Ellis, Ronald E. et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "Evolution of Self-Fertility in Nematodes"

Lin, Shin-Yi, Shen, Yongquan, Zhao, Yanmei, Chen, Xiangmei, Guo, Yiqing, Ellis, Ronald E., Wei, Qing

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

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 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.By comparing C. elegans and C. briggsae hermaphrodites, our group and others found that the ancestral sex-determination pathway has been altered in multiple, unique ways in each species. New genes like she-1 have been recruited to the pathway. Genes active in other germline processes, like those encoding the Nucleosome Remodeling Factor complex, now play a role in the sperm/oocyte decision in some species. Finally, the structure of the core pathway has been altered, so that the relative importance of the Tip60 Histone Acetyl Transferase Complex and the FEM proteins has shifted. Some of these changes may have precipitated the production of sperm in XX animals, and others are probably modifying mutations that increased the efficiency of hermaphroditic reproduction.We are using gene editing to learn how XX animals acquired the ability to activate sperm. In each of nine Caenorhabditis genomes, we found orthologs of all five of the spe-8 group of genes, which control sperm activation in C. elegans hermaphrodites, as well as of try-5 and swm-1, which control sperm activation in males. We used TALENs to knock out each pathway in C. briggsae and C. tropicalis (formerly C. sp. 11). C. briggsae hermaphrodites require the spe-8 pathway for sperm activation, whereas C. tropicalis hermaphrodites require try-5. Because the two pathways are redundant in C. elegans and C. briggsae males, we propose that both were used in the ancestral males. As species began the transition to androdioecy, they co-opted one or the other of these male pathways to controlhermaphrodite sperm activation.Analysis of these evolutionary transitions shows that co-option was critical for the origin of self-fertility. Furthermore, several traits found in nematodes but absent from other groups suggest that developmental biases could explain the frequent evolution of hermaphrodites in worms.

Ronald E Ellis et al. (2005) International Worm Meeting "Rapid Evolution of Mating Systems"

Ronald E Ellis, Chris Baldi

[International Worm Meeting, 2005]

XX worms become self-fertile hermaphrodites in some species, but females in others. Hermaphrodites and females have similar bodies, but hermaphrodites make sperm early in life and oocytes as adults. Thus, studying germline development is the key to learning how mating systems evolve. Our phylogeny showed that C. remanei and C. briggsae are sister species, though one makes females and the other makes hermaphrodites. Furthermore, it implied that hermaphroditism evolved separately in C. elegans and C. briggsae. If so, underlying features of nematode sex-determination might favor this change. Since fog-1 and fog-3 control which cells make sperm in C. elegans, we cloned their homologs from other nematodes. RNAi shows that their functions have been conserved. Why do fog-1 and fog-3 cause XX animals to make sperm in some species, but not in others? We found that fog-3 transcripts are expressed during larval development in XX hermaphrodites, but not in XX females. By contrast, fog-1 is expressed broadly. The expression of fog-3 is controlled by upstream sex-determination genes, which are likely to act through conserved TRA-1 binding sites in its promoter. Knocking down tra-1 alters both fog-3 levels and germ cell fates.We are now studying these upstream regulators. In C. elegans, fog-2 mutations transform XX animals into females. We found a similar mutation, glf-1(v35), in C. briggsae. Genetic mapping and sequence analyses imply that this gene is a novel regulator of sexual fate. We are using SNP mapping to clone it, and recentl;y identified linked super-contigs. We conclude that single mutations can transform a hermaphroditic species into a female one.In a reciprocal experiment, we used RNAi to produce C. remanei XX animals that make sperm and oocytes. Surprisingly, these 'pseudo-hermaphrodites' are not self-fertile. We know their oocytes are normal, because they can be fertilized by sperm from males. However, their own sperm neither activate nor track to the spermatheca, although they can be activated in vitro by pronase. Based on this fact, and the existence of C. elegans mutations that block the activation of hermaphrodite sperm, we propose that the evolution of hermaphrodites requires at least two steps. Furthermore, we suspect that the simple circuit of tra-1, fog-1, and fog-3 we identified in nematodes allows rapid evolution of new mating systems, since genes that control germ cell fate in other animals often have pleiotropic functions.

