Rewiring the Sex-Determination Pathway During the Evolution of Self-Fertility.

Although evolution is driven by changes in how regulatory pathways control development, we know little about the molecular details underlying these transitions. The TRA-2 domain that mediates contact with TRA-1 is conserved in Caenorhabditis. By comparing the interaction of these proteins in two species, we identified a striking change in how sexual development is controlled. Identical mutations in this domain promote oogenesis in Caenorhabditis elegans but promote spermatogenesis in Caenorhabditis briggsae. Furthermore, the effects of these mutations involve the male-promoting gene fem-3 in C. elegans but are independent of fem-3 in C. briggsae. Finally, reciprocal mutations in these genes show that C. briggsae TRA-2 binds TRA-1 to prevent expression of spermatogenesis regulators. By contrast, in C. elegans TRA-1 sequesters TRA-2 in the germ line, allowing FEM-3 to initiate spermatogenesis. Thus, we propose that the flow of information within the sex determination pathway has switched directions during evolution. This result has important implications for how evolutionary change can occur.

The zinc transporter ZIPT-7.1 regulates sperm activation in nematodes.

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating signals. The zipt-7.1 gene is expressed in the germ line and functions in germ cells to promote sperm activation. When expressed in mammalian cells, ZIPT-7.1 mediates zinc transport with high specificity and is predominantly located on internal membranes. Finally, genetic epistasis places zipt-7.1 at the end of the spe-8 sperm activation pathway, and ZIPT-7.1 binds SPE-4, a presenilin that regulates sperm activation. Based on these results, we propose a new model for sperm activation. In spermatids, inactive ZIPT-7.1 is localized to the membranous organelles, which contain higher levels of zinc than the cytoplasm. When sperm activation is triggered, ZIPT-7.1 activity increases, releasing zinc from internal stores. The resulting increase in cytoplasmic zinc promotes the phenotypic changes characteristic of activation. Thus, zinc signaling is a key step in the signal transduction process that mediates sperm activation, and we have identified a zinc transporter that is central to this activation process.

"The persistence of memory"-Hermaphroditism in nematodes.

Self-fertility has evolved many times in nematodes. This transition often produces an androdioecious species, with XX hermaphrodites and XO males. Although these hermaphrodites resemble females in most respects, early germ cells differentiate as sperm, and late ones as oocytes. The sperm then receive an activation signal, populate the spermathecae, and are stored for later use in self-fertilization. These traits are controlled by complex modifications to the sex-determination and sperm activation pathways, which have arisen independently during the evolution of each hermaphroditic species. This transformation in reproductive strategy then promotes other major changes in the development, evolution, and population structure of these animals. Mol. Reprod. Dev. 84: 144-157, 2017. © 2016 Wiley Periodicals, Inc.

Rogue sperm indicate sexually antagonistic coevolution in nematodes.

Intense reproductive competition often continues long after animals finish mating. In many species, sperm from one male compete with those from others to find and fertilize oocytes. Since this competition occurs inside the female reproductive tract, she often influences the outcome through physical or chemical factors, leading to cryptic female choice. Finally, traits that help males compete with each other are sometimes harmful to females, and female countermeasures may thwart the interests of males, which can lead to an arms race between the sexes known as sexually antagonistic coevolution. New studies from Caenorhabditis nematodes suggest that males compete with each other by producing sperm that migrate aggressively and that these sperm may be more likely to win access to oocytes. However, one byproduct of this competition appears to be an increased probability that these sperm will go astray, invading the ovary, prematurely activating oocytes, and sometimes crossing basement membranes and leaving the gonad altogether. These harmful effects are sometimes observed in crosses between animals of the same species but are most easily detected in interspecies crosses, leading to dramatically lowered fitness, presumably because the competitiveness of the sperm and the associated female countermeasures are not precisely matched. This mismatch is most obvious in crosses involving individuals from androdioecious species (which have both hermaphrodites and males), as predicted by the lower levels of sperm competition these species experience. These results suggest a striking example of sexually antagonistic coevolution and dramatically expand the value of nematodes as a laboratory system for studying postcopulatory interactions.

