David Greenstein

The Caenorhabditis elegans Gonad: A Test Tube for Cell and Developmental Biology

Sexual reproduction of multicellular organisms depends critically on the coordinate development of the germ line and somatic gonad, a process known as gonadogenesis. Together these tissues ensure the formation of functional gametes and, in the female of many species, create a context for production and further development of the zygote. Since the future of the species hangs in the balance, it is not surprising that gonadogenesis is a complex process involving conserved and multi‐faceted developmental mechanisms. Genetic, anatomical, cell biological, and molecular experiments have established the nematode Caenorhabditis elegans as a paradigm for studying gonadogenesis. Furthermore, these studies demonstrate the utility of C. elegans gonadogenesis for exploring broad issues in cell and developmental biology, such as cell fate specification, morphogenesis, cell signaling, cell cycle control, and programmed cell death. The synergy of molecular genetics and cell biology conducted at single‐cell resolution in real time permits an extraordinary depth of analysis in this organism. In this review, we first describe the embryonic and post‐embryonic development and morphology of the C. elegans gonad. Next we recount seminal experiments that established the field, highlight recent results that provide insight into conserved developmental mechanisms, and present future prospects for the field. Dev Dyn;218:2–22.

C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes

The major sperm protein (MSP) is the central cytoskeletal element required for actin-independent motility of nematode spermatozoa. MSP has a dual role in Caenorhabditis elegans reproduction, functioning as a hormone for both oocyte meiotic maturation and ovarian muscle contraction. The identification of the signaling function of MSP raised the question, how do spermatozoa, which are devoid of ribosomes, ER and Golgi, release a cytoplasmic protein lacking a signal sequence? Here, we provide evidence that MSP export occurs by the budding of novel vesicles that have both inner and outer membranes with MSP sandwiched in between. MSP vesicles are apparently labile structures that generate long-range MSP gradients for signaling at the oocyte cell surface. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly. We propose that MSP generates the protrusive force for its own vesicular export.

MSP and GLP-1/Notch signaling coordinately regulate actomyosin-dependent cytoplasmic streaming and oocyte growth in C. elegans

Fertility depends on germline stem cell proliferation, meiosis and gametogenesis, yet how these key transitions are coordinated is unclear. In C. elegans, we show that GLP-1/Notch signaling functions in the germline to modulate oocyte growth when sperm are available for fertilization and the major sperm protein (MSP) hormone is present. Reduction-of-function mutations in glp-1 cause oocytes to grow abnormally large when MSP is present and Gαs-adenylate cyclase signaling in the gonadal sheath cells is active. By contrast, gain-of-function glp-1 mutations lead to the production of small oocytes. Surprisingly, proper oocyte growth depends on distal tip cell signaling involving the redundant function of GLP-1 ligands LAG-2 and APX-1. GLP-1 signaling also affects two cellular oocyte growth processes, actomyosin-dependent cytoplasmic streaming and oocyte cellularization. glp-1 reduction-of-function mutants exhibit elevated rates of cytoplasmic streaming and delayed cellularization. GLP-1 signaling in oocyte growth depends in part on the downstream function of the FBF-1/2 PUF RNA-binding proteins. Furthermore, abnormal oocyte growth in glp-1 mutants, but not the inappropriate differentiation of germline stem cells, requires the function of the cell death pathway. The data support a model in which GLP-1 function in MSP-dependent oocyte growth is separable from its role in the proliferation versus meiotic entry decision. Thus, two major germline signaling centers, distal GLP-1 activation and proximal MSP signaling, coordinate several spatially and temporally distinct processes by which germline stem cells differentiate into functional oocytes.

Gαo/i and Gαs Signaling Function in Parallel with the MSP/Eph Receptor to Control Meiotic Diapause in C. elegans

Summary Background A conserved biological feature of sexual reproduction in animals is that oocytes arrest in meiotic prophase and resume meiosis in response to extraovarian signals. In C. elegans , sperm trigger meiotic resumption by means of the major sperm protein (MSP) signal. MSP promotes meiotic resumption by functioning as an ephrin-signaling antagonist and by counteracting inhibitory inputs from the somatic gonadal sheath cells. Results By using a genome-wide RNAi screen in a female-sterile genetic background, we identified 17 conserved genes that maintain meiotic arrest in the absence of the MSP signal. In vitro binding experiments show that MSP promotes oocyte mitogen-activated protein kinase activation and meiotic maturation in part through direct interaction with the VAB-1 Eph receptor. Four conserved proteins, including a disabled protein (DAB-1), a vav family GEF (VAV-1), a protein kinase C (PKC-1), and a STAM homolog (PQN-19), function with the VAB-1 Eph/MSP receptor in oocytes. We show that antagonistic Gα o/i and Gα s signaling pathways function in the soma to regulate meiotic maturation in parallel to the VAB-1 pathway. Gα s activity is necessary and sufficient to promote meiotic maturation, which it does in part by antagonizing inhibitory sheath/oocyte gap-junctional communication. Conclusions Our findings show that oocyte Eph receptor and somatic cell G protein signaling pathways control meiotic diapause in C. elegans , highlighting contrasts and parallels between MSP signaling in C. elegans and luteinizing hormone signaling in mammals.