Ronald E Ellis et al. (2003) International Worm Meeting "Rapid evolution of nematode mating systems."

Soochin Cho, Ronald E Ellis

[International Worm Meeting, 2003]

In animals, sexual traits evolve rapidly. To learn why, we are studying nematode mating systems. C. elegans (Ce) and C. briggsae (Cb) have male and hermaphrodite sexes, whereas C. remanei (Cr) has male and female sexes. We want to learn (1) why XX animals become self-fertile hermaphrodites in some species, but females in others, and (2) how these different systems arose. The essential difference between hermaphrodites and females is that the former produce sperm and oocytes, but the latter only make ooctytes. In Ce, we showed that FOG-3 and the CPEB protein FOG-1 are required for germ cells to initiate spermatogenesis. To study the control of germ cell fate, we cloned homologs of these genes from Cb and Cr. We found that each species has four CPEB genes, just like Ce. In all three species, dsRNAi against the fog-1 homolog causes germ cells to become oocytes rather than sperm. The requirement for cpb-1 in early spermatogenesis has been conserved as well. Thus, the divergence of these proteins pre-dates the origin of this genus. All three species have one fog-3 gene, which is required for germ cells to become sperm rather than oocytes. Since the levels of fog-3 transcripts are correlated with spermatogenesis, the control of fog-3 expression could be responsible for determining if XX animals become females or hermaphrodites. How might this work? Experiments with chimeric transgenes show that the fog-3 promoters from all three species can drive expression of fog-3 in Ce XX larvae. Furthermore, these promoters each contain multiple binding sites for the sex-determination protein TRA-1A. Thus, we propose that fog-3 controls germ cell fate in all caenorhabditids, and that the activity of TRA-1A is modulated in hermaphrodite species to allow fog-3 expression in XX larvae. To test this hypothesis, we cloned tra-1 from Cr. Surprisingly, in Cr, tra-1(RNAi) males only produce oocytes. Furthermore, we observed similar results in Cb. These results suggest that TRA-1 plays an important role promoting spermatogenesis in these species. In Ce, the activity of TRA-1A is modulated by upstream factors like FOG-2, to allow hermaphrodite spermatogenesis. We screened for Fog mutants in Cb, and identified a mutation, v35, with a similar phenotype XX animals are female, and XO animals are male. We are mapping this locus in preparation for positional cloning. Finally, we used sequence analysis of fog-1, fog-3, cpb-1, cpb-2, and cpb-3 to establish a phylogeny for all caenorhabditids. We find that Ce and Cb are not sister species, and that mating systems must have switched multiple times during the evolution of this genus. Furthermore, we observed rapid evolution of intron number in these genes.

Ronald E Ellis et al. (2004) East Coast Worm Meeting "A New Phylogeny Reveals Frequent Loss of Introns During Nematode Evolution"

Ronald E Ellis, Soochin Cho

[East Coast Worm Meeting, 2004]

Since introns were discovered 26 years ago, people have wondered how changes in intron/exon structure occur, and what role these changes play in evolution. To answer these questions, we have begun studying gene structure in nematodes related to C. elegans. As a first step, we cloned a set of five genes from six different Caenorhabditis species, and used their amino acid sequences to construct a detailed phylogeny of the genus. Our results show that C. briggsae and C. remanei are sister species, and imply that mating systems have changed frequently during recent nematode evolution. Using this phylogeny, and the species C. sp. PS1010 as an outgroup , we were able to determine which changes in intron/exon structure were caused by the deletion of ancestral introns, and which were caused by the insertion of new introns. Our results show that nematode introns are lost at a very high rate during evolution, almost 400-fold higher than in mammals. These losses do not occur randomly, but instead favor some introns and do not affect others. By contrast, intron gains are far less common than losses in these genes. For years, people have focused on recombination between a gene and a cDNA copy of its transcript as the primary cause of intron loss. However, we found that adjacent introns were not likely to be lost together, as one might expect from this model, which implies that other mechanisms are also important. Based on sequence data, we suggest that both simple deletions and mutations at splice donor sites might play a significant role in causing the loss of introns during evolution. Furthermore, each of these species has small introns, much like those in C. elegans. The small size of these introns should increase the rate at which each type of loss occurs, and could account for the dramatic difference in loss rate between nematodes and mammals. Because of the wealth of genomic data that will be generated for these species, we should soon be able to expand our studies and determine the relative importance of these mechanisms during evolution.