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.

Evolutionary change within a bipotential switch shaped the sperm/oocyte decision in hermaphroditic nematodes.

A subset of transcription factors like Gli2 and Oct1 are bipotential--they can activate or repress the same target, in response to changing signals from upstream genes. Some previous studies implied that the sex-determination protein TRA-1 might also be bipotential; here we confirm this hypothesis by identifying a co-factor, and use it to explore how the structure of a bipotential switch changes during evolution. First, null mutants reveal that C. briggsae TRR-1 is required for spermatogenesis, RNA interference implies that it works as part of the Tip60 Histone Acetyl Transferase complex, and RT-PCR data show that it promotes the expression of Cbr-fog-3, a gene needed for spermatogenesis. Second, epistasis tests reveal that TRR-1 works through TRA-1, both to activate Cbr-fog-3 and to control the sperm/oocyte decision. Since previous studies showed that TRA-1 can repress fog-3 as well, these observations demonstrate that it is bipotential. Third, TRR-1 also regulates the development of the male tail. Since Cbr-tra-2 Cbr-trr-1 double mutants resemble Cbr-tra-1 null mutants, these two regulatory branches control all tra-1 activity. Fourth, striking differences in the relationship between these two branches of the switch have arisen during recent evolution. C. briggsae trr-1 null mutants prevent hermaphrodite spermatogenesis, but not Cbr-fem null mutants, which disrupt the other half of the switch. On the other hand, C. elegans fem null mutants prevent spermatogenesis, but not Cel-trr-1 mutants. However, synthetic interactions confirm that both halves of the switch exist in each species. Thus, the relationship between the two halves of a bipotential switch can shift rapidly during evolution, so that the same phenotype is produce by alternative, complementary mechanisms.

Dependence of the sperm/oocyte decision on the nucleosome remodeling factor complex was acquired during recent Caenorhabditis briggsae evolution.

The major families of chromatin remodelers have been conserved throughout eukaryotic evolution. Because they play broad, pleiotropic roles in gene regulation, it was not known if their functions could change rapidly. Here, we show that major alterations in the use of chromatin remodelers are possible, because the nucleosome remodeling factor (NURF) complex has acquired a unique role in the sperm/oocyte decision of the nematode Caenorhabditis briggsae. First, lowering the activity of C. briggsae NURF-1 or ISW-1, the core components of the NURF complex, causes germ cells to become oocytes rather than sperm. This observation is based on the analysis of weak alleles and null mutations that were induced with TALENs and on RNA interference. Second, qRT-polymerase chain reaction data show that the C. briggsae NURF complex promotes the expression of Cbr-fog-1 and Cbr-fog-3, two genes that control the sperm/oocyte decision. This regulation occurs in the third larval stage and affects the expression of later spermatogenesis genes. Third, double mutants reveal that the NURF complex and the transcription factor TRA-1 act independently on Cbr-fog-1 and Cbr-fog-3. TRA-1 binds both promoters, and computer analyses predict that these binding sites are buried in nucleosomes, so we suggest that the NURF complex alters chromatin structure to allow TRA-1 access to Cbr-fog-1 and Cbr-fog-3. Finally, lowering NURF activity by mutation or RNA interference does not affect this trait in other nematodes, including the sister species C. nigoni, so it must have evolved recently. We conclude that altered chromatin remodeling could play an important role in evolutionary change.

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.

Otx-dependent expression of proneural bHLH genes establishes a neuronal bilateral asymmetry in C. elegans.

