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Enigma variations: control of sexual fate in nematode germ cells
Genome Biology volume 7, Article number: 227 (2006)
A new study showing that neither FEM-2 nor FEM-3 is required for spermatogenesis in Caenorhabditis briggsae, unlike in Caenorhabditis elegans, implies that the sex-determination pathway in these species is evolving rapidly, and supports the proposal that they evolved hermaphroditism independently.
Years ago, French pop star Patrick Juvet raised a question that evolutionary biologists are pondering anew in the aftermath of a paper about nematode sex determination from Eric Haag, David Pilgrim and their colleagues published recently in Developmental Cell : "Où sont les femmes?" - Where are the fems? By showing that the fem genes, which are essential for spermatogenesis in Caenorhabditis elegans, are dispensable in Caenorhabditis briggsae germ cells, they proved that the regulatory pathways in these species have undergone recent and dramatic change.
Regulation of sexual traits evolves rapidly
During the 1980s and 1990s, researchers cloned many of the genes that control sexual development in nematodes and fruit flies, and found that none of them resembled each other (reviewed in ). Since many other regulatory pathways were conserved, these results took everyone by surprise. Later, Raymond et al.  found that a single downstream gene, mab-3/doublesex, had been conserved between nematodes and insects. Taken together, these data implied that the regulatory pathways that control sexual development are derived from a common ancestor, but have been evolving rapidly.
As an example of just how rapid this process can be, consider the nematode family Rhabditidae. In most of its species, XO animals are male and XX animals are female. Some species, however, feature XO males and XX hermaphrodites. Furthermore, all these hermaphrodites are essentially female animals that make their own sperm during larval development, which they use for self-fertilization. Surprisingly, self-fertile hermaphrodites have evolved independently many times in the Rhabditidae . Even during recent evolution, these mating systems have changed multiple times within a small subgroup of the genus Caenorhabditis [5, 6]. Thus, these nematodes provide a terrific model for studying the rapid evolution of sexual traits, and the recent work by Hill et al.  is the first major advance in this developing field.
The fem genes promote male sexual fates in C. elegans
Genetic analysis of C. elegans revealed that male sexual fates are coordinated by a secreted protein, HER-1, that binds to and inactivates the receptor protein TRA-2 (reviewed in ). Three intracellular proteins, FEM-1, FEM-2, and FEM-3, help transmit this inhibitory signal to the transcription factor TRA-1, which controls cell fate (Figure 1). Inactivation of any of these fem genes has two effects: all somatic cells choose female fates; and all germ cells choose oogenesis. How the FEM proteins work is unclear. FEM-1 contains ankyrin repeats , which often mediate interactions with other proteins. In vitro assays show that it binds FEM-2 . FEM-2 is a PP2C-type protein phosphatase [10, 11], but its targets remain unknown. FEM-3 is a novel protein  that can bind FEM-2  and TRA-2 . Perhaps FEM-3 forms the core of a complex that promotes male development by inhibiting TRA-1 activity (see Figure 1). In the soma, the three FEM proteins appear to be the major pathway for information flow between the receptor TRA-2 and the transcription factor TRA-1. Protein-protein interaction studies using the yeast two-hybrid system showed, however, that an intracellular fragment derived from TRA-2 (TRA-2ic; see Figure 1) can bind directly to TRA-1 [14, 15]. Furthermore, mutations that disrupt this interaction cause all germ cells to choose oogenesis [14–17]. These findings raise several questions. Why are there two information paths? How did this system arise? And are parallel, redundant pathways stable during evolution? The logical place to turn for answers was the other hermaphroditic species in the Elegans group, C. briggsae.
The role of the fem genes in C. briggsae
Both tra-1 and tra-2 are conserved in C. briggsae, and both regulate sexual development much as in C. elegans [18, 19]. A third gene, fog-3, is a major target of TRA-1 in C. elegans  (see Figure 1), and its promoter, coding sequence and function are conserved in C. briggsae . This result suggested that the entire sex-determination pathway might be the same in these nematodes, so Nayak et al.  used the C. briggsae genome sequence to search for the other factors. And this is where the surprises started. They found no clear homolog of fog-2, a gene that regulates tra-2 translation in C. elegans hermaphrodites. They had suspected that this might be so, as fog-2 seemed to have evolved recently in C. elegans from a duplicated F-box gene . And Nayak et al.  also showed that the partner of FOG-2 in C. elegans, an RNA-binding protein called GLD-1, has an entirely different function in C. briggsae - it promotes oogenesis rather than spermatogenesis. Given these findings, and the likelihood that C. briggsae and C. elegans had evolved self-fertile hermaphroditism independently, Hill et al.  focused on the germline, and the role of the three fem genes, which are required for spermatogenesis in both sexes of C. elegans.
