP‐dependent protein kinases (cGKs) have been implicated in the regulation of many neuronal functions including long‐term potentiation and long‐term depression of synaptic efficacy. To develop Caenorhabditis elegans as a model system for studying the neuronal function of the cGKs, we cloned and characterized the cgk‐1 gene. A combination of approaches showed that cgk‐1 produces three transcripts, which differ in their first exon but are similar in length. Northern analysis of C. elegans RNA, performed with a probe designed to hybridize to all three transcripts, confirmed that a major 3.0 kb cgk‐1 transcript is present at all stages of development. To determine if the CGK‐1C protein was a cGMP‐dependent protein kinase, CGK‐1C was expressed in Sf9 cells and purified. CGK‐1C shows a Ka of 190 ± 14 nm for cGMP and 18.4 ± 2 µm for cAMP. Furthermore, CGK‐1C undergoes autophosphorylation in a cGMP‐dependent manner and is inhibited by the commonly used cGK inhibitor, KT5823. To determine which cells expressed CGK‐1C, a 2.4‐kb DNA fragment from the promoter of CGK‐1C was used to drive GFP expression. The CGK‐1C reporter construct is strongly expressed in the ventral nerve cord and in several other neurons as well as the marginal cells of the pharynx and intestine. Finally, RNA‐mediated interference of CGK‐1 resulted in movement defects in nematode larvae. These results provide the first demonstration that cGMP‐dependent protein kinase is present in neurons of C.elegans and show that this kinase is required for normal motility.

The evolution of spermatogenesis

Publisher Summary This chapter aims to discern the evolutionary histories leading to the varied types of spermatogenesis seen today. It focuses on three model systems: the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the mouse Mus musculus. In these and all other animals, the production of sperm has four steps: (1) Establishment of the germline—in C. elegans and D. melanogaster, primordial germ cells are specified by determinants present in the egg. By contrast, in M. musculus they are specified by signals from neighboring cells. In either case, once formed, the primordial germ cells migrate toward the somatic gonad precursors, and together form the gonad. (2) Proliferation of germ cells—in all three species, a somatic stem cell niche stimulates germ cells to proliferate. However, once they lose access to this signal, germ cells eventually enter meiosis and begin the process of spermatogenesis. In nematodes, this transition happens almost immediately—germ cells enter meiosis, become primary spermatocytes, and separate from the rachis, which is a core of cytoplasm that runs through the center of the male germline. In mammals and fruit flies, germ cells first undergo a further series of mitotic divisions. (3) Production of spermatids by meiosis—in all three species, once germ cells become primary spermatocytes, they go through two meiotic divisions, resulting in four haploid spermatids and a residual body, which contains leftover material. (4) Differentiation of mature sperm—at the appropriate time, the spermatids undergo spermiogenesis, producing amoeboid sperm in nematodes or flagellated sperm in most other species. It is only during this final step that spermatids become fully cellularized in fruit flies or mammals. This review explores how these complex processes are evolved.

Germline Quality Control: eEF2K Stands Guard to Eliminate Defective Oocytes

The control of germline quality is critical to reproductive success and survival of a species; however, the mechanisms underlying this process remain unknown. Here, we demonstrate that elongation factor 2 kinase (eEF2K), an evolutionarily conserved regulator of protein synthesis, functions to maintain germline quality and eliminate defective oocytes. We show that disruption of eEF2K in mice reduces ovarian apoptosis and results in the accumulation of aberrant follicles and defective oocytes at advanced reproductive age. Furthermore, the loss of eEF2K in Caenorhabditis elegans results in a reduction of germ cell death and significant decline in oocyte quality and embryonic viability. Examination of the mechanisms by which eEF2K regulates apoptosis shows that eEF2K senses oxidative stress and quickly downregulates short-lived antiapoptotic proteins, XIAP and c-FLIPL by inhibiting global protein synthesis. These results suggest that eEF2K-mediated inhibition of protein synthesis renders cells susceptible to apoptosis and functions to eliminate suboptimal germ cells.

Rapid Creation of Forward-Genetics Tools for C. briggsae Using TALENs: Lessons for Nonmodel Organisms

Although evolutionary studies of gene function often rely on RNA interference, the ideal approach would use reverse genetics to create null mutations for cross-species comparisons and forward genetics to identify novel genes in each species. We have used transcription activator-like effector nucleases (TALENs) to facilitate both approaches in Caenorhabditis nematodes. First, by combining golden gate cloning and TALEN technology, we can induce frameshifting mutations in any gene. Second, by combining this approach with bioinformatics we can predict and create the resources needed for forward genetic analysis in species like Caenorhabditis briggsae. Although developing genetic model organisms used to require years to isolate marker mutations, balancers, and tools, with TALENs, these reagents can now be produced in months. Furthermore, the analysis of nonsense mutants in related model organisms allows a directed approach for making these markers and tools. When used together, these methods could simplify the adaptation of other organisms for forward and reverse genetics.

The evolutionary origins and consequences of self-fertility in nematodes.

Self-fertile hermaphrodites have evolved from male/female ancestors in many nematode species, and this transition occurred on three independent occasions in the genus Caenorhabditis. Genetic analyses in Caenorhabditis show that the origin of hermaphrodites required two types of changes: alterations to the sex-determination pathway that allowed otherwise female animals to make sperm during larval development, and the production of signals from the gonad that caused these sperm to activate and fertilize oocytes. Comparisons of C. elegans and C. briggsae hermaphrodites show that the ancestral sex-determination pathway has been altered in multiple unique ways. Some of these changes must have precipitated the production of sperm in XX animals, and others were modifying mutations that increased the efficiency of hermaphroditic reproduction. Reverse genetic experiments show that XX animals acquired the ability to activate sperm by co-opting one of the two redundant pathways that normally work in males. Finally, the adoption of a hermaphroditic lifestyle had profound effects on ecological and sexual interactions and genomic organization. Thus, nematode mating systems are ideal for elucidating the origin of novel traits, and studying the influence of developmental processes on evolutionary change.