Bilateral asymmetry in Caenorhabditis elegans arises in part from cell lineages that differ on the left and right sides of the animal. The unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that generates the MI neuron on the right and the e3D epithelial cell on the left. We isolated mutations in three genes that caused left-right symmetry in this normally asymmetric cell lineage by transforming MI into an e3D-like cell. These genes encode the proneural bHLH proteins NGN-1 and HLH-2 and the Otx homeodomain protein CEH-36. We identified the precise precursor cells in which ceh-36 and ngn-1 act, and showed that CEH-36 protein is asymmetrically expressed and is present in an MI progenitor cell on the right but not in its bilateral counterpart. This asymmetric CEH-36 expression promotes asymmetric ngn-1 and hlh-2 expression, which in turn induces asymmetric MI neurogenesis. Our results indicate that this left-right asymmetry is specified within the two sister cells that first separate the left and right branches of the cell lineage. We conclude that the components of an evolutionarily conserved Otx/bHLH pathway act sequentially through multiple rounds of cell division on the right to relay an initial apparently cryptic asymmetry to the presumptive post-mitotic MI neuron, thereby creating an anatomical bilateral asymmetry in the C. elegans nervous system.

Efficient production of CRISPR/Cas9 gene knockouts in the male/female nematode Caenorhabditis nigoni.

Although nematode genetics was founded on the use of hermaphrodite genetics for studying animal development and behavior, there is a growing need to extend this work to male/female species. One of the most promising species is C. nigoni, because it is so closely related to the model hermaphroditic C. briggsae. We present methods for using CRISPR/Cas9 gene editing to create mutations, and techniques for balancing, maintaining and studying these mutations.

Sex Determination in Nematode Germ Cells.

BACKGROUND: Animal germ cells differentiate as sperm or as oocytes. These sexual fates are controlled by complex regulatory pathways to ensure that the proper gametes are made at the appropriate times. SUMMARY: Nematodes like Caenorhabditis elegans and its close relatives are ideal models for studying how this regulation works, because the XX animals are self-fertile hermaphrodites that produce both sperm and oocytes. In these worms, germ cells use the same signal transduction pathway that functions in somatic cells. This pathway determines the activity of the transcription factor TRA-1, a Gli protein that can repress male genes. However, the pathway is extensively modified in germ cells, largely by the action of translational regulators like the PUF proteins. Many of these modifications play critical roles in allowing the XX hermaphrodites to make sperm in an otherwise female body. Finally, TRA-1 cooperates with chromatin regulators in the germ line to control the activity of fog-1 and fog-3, which are essential for spermatogenesis. FOG-1 and FOG-3 work together to determine germ cell fates by blocking the translation of oogenic transcripts. KEY MESSAGES: Although there is great diversity in how germ cell fates are controlled in other animals, many of the key nematode genes are conserved, and the critical role of translational regulators may be universal.

Functional divergence of orthologous temperature-sensitive mutations in C. elegans and C. briggsae.

To learn if orthologous mutations are temperature-sensitive in related species, we studied four C. briggsae mutations orthologous to alleles of important C. elegans genes. Both Cel-glp-4(bn2) and Cbr-glp-4(v473) are temperature-sensitive, causing sterility at 25°C. By contrast, Cel-fog-1 ( q253) is strongly ts , but its ortholog Cbr-fog-1(v442) causes a loss-of-function at all temperatures. Finally, the C. elegans glp-1 alleles bn18 and e2141 are ts sterile. However, their C. briggsae orthologs, Cbr-glp-1(v429) and Cbr-glp-1(v438) respectively, are wild-type at all temperatures. Thus, a ts mutation in one species provides clues about how to design ts alleles in another, but all theoretical outcomes are possible.

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.

Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue.