Shen, Yongquan et al. (2013) International Worm Meeting "PQN-94 regulates hermaphrodite development by interacting with SHE-1."

Ellis, Ronald E, Shen, Yongquan

[International Worm Meeting, 2013]

F-box proteins play critical roles promoting hermaphrodite development in two different species of Caenorhabditis. In C. elegans, the F-box protein FOG-2 interacts with GLD-1 to repress the translation of tra-2 mRNAs. Low TRA-2 activity allows the XX hermaphrodites to make sperm. By contrast, C. briggsae lacks an ortholog of FOG-2, but the F-box protein SHE-1 down-regulates TRA-2; and does so without help from GLD-1. We want to learn how SHE-1 alters the sex-determination process. Because a missense mutation in the F-box inactivates SHE-1, it might act as a classical F-box protein, bringing a target to the E3 ubiquitin ligase complex to be marked for degradation. To identify potential SHE-1 targets, we used the yeast two-hybrid system. From a screen of about 340,000 cDNAs, we identified three genes that that were represented by multiple, independent clones. The pqn-94 gene passed two further tests. First, PQN-94 also interacts with SHE-1 when used as bait. Second, pqn-94(RNAi) partially suppresses she-1. At 25 deg C, she-1(v49); control(RNAi) mothers did not produce hermaphrodites, but she-1(v49); pqn-94(RNAi) mothers produced 7% hermaphrodites. Tests with she-1(v35) showed the same pattern of suppression, whereas knocking down pqn-94 on its own had no phenotype. To confirm that SHE-1 controls germ cell fates by interacting with PQN-94, we used TALEN technology to generate a pqn-94 null mutant with a frame-shifting deletion. These pqn-94(v203) animals are healthy, but she-1(v35); pqn-94(v203) mothers produced 12% hermaphrodites at 25 deg C, whereas she-1(v35) controls were all female. Since experiments with the null allele resembled those done with RNAi, we conclude that the absence of PQN-94 partially compensates for a loss of SHE-1. This result supports models in which SHE-1 targets PQN-94 for degradation. However, PQN-94 cannot be the sole target of SHE-1, since pqn-94(v203) is not completely epistatic to she-1.

Guo, Yiqing et al. (2009) International Worm Meeting "The Tip60/NuA4 HAT complex promotes spermatogenesis in C. briggsae."

Guo, Yiqing, Ellis, Ronald E

[International Worm Meeting, 2009]

In a general screen for female mutants in C.briggsae, we found two alleles of a new gene required for spermatogenesis in both hermaphrodites and males. We named this gene cb-fog-4 because of its Fog phenotype. Epistasis studies showed that fog-4 acts downstream of tra-2, and upstream of or in parallel to tra-1. Genetic mapping located cb-fog-4 on Chromosome II, and fine scale mapping placed it in a 160kb region between the SNPs cb43333 and cb43262. There are only 25 predicted genes in this region. One of these genes, cb-trr-1, is fog-4, since cb-trr-1(RNAi) animals are Fog, and both mutants have lesions in cb-trr-1. TRR-1 is a homolog of TRRAP in humans. Since both mutations are missense alleles, we set up a non-complementation screen using TMP/UV to isolate null alleles, and found 3 Fog alleles that have deletions in cb-trr-1. TRRAP is highly conserved. It is a common subunit of several HAT complexes, including Tip60/NuA4 and PCAF/GCN5. RNAi against cb-mys-1, the catalytic subunit of Tip60/NuA4, also causes a Fog phenotype, but RNAi against cb-pcaf-1, the catalytic subunit of PCAF1/GCN5, does not. Furthermore, RNAi targeting four other components of the Tip60/NuA4 complex, gfl-1, yl-1, epc-1, and ssl-1, also causes a Fog phenotype. Thus, we propose that the Tip60/NuA4 complex controls spermatogenesis in C. briggsae. In C. elegans, trr-1 mutations cause a weakly penetrant vulval defect on their own, as well as slow growth and sterility, but do not create Fog animals. RNAi against either cb-trr-1 or cb-mys-1 in a fem-2 mutant background caused embryonic lethality and L1 arrest. Moreover, RNAi showed that epc-1 and ssl-1 are also essential for the viability of embryos. By contrast, cb-trr-1; cb-fem-3 animals produce normal oocytes. These results suggest that the Tip60/NuA4 complex is essential for embryonic development and is redundant with FEM-2. We propose that cb-trr-1 might provide a model for the recruitment of chromatin regulators into a development pathway.