Initial experiments using RNA interference (RNAi) suggested that the fem genes were not required for spermatogenesis in C. briggsae [24, 25]. RNAi usually lowers but does not eliminate gene activity, however, so the meaning of these results remained in doubt. To resolve this dilemma, Hill et al.  decided to look for null alleles of both genes, using two clever approaches. First, they screened for deletions of fem-2 and fem-3 using the same PCR-based methods that had been developed for C. elegans. Despite the effort involved, this method is ideal for finding null alleles. Second, they screened for suppressors of tra-2, a method that yields lots of mutations in the fem genes in C. elegans.
Having isolated deletion mutants of both fem-2 and fem-3 in C. briggsae, Hill et al.  found that mutant XX animals were able to make sperm just fine, unlike the analogous mutants in C. elegans. Since C. elegans hermaphrodite development depends on a competition between TRA-2 and FEM-3 activity (reviewed in ), these results showed that something fundamentally different must be happening in C. briggsae. Given these findings from XX hermaphrodites, one might imagine that the FEM pathway plays no role at all in the C. briggsae germline. But Hill et al.  went on to show that C. briggsae XO males require both fem-2 and fem-3 to continue producing sperm. Mutations in either gene cause males to switch to oogenesis late in life. And although tra-2 mutants normally produce only sperm, the addition of a fem mutation caused these animals to switch to oogenesis later in life, suggesting that in hermaphrodites too the FEM proteins are required to maintain the ability to produce sperm. The FEM proteins are therefore active in the germlines of both sexes in C. briggsae. But where in the developmental pathway? And how?
If the role of the fem genes in primary sex determination had been supplanted by new genes in C. briggsae, one might expect to find mutations in those genes among the pool of tra-2 suppressors. A large screen for tra-2 suppressors did not, however, yield any mutations that caused C. briggsae XX tra-2 mutants to develop as females . Thus, the sex-determination pathway appears to work differently in C. briggsae and C. elegans.
What are the alternatives?
The new results raise many questions. First, how does TRA-2 interact with TRA-1? By showing that the FEM proteins have a different role in the C. briggsae germline than expected, Hill et al.  underscore the importance of the direct interaction between TRA-2 and TRA-1, as this represents the other known pathway from receptor to nucleus (Figure 1). Wang and Kimble  have shown that this interaction is conserved in C. briggsae. However, the exact nature and function of this interaction remain unknown in either species, so much work remains to be done.
Second, has the germline sex-determination pathway recruited somatic genes? Were the fem genes originally somatic regulators that were expressed in the germline only because maternal product was needed in embryos? In this scenario, the fem genes once played no role in spermatogenesis, as suggested by RNAi experiments that show no requirement for them in Caenorhabditis remanei males . One could imagine that ectopic expression of fem transcripts in the maternal germline led first to a small role for FEM proteins in spermatogenesis (as in C. briggsae) and later to an absolute requirement for FEMs for male sex determination (as in C. elegans). This type of change could also have worked in reverse.
Third, what constitutes the switch that controls spermatogenesis and oogenesis in C. briggsae? That C. briggsae fem-2(lf), fem-3(lf) and tra-1(lf) mutants (where lf indicates loss of function) all produce sperm when young and oocytes when old (D. Keller and E. Haag, personal communication) suggests that the activity of the genes that specify spermatogenesis or oogenesis changes naturally during aging. If so, the sex-determination pathway modulates this rate of change to produce males or females, and the ground state would be hermaphrodite development.
Finally, could binary pathways, such as those that regulate sex, be inherently unstable? Since C. elegans has two pathways that transmit information from TRA-2 to the nucleus, the requirement for the fem genes might have arisen recently from a subsidiary role like that in C. briggsae. If so, in other nematode species the TRA-2/TRA-1 interaction might have become completely superfluous, and been lost. As the output of a binary pathway is always one of two states (like male or female), it is easy to imagine upstream regulators constantly being added to or subtracted from binary pathways during evolution. Perhaps that feature explains why the downstream regulator mab-3/doublesex is the only sex-determination gene conserved between worms and flies.
Thus, recent studies comparing sex-determination pathways in C. elegans and C. briggsae have raised numerous fascinating questions, many of which can be answered by studying other Caenorhabditis species. Since the genomes of many of these species are now being sequenced, the future should be exciting.
Hill RC, de Carvalho CE, Salogiannis J, Schlager B, Pilgrim D, Haag ES: Genetic flexibility in the convergent evolution of hermaphroditism in Caenorhabditis nematodes. Dev Cell. 2006, 10: 531-538.
Cline TW, Meyer BJ: Vive la difference: males vs females in flies vs worms. Annu Rev Genet. 1996, 30: 637-702. 10.1146/annurev.genet.30.1.637.
Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J, Zarkower D: Evidence for evolutionary conservation of sex-determining genes. Nature. 1998, 391: 691-695. 10.1038/35618.
Fitch DH: Phylogeny. Wormatlas Handbook. Wormatlas. 2002, [http://www.wormatlas.org/handbook/contents.htm]
Cho S, Jin SW, Cohen A, Ellis RE: A phylogeny of Caenorhabditis reveals frequent loss of introns during nematode evolution. Genome Res. 2004, 14: 1207-1220. 10.1101/gr.2639304.
