DYRK2 and GSK-3 phosphorylate and promote the timely degradation of OMA-1, a key regulator of the oocyte-to-embryo transition in C. elegans

Oocyte maturation and fertilization initiates a dynamic and tightly regulated process in which a non-dividing oocyte is transformed into a rapidly dividing embryo. We have shown previously that two C. elegans CCCH zinc finger proteins, OMA-1 and OMA-2, have an essential and redundant function in oocyte maturation. Both OMA-1 and OMA-2 are expressed only in oocytes and 1-cell embryos, and need to be degraded rapidly after the first mitotic division for embryogenesis to proceed normally. We report here a distinct redundant function for OMA-1 and OMA-2 in the 1-cell embryo. Depletion of both oma-1 and oma-2 in embryos leads to embryonic lethality. We also show that OMA-1 protein is directly phosphorylated at T239 by the DYRK kinase MBK-2, and that phosphorylation at T239 is required both for OMA-1 function in the 1-cell embryo and its degradation after the first mitosis. OMA-1 phosphorylated at T239 is only detected within a short developmental window of 1-cell embryos, beginning soon after the proposed activation of MBK-2. Phosphorylation at T239 facilitates subsequent phosphorylation of OMA-1 by another kinase, GSK-3, at T339 in vitro. Phosphorylation at both T239 and T339 are essential for correctly-timed OMA-1 degradation in vivo. We propose that a series of precisely-timed phosphorylation events regulates both the activity and the timing of degradation for OMA proteins, thereby allowing restricted and distinct functions of OMA-1 and OMA-2 in the maturing oocyte and 1-cell embryo, ensuring a normal oocyte-to-embryo transition in C. elegans.     

The role of autophagy during the oocyte-to-embryo transition

After fertilization, the maternal proteins stored in oocytes are degraded and new proteins encoded by the zygotic genome are synthesized. Although several proteins are degraded by the ubiquitin-proteasome system, the mechanism underlying the dynamic protein turnover during this process remains largely unknown. We recently reported that autophagy plays a critical role during preimplantation embryonic development. We found that the level of autophagy was low in unfertilized oocytes; however, autophagy was activated shortly after fertilization. The function of autophagy was further analyzed using oocyte-specific Atg5 (autophagy-related 5) knockout mice. Atg5-null oocytes could develop if they were fertilized with wild-type sperm, but could not develop beyond the four- and eight-cell stages if they were fertilized with Atg5-null sperm. Furthermore, protein synthesis rates were reduced in the autophagy-deficient embryos. We have previously reported that Atg5-null oocytes derived from Atg5+/- mice, which should contain maternally inherited Atg5 protein in the oocyte, were able to produce Atg5-/- neonates, emphasizing the specific importance of autophagy during very early embryogenesis. Thus, the degradation of maternal factors by autophagy is essential for preimplantation development in mammals.

Addendum to: Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, Mizushima N. Autophagy is essential for preimplantation development of mouse embryos. Science 2008; 321:117-20.     

The role of autophagy during the oocyte-to-embryo transition

After fertilization, the maternal proteins stored in oocytes are degraded and new proteins encoded by the zygotic genome are synthesized. Although several proteins are degraded by the ubiquitin-proteasome system, the mechanism underlying the dynamic protein turnover during this process remains largely unknown. We recently reported that autophagy plays a critical role during preimplantation embryonic development. We found that the level of autophagy was low in unfertilized oocytes; however, autophagy was activated shortly after fertilization. The function of autophagy was further analyzed using oocyte-specific Atg5 (autophagy-related 5) knockout mice. Atg5-null oocytes could develop if they were fertilized with wild-type sperm, but could not develop beyond the four- and eight-cell stages if they were fertilized with Atg5-null sperm. Furthermore, protein synthesis rates were reduced in the autophagy-deficient embryos. We have previously reported that Atg5-null oocytes derived from Atg5(+/-) mice, which should contain maternally inherited Atg5 protein in the oocyte, were able to produce Atg5(-/-) neonates, emphasizing the specific importance of autophagy during very early embryogenesis. Thus, the degradation of maternal factors by autophagy is essential for preimplantation development in mammals.     