Shen, Y et al. (2011) International Worm Meeting "Regulation of Hermaphrodite Development by the F-box Protein SHE-1."

Shen, Y, Ellis, Ronald E

[International Worm Meeting, 2011]

In two androdioecious species of Caenorhabditis, F-box proteins play critical roles in hermaphrodite development. In C. elegans, FOG-2 interacts with GLD-1 to repress the translation of tra-2 mRNAs. Although C. briggsae lacks an ortholog of FOG-2, the F-box protein SHE-1 acts independently of GLD-1 to down-regulate TRA-2. Because a missense mutation in the F-box domain inactivates SHE-1, it might function as a classical F-box protein, bringing a target to the E3 ubiquitin ligase complex, where it is marked for degradation. To identify possible SHE-1 targets, we are using the yeast two-hybrid system. From a screen of about 340,000 cDNAs, we identified three genes that gave multiple, independent clones. One of these clones passed two further tests. First, PQN-94 interacts with SHE-1 when used as bait in the yeast two-hybrid system. Second, using RNA interference to knock down pqn-94 partially suppresses the she-1 phenotype. At 25 deg C, she-1(v49); control(RNAi) mothers never produced hermaphrodites, but she-1(v49); pqn-94(RNAi) mothers produced 7% hermaphrodites. Additional tests with she-1(v35) showed the same pattern of suppression. However, knocking down pqn-94 on its own had no phenotype. To see if SHE-1 controls germ cell fates by targeting PQN-94 for degradation, we are carrying out three sets of experiments. (1) We are testing both proteins for interaction when expressed in HEK 293 cells. (2) We are preparing antibodies to each protein, to test their in vivo activities and distributions. And (3) we are looking for other proteins that interact with PQN-94, to learn how it might influence germ cell fates.

Chen, Xiangmei et al. (2013) International Worm Meeting "The Nucleosome Remodeling Factor complex controls germ cell fates in C. briggsae."

Ellis, Ronald E, Chen, Xiangmei

[International Worm Meeting, 2013]

In Caenorhabditis nematodes, the transcription factor TRA-1 controls the sperm/oocyte decision by regulating fog-1 and fog-3, which are needed for spermatogenesis. Recent work from our lab indicates that the Tip60 Histone Acetyl Transferase complex works with TRA-1 to control fog-3. Thus, we have been investigating the role of other chromatin remodelers in the sperm/oocyte decision. In C. briggsae, knocking down the activity of NURF-1.1 or ISW-1, the core components of the Nucleosome Remodeling Factor (NURF) complex, caused germ cells in both sexes to differentiate as oocytes rather than as sperm. This phenotype was confirmed using deletion mutants that were isolated with TALENs. Molecular tests revealed that these NURF genes are predominantly expressed in the germ line, and that they control the transcript levels of fog-1 and fog-3. Furthermore, the analysis of double mutants showed that the NURF complex acts downstream of TRA-1 to control this decision. We propose that the NURF complex alters chromatin structure in the germ line of L3 larvae, which allows the transcription factor TRA-1 to access the fog-1 and fog-3 promoters. These results imply that the NURF complex acts downstream of the Tip60 complex to control germ cell fates in C. briggsae, just as it does to regulate vulval cell fates in C. elegans. However, the Tip60 and NURF complexes cooperate in C. briggsae, but oppose each other in C. elegans. Furthermore, knocking down the activity of the NURF complex did not alter the sperm/oocyte decision in C. elegans, C. remanei or C. sp. 9. Hence, the role that the NURF complex plays in sex determination appears to have changed dramatically during recent evolution.

Guo, Yiqing et al. (2009) International Worm Meeting "Independent Recruitment of F-box Genes to Specify Hermaphrodite Development."