Somatic cAMP signaling regulates MSP-dependent oocyte growth and meiotic maturation in C. elegans

Soma-germline interactions control fertility at many levels, including stem cell proliferation, meiosis and gametogenesis, yet the nature of these fundamental signaling mechanisms and their potential evolutionary conservation are incompletely understood. In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and fertilization. Sperm release the major sperm protein (MSP) signal to trigger meiotic resumption (meiotic maturation) and to promote contraction of the follicle-like gonadal sheath cells that surround oocytes. Using genetic mosaic analysis, we show that all known MSP-dependent meiotic maturation events in the germline require Gαs-adenylate cyclase signaling in the gonadal sheath cells. We show that the MSP hormone promotes the sustained actomyosin-dependent cytoplasmic streaming that drives oocyte growth. Furthermore, we demonstrate that efficient oocyte production and cytoplasmic streaming require Gαs-adenylate cyclase signaling in the gonadal sheath cells, thereby providing a somatic mechanism that coordinates oocyte growth and meiotic maturation with sperm availability. We present genetic evidence that MSP and Gαs-adenylate cyclase signaling regulate oocyte growth and meiotic maturation in part by antagonizing gap-junctional communication between sheath cells and oocytes. In the absence of MSP or Gαs-adenylate cyclase signaling, MSP binding sites are enriched and appear clustered on sheath cells. We discuss these results in the context of a model in which the sheath cells function as the major initial sensor of MSP, potentially via multiple classes of G-protein-coupled receptors. Our findings highlight a remarkable similarity between the regulation of meiotic resumption by soma-germline interactions in C. elegans and mammals.

Clustered Organization of Reproductive Genes in the C. elegans Genome

Defining the forces that sculpt genome organization is fundamental for understanding the origin, persistence, and diversification of species. The genomic sequences of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae provide an excellent opportunity to explore the dynamics of chromosome evolution. Extensive chromosomal rearrangement has accompanied divergence from their common ancestor, an event occurring roughly 100 million years ago (Mya); yet, morphologically, these species are nearly indistinguishable and both reproduce primarily by self-fertilization. Here, we show that genes expressed during spermatogenesis (sperm genes) are nonrandomly distributed across the C. elegans genome into three large clusters located on two autosomes. In addition to sperm genes, these chromosomal regions are enriched for genes involved in the hermaphrodite sperm/oocyte switch and in the reception of sperm signals that control fertilization. Most loci are present in single copy, suggesting that cluster formation is largely due to gene aggregation and not to tandem duplication. Comparative mapping indicates that the C. briggsae genome differs dramatically from the C. elegans genome in clustering. Because clustered genes have a direct role in reproduction and thus fitness, their aggregated pattern might have been shaped by natural selection, perhaps as hermaphroditism evolved.

Start me up: Cell signaling and the journey from oocyte to embryo in C. elegans

Intercellular communication plays a pivotal role in regulating and coordinating oocyte meiosis and fertilization, key triggers for embryonic development. The nematode Caenorhabaditis elegans has emerged as an important experimental paradigm for exploring these fundamental reproductive processes and their regulation. The oocytes of most animal species arrest during meiotic prophase and complete meiosis in response to intercellular signaling in the process of meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase of meiosis I and is accompanied by nuclear envelope breakdown and meiotic spindle assembly. As such, the meiotic maturation process is essential for completing meiosis and a prerequisite for successful fertilization. In C. elegans, the processes of meiotic maturation, ovulation, and fertilization are temporally coupled: sperm utilize the major sperm protein as a hormone to trigger oocyte meiotic maturation, and, in turn, the maturing oocyte signals its own ovulation, leading to fertilization. The powerful genetic screens possible in C. elegans have led to the identification of several sperm cell surface proteins that are required for the interaction and fusion of gametes at fertilization. The study of these proteins provides fundamental insights into fertilization mechanisms, their role in speciation, and their potential conservation across phyla. Signaling processes sparked by fertilization are required for meiotic chromosome segregation and initiating the embryonic program. Here we review recent advances in understanding how signaling mechanisms contribute to the oocyte‐to‐embryo transition in C. elegans. Developmental Dynamics 235:571–585, 2006.

Control of Oocyte Growth and Meiotic Maturation in Caenorhabditis elegans

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. Caenorhabditis elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gα(s)-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition.

Regulated Trafficking of the MSP/Eph Receptor during Oocyte Meiotic Maturation in C. elegans

BACKGROUND In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. RESULTS Here, we present evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, we show that Galpha(s) signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. CONCLUSIONS Regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells.

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.