Inducible antibacterial defense system in C. elegans

The term innate immunity refers to a number of evolutionary ancient mechanisms that serve to defend animals and plants against infection. Genetically tractable model organisms, especially Drosophila, have contributed greatly to advances in our understanding of mammalian innate immunity. Essentially, nothing is known about immune responses in the nematode Caenorhabditis elegans. Using high-density cDNA microarrays, we show here that infection of C. elegans by the Gram-negative bacterium Serratia marcescens provokes a marked upregulation of the expression of many genes. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Certain infection-inducible genes are under the control of the DBL-1/TGFbeta pathway. We found that dbl-1 mutants exhibit increased susceptibility to infection. Conversely, overexpression of the lysozyme gene lys-1 augments the resistance of C. elegans to S. marcescens. These results constitute the first demonstration of inducible antibacterial defenses in C. elegans and open new avenues for the investigation of evolutionary conserved mechanisms of innate immunity.     

Cyclin D involvement demarcates a late transition in C. elegans embryogenesis

During development, progression through the cell cycle must be coordinately regulated with cellular differentiation. Despite significant progress in identifying genes required independently for each of these processes, the molecules which facilitate this cross talk have for the most part been elusive. Using the six macrophage-like coelomocytes of the nematode Caenorhabditis elegans as a model system to gain insight into the mesodermal differentiation pathway, we have isolated a set of mutants that alter coelomocyte numbers. One of these mutations, cc600, apparently results from a partial loss-of-function in the C. elegans cyclin D gene, cyd-1. The mutant has coelomocyte-specific defects without changes in other lineages. The mutants show that cell growth, terminal differentiation and cellular function proceed in the absence of cyd-1 activity and cell division. The results suggest that certain mesodermal lineages may be uniquely affected by changes in cyd-1 activity.     

Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1] and [2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4] and [5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition.     

Control of embryonic spindle positioning and Galpha activity by C. elegans RIC-8

Asymmetric spindle positioning is of fundamental importance for generating cell diversity during development. In the C. elegans 1 cell embryo, spindle positioning has been shown to depend on heterotrimeric G protein signaling. Two Galpha subunits, GOA-1 and GPA-16 (hereafter Galpha), and receptor independent activators of G protein signaling GPR-1 and GPR-2 (GPR-1/2) are required for proper regulation of spindle positioning . However, it remains unclear whether Galpha regulates spindle positioning in its GDP or GTP bound form. Here, we investigate the role of RIC-8 in this pathway. RIC-8 was genetically shown to act in concert with goa-1 to regulate centrosome movements in C. elegans . Interestingly, mammalian RIC-8 was recently found to behave as a GEF for Galpha subunits in vitro . We show that reduction of function of ric-8 results in a 1 cell embryo phenotype very similar to the phenotype of embryos depleted of Galpha. RIC-8 is able to directly bind to GOA-1, preferentially to GOA-1-GDP, consistent with a GEF role. RIC-8 is localized at the embryo cortex, and its activity is essential for the asymmetric localization of GPR-1/2. We suggest that RIC-8 directly modulates Galpha activity and that Galpha-GTP is the signaling molecule regulating spindle positioning in the early embryo.     

Control of nuclear centration in the C. elegans zygote by receptor-independent Galpha signaling and myosin II

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus-centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Galpha proteins GOA-1/GPA-16. In centration- defective let-99(-) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Galpha signaling and actomyosin. During wild-type centration, NMY-2-GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16-depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(-) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Galpha signaling and LET-99 control centration by regulating polarized actomyosin contraction.     

Control of cell fate in the tail region of C. elegans by the gene egl-5

The tail region of C. elegans contains a number of blast cell and neuron types that either are found only in the tail, or are different from more anterior homologues. In egl-5 mutants, the fates of many of these tail cells are abnormal or transformed to those of anterior homologues. The affected cells are related only by position and not by ancestry. egl-5 is also required for normal development of the somatic gonad and sex muscles in males. The function of egl-5 in all these tissues is cell autonomous. By genetic mapping, egl-5 lies very close to mab-5, a gene with an analogous role in the immediately anterior body region. egl-5 and mab-5 may constitute a 'mini-cluster' of regional determination genes, similar to those described in other animal phyla.     