Ellis, Ronald E, Guo, Yiqing

[International Worm Meeting, 2009]

Although sexual reproduction is found throughout the animal kingdom, the mechanisms that control it change rapidly. To elucidate how sex-determination pathways evolve, we are comparing two related nematodes, C. elegans and C. briggsae. Both species make XX hermaphrodites, which are female in appearance, but produce sperm during larval development that can be used for self-fertilization. Phylogenies suggest that each species evolved hermaphroditism independently. In C. elegans, fog-2 is essential for XX animals to become hermaphrodites. However, the Schedl group showed that FOG-2 is a novel F-box protein with no homolog in C. briggsae. This result implied that C. briggsae must use another mechanism to specify hermaphrodite development. Thus, we screened for C. briggsae mutants that became female rather than hermaphrodite. Four mutations created XX females but did not affect males, and all failed to complement each other, so they define the new gene she-1, for spermless hermaphrodites. Two alleles isolated in non-complementation screens have the same phenotype. Genetic tests place she-1 upstream of tra-2 in the sex-determination pathway, at the same position that fog-2 occupies in C. elegans. Furthermore, both she-1 and fog-2 are sensitive to changes in tra-2 dosage. These results imply that tra-2 acts at a key point in the sex-determination pathway, and that two species have evolved hermaphroditism by modulating its activity. Positional cloning revealed that SHE-1 is an F-box protein that is unrelated in structure to FOG-2. Since SHE-1 can bind SKR-1, a component of the E3 ubiquitin ligase complex, and a missense mutation in the F-box domain inactivates SHE-1, it is likely to promote hermaphrodite development by regulating the stability of a target protein. We are currently using the yeast two-hybrid system to find the target. Thus, two different species have recruited novel members of the F-box family of genes into the sex-determination pathway to create hermaphrodites. Since FOG-2 regulates the translation of tra-2 messages by interacting with GLD-1, and SHE-1 does not bind GLD-1, these proteins act by different molecular mechanisms.

Shen, Yongquan et al. (2021) International Worm Meeting "C. elegans has lost a regulatory motif that represses fog-3 transcription in C. briggase"

Ellis, Ronald E, Shen, Yongquan

[International Worm Meeting, 2021]

In C. elegans, FOG-1 and FOG-3 directly promote spermatogenesis, and the activity of each gene is regulated by the TRA-1 transcription factor. as we reported earlier, the C.briggase fog-3 promoter also has several TRA-1 binding sites and can work in C. elegans. To begin studying its native functuion, we made cbr-fog-3(v501), a frameshift mutation that creates a recessive null allele. It feminizes the germline in XX and XO animals but does not affect the soma. These phenotypes are just like those seen in C.elegans. Our phylogenetic analysis revealed that C. briggase fog-3 promoter contains a second regulatory element, that is the unique 26 bp sequence is located near the first TRA-1 binding site, and is conserved in most Caenorhabditis species, but is not found in C.elegans. to study this site, we made the cbr-fog-3(v506) mutation, which scrambles the 26 bp conserved element. Surprisingly, fog-3(v506) causes dominant feminization of germ cells in both XX and XO animals. This phenotype is not suppressed by mutations in tra-2 or tra-1, and we can only maitian the strain by mating with wildtype males. To learn how this 26 bp element works, we used single worm RT-PCR to detect fog-3 transcript levels in young XX animals. Our prelimary results suggested that v506 mutants produce higher levels of fog-3 transcripts than the wild type. this surprising result indicated that too much FOG-3 might disrupt function of the FOG-3/FOG-1 complex recently described by the Kimble lab. To test this model, we greated a cbr-fog-3(v501v506) double mutant. The addition of the v501 frameshift mutation makes this double mutant recessive. we infer that v506 normally causes a dominant Fog phenotype through the production of too much FOG-3 protein. Thus, all our data indicate the 26 bp element mediates repression of fog-3, in addition to nearby TRA-1 binding sites. Furthermore, the loss of repression results in too much FOG-3, which might disrupt FOG-3/FOG-1 complex formation and function. The C. elegans fog-3 promoter might compensate for the lack of this site by the presence of extra TRA-1 binding sites.