Kiontke K, Gavin NP, Raynes Y, Roehrig C, Piano F, Fitch DH: Caenorhabditis phylogeny predicts convergence of hermaphroditism and extensive intron loss. Proc Natl Acad Sci USA. 2004, 101: 9003-9008. 10.1073/pnas.0403094101.
Goodwin EB, Ellis RE: Turning clustering loops: sex determination in Caenorhabditis elegans. Curr Biol. 2002, 12: R111-R120. 10.1016/S0960-9822(02)00675-9.
Spence AM, Coulson A, Hodgkin J: The product of fem-1, a nematode sex-determining gene, contains a motif found in cell cycle control proteins and receptors for cell-cell interactions. Cell. 1990, 60: 981-990. 10.1016/0092-8674(90)90346-G.
Tan KM, Chan SL, Tan KO, Yu VC: The Caenorhabditis elegans sex-determining protein FEM-2 and its human homologue, hFEM-2, are Ca2+/calmodulin-dependent protein kinase phosphatases that promote apoptosis. J Biol Chem. 2001, 276: 44193-44202. 10.1074/jbc.M105880200.
Chin-Sang ID, Spence AM: Caenorhabditis elegans sex-determining protein FEM-2 is a protein phosphatase that promotes male development and interacts directly with FEM-3. Genes Dev. 1996, 10: 2314-2325.
Pilgrim D, McGregor A, Jackle P, Johnson T, Hansen D: The C. elegans sex-determining gene fem-2 encodes a putative protein phosphatase. Mol Biol Cell. 1995, 6: 1159-1171.
Ahringer J, Rosenquist TA, Lawson DN, Kimble J: The Caenorhab-ditis elegans sex determining gene fem-3 is regulated post-transcriptionally. EMBO J. 1992, 11: 2303-2310.
Mehra A, Gaudet J, Heck L, Kuwabara PE, Spence AM: Negative regulation of male development in Caenorhabditis elegans by a protein-protein interaction between TRA-2A and FEM-3. Genes Dev. 1999, 13: 1453-1463.
Wang S, Kimble J: The TRA-1 transcription factor binds TRA-2 to regulate sexual fates in Caenorhabditis elegans. EMBO J. 2001, 20: 1363-1372. 10.1093/emboj/20.6.1363.
Lum DH, Kuwabara PE, Zarkower D, Spence AM: Direct protein-protein interaction between the intracellular domain of TRA-2 and the transcription factor TRA-1A modulates feminizing activity in C. elegans. Genes Dev. 2000, 14: 3153-3165.
Doniach T: Activity of the sex-determining gene tra-2 is modulated to allow spermatogenesis in the C. elegans hermaphrodite. Genetics. 1986, 114: 53-76.
Kuwabara PE, Okkema PG, Kimble J: tra-2 encodes a membrane protein and may mediate cell communication in the Caenorhabditis elegans sex determination pathway. Mol Biol Cell. 1992, 3: 461-473.
Kuwabara PE: Interspecies comparison reveals evolution of control regions in the nematode sex-determining gene tra-2. Genetics. 1996, 144: 597-607.
de Bono M, Hodgkin J: Evolution of sex determination in Caenorhabditis: unusually high divergence of tra-1 and its functional consequences. Genetics. 1996, 144: 587-595.
Chen P, Ellis RE: TRA-1A regulates transcription of fog-3, which controls germ cell fate in C. elegans. Development. 2000, 127: 3119-3129.
Chen PJ, Cho S, Jin SW, Ellis RE: Specification of germ cell fates by fog-3 has been conserved during nematode evolution. Genetics. 2001, 158: 1513-1525.
Nayak S, Goree J, Schedl T: fog-2 and the evolution of self-fertile hermaphroditism in Caenorhabditis. PLoS Biol. 2005, 3: e6-10.1371/journal.pbio.0030006.
Clifford R, Lee MH, Nayak S, Ohmachi M, Giorgini F, Schedl T: FOG-2, a novel F-box containing protein, associates with the GLD-1 RNA binding protein and directs male sex determination in the C. elegans hermaphrodite germline. Development. 2000, 127: 5265-5276.
Haag ES, Wang S, Kimble J: Rapid coevolution of the nematode sex-determining genes fem-3 and tra-2. Curr Biol. 2002, 12: 2035-2041. 10.1016/S0960-9822(02)01333-7.
Stothard P, Hansen D, Pilgrim D: Evolution of the PP2C family in Caenorhabditis: rapid divergence of the sex-determining protein FEM-2. J Mol Evol. 2002, 54: 267-282. 10.1007/s0023901-0008-y.
I thank E. Moss, Y. Guo and E. Haag for critical reading of this manuscript. This review was funded by an ACS Research Scholar grant.
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Ellis, R.E. Enigma variations: control of sexual fate in nematode germ cells. Genome Biol 7, 227 (2006). https://doi.org/10.1186/gb-2006-7-7-227
- Sexual Trait
- Caenorhabditis Briggsae
- Caenorhabditis Remanei
- Binary Pathway