Establishment of a tissue-specific RNAi system in C. elegans

In C. elegans, mosaic analysis is a powerful genetic tool for determining in which tissue or specific cells a gene of interest is required. For traditional mosaic analysis, a loss-of-function mutant and a genomic fragment that can rescue the mutant phenotype are required. Here we establish an easy and rapid mosaic system using RNAi (RNA mediated interference), using a rde-1 mutant that is resistant to RNAi. Tissue-specific expression of the wild type rde-1 cDNA in rde-1 mutants limits RNAi sensitivity to a specific tissue. We established hypodermal-and muscle-specific RNAi systems by expressing rde-1 cDNA under the control of the lin-26 and hlh-1 promoters, respectively. We confirmed tissue-specific RNAi using two assays: (1) tissue-specific knockdown of GFP expression, and (2) phenocopy of mutations in essential genes that were previously known to function in a tissue-specific manner. We also applied this system to an essential gene, ajm-1, expressed in hypodermis and gut, and show that lethality in ajm-1 mutants is due to loss of expression in hypodermal cells. Although we demonstrate tissue-specific RNAi in hypodermis and muscle, this method could be easily applied to other tissues.     

Hierarchical guidance cues in the developing nervous system of C. elegans

During embryogenesis, the basic axon scaffold of the nervous system is formed by special axons that pioneer pathways between groups of cells. To find their way, the pioneer growth cones detect specific cues in their extracellular environment. One of these guidance cues is netrin. Observations and experimental manipulations in vertebrates and nematodes have shown that netrin is a bifunctional guidance cue that can simultaneously attract and repel axons. During the formation of this basic axon scaffold in Caenorhabditis elegans, the netrin UNC-6 is expressed by neuroglia and pioneer neurons, providing hierarchical guidance cues throughout the animal. Each cue has a characteristic role depending on the cell type, its position and the developmental stage. These roles include activities as global, decussation and labeled-pathway cues. This hierarchical model of UNC-6 netrin-mediated guidance suggests a method by which guidance cues can direct formation of basic axon scaffolds in developing nervous systems.     

Control of cell cycle timing during C. elegans embryogenesis

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P₃ (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.     

An efficient transgenic system by TA cloning vectors and RNAi for C. elegans

In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusion gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.     

Cell interactions coordinate the development of the C. elegans egg-laying system

Egg laying by the nematode Caenorhabditis elegans requires the functioning of the vulva, the gonad, the egg-laying muscles, and the two HSN neurons, which innervate these muscles. By analyzing a newly isolated mutant (dig-1) that displaces the gonad, we discovered that cell interactions coordinate the spatial relationships among the different components of the egg-laying system. First, the gonad induces the formation of the vulva, and vulval induction by dorsal gonads strongly suggests that the inductive signal can act at a distance. Second, the gonad acts at a distance to regulate the migrations of the sex myoblasts that generate the egg-laying musculature. Third, the positions of the axonal branch and synapses of each HSN neuron are displaced correspondingly with the rest of the egg-laying system in dig-1 animals, which suggests that cell interactions also control aspects of HSN development.     

Organogenesis in C. elegans: positioning of neurons and muscles in the egg-laying system

One of the final stages in the development of egg-laying behavior in the nematode C. elegans is the organization of 8 motor neurons (2 HSN and 6 VC cells) and 8 muscles into a motor system to control the opening of the vulva. Using mutations that disrupt the development of specific components of the egg-laying system and laser microsurgery to ablate selected precursor cells, we have determined that the guidance of the egg-laying neurons and muscles, and in particular the VC neurons and vulval muscles, into the vulval region is dependent on interactions with surrounding epithelial and gonadal tissue and appears to be independent of neuron-neuron and neuron-muscle interactions. The development of the egg-laying system can be described as a series of cell interactions in which certain cells arise through induction and subsequently provide inductive cues themselves.