Specialized oxygen-sensing cells in the nervous system generate rapid behavioural responses to oxygen. We show here that the nematode Caenorhabditis elegans exhibits a strong behavioural preference for 5-12% oxygen, avoiding higher and lower oxygen levels. 3',5'-cyclic guanosine monophosphate (cGMP) is a common second messenger in sensory transduction and is implicated in oxygen sensation. Avoidance of high oxygen levels by C. elegans requires the sensory cGMP-gated channel tax-2/tax-4 and a specific soluble guanylate cyclase homologue, gcy-35. The GCY-35 haem domain binds molecular oxygen, unlike the haem domains of classical nitric-oxide-regulated guanylate cyclases. GCY-35 and TAX-4 mediate oxygen sensation in four sensory neurons that control a naturally polymorphic social feeding behaviour in C. elegans. Social feeding and related behaviours occur only when oxygen exceeds C. elegans' preferred level, and require gcy-35 activity. Our results suggest that GCY-35 is regulated by molecular oxygen, and that social feeding can be a behavioural strategy for responding to hyperoxic environments.

Evolution: A Developmental Tradeoff that Wins in Changing Environments.

Changing how hormones regulate development can influence the complex trade-offs involved in the competition to survive and reproduce. A new report identifies a molecular variant of eak-3 that trades lower growth rates for the ability to survive sudden temperature extremes.

Sex Determination: TRA-1 Is a Non-binary Regulator of Sexual Identity.

In nematodes, TRA-1 represses the transcription of genes involved in male differentiation, allowing XX animals to undergo normal hermaphrodite development. New reports show that this transcription factor also acts in XO males, to control the differentiation of many neurons.

Evolution of Yin and Yang isoforms of a chromatin remodeling subunit precedes the creation of two genes.

Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new environments. Here we show that de novo, beneficial alleles in the nurf-1 gene became fixed in two laboratory lineages of C. elegans after isolation from the wild in 1951, before methods of cryopreservation were developed. nurf-1 encodes an ortholog of BPTF, a large (>300 kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in C. elegans, nurf-1 has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of Caenorhabditis nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional nurf-1 transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.

Genetic interactions due to constitutive and inducible gene regulation mediated by the unfolded protein response in C. elegans.

The unfolded protein response (UPR) is an adaptive signaling pathway utilized to sense and alleviate the stress of protein folding in the endoplasmic reticulum (ER). In mammals, the UPR is mediated through three proximal sensors PERK/PEK, IRE1, and ATF6. PERK/PEK is a protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 to inhibit protein synthesis. Activation of IRE1 induces splicing of XBP1 mRNA to produce a potent transcription factor. ATF6 is a transmembrane transcription factor that is activated by cleavage upon ER stress. We show that in Caenorhabditis elegans, deletion of either ire-1 or xbp-1 is synthetically lethal with deletion of either atf-6 or pek-1, both producing a developmental arrest at larval stage 2. Therefore, in C. elegans, atf-6 acts synergistically with pek-1 to complement the developmental requirement for ire-1 and xbp-1. Microarray analysis identified inducible UPR (i-UPR) genes, as well as numerous constitutive UPR (c-UPR) genes that require the ER stress transducers during normal development. Although ire-1 and xbp-1 together regulate transcription of most i-UPR genes, they are each required for expression of nonoverlapping sets of c-UPR genes, suggesting that they have distinct functions. Intriguingly, C. elegans atf-6 regulates few i-UPR genes following ER stress, but is required for the expression of many c-UPR genes, indicating its importance during development and homeostasis. In contrast, pek-1 is required for induction of approximately 23% of i-UPR genes but is dispensable for the c-UPR. As pek-1 and atf-6 mainly act through sets of nonoverlapping targets that are different from ire-1 and xbp-1 targets, at least two coordinated responses are required to alleviate ER stress by distinct mechanisms. Finally, our array study identified the liver-specific transcription factor CREBh as a novel UPR gene conserved during metazoan evolution.

Reproduction: Sperm with Two X Chromosomes and Eggs with None.

A new study shows that the nematode Auanema rhodensis manipulates X chromosome segregation in surprising ways that depend on both the sex of the parent and the type of gamete. The result is a complex mating system that produces unusual sex ratios and inheritance patterns.

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.