Sex determination by multiple sex chromosomes in Xenopus tropicalis

Sex determination shows a great diversity of mechanisms, ranging from temperature and other environmental cues to strict chromosomal control over this process. In the case of genetic sex determination, this variability is linked to a similarly high variation of sex chromosome differentiation. In vertebrates the most widespread situation is that a pair of sex chromosomes differs in males and females. Either females can be XX and produce only one type of gamete, being the “homogametic” sex, and then males are XY and thus heterogametic, like most mammals; or if females are heterogametic (in analogy: ZW), then males have two copies of the same sex chromosome and are ZZ, like in birds. Curiously, outside of mammals and birds, the phylogenetic patterns of these two obviously contrary modes of sex determination indicate many transitions (1, 2). Many cases have been documented where closely related lineages switch between XY or ZW sex chromosomes, demonstrating that transitions between male and female heterogamety have occurred quite frequently in the evolutionary past of vertebrates (1–4). A large body of literature explains theoretically the “how” and “why” of such changes (e.g., refs. 5–7), but systems to study sex chromosome transitions experimentally or observe sex chromosome evolution in action are extremely rare. Filling up this gap, Roco et al. report in PNAS (8) the intriguing situation of the simultaneous presence of three different sex chromosomes, W, Z, and Y, in the Western clawed frog, Xenopus tropicalis.

Turnovers and Transitions of Sex Determination

When sex is determined by a single locus or chromosomal region, the corresponding chromosome pair becomes the sex chromosomes. However, the affiliation of the master sex-determining gene and a chromosome are not always forever. The same sex-determining gene can reside on different chromosomes in closely related species, for example in the salmonids (rainbow trout, salmon, and relatives). Here, recent evidence assigns transposable elements a role in having moved the male sex-determining gene to other chromosomes (9). As result, the Y chromosome is a different linkage group in several salmonid species. If large pieces or the entire Y are translocated to an autosome, so-called “neo-Y chromosomes” and multiple sex-chromosome systems are created. Such situations are

Roco et al. report in PNAS the intriguing situation of the simultaneous presence of three different sex chromosomes, W, Z, and Y, in the Western clawed frog, Xenopus tropicalis.

particularly common in fishes (10). These changes, however, do not necessarily lead to turnovers of the master regulators and the molecular control of the processes that determine whether the undifferentiated gonad anlage of the embryo will develop into ovary or testis.

Even more intriguingly, the master sex-determining gene itself can change; and depending on the type of change, this has different chromosomal consequences. If for example, the function of a gene as a male sex-determining gene is turned over to a different gene, which resides nearby on the same chromosome as the previous master gene, there is no change of the Y chromosome (homologous turnover). This scenario is only theoretical, as no example has been reported yet. If a gene from elsewhere in the genome becomes the new male sex-determining gene, then a new chromosome will evolve to become the Y (nonhomologous turnover). An increasing number of systems are described where this may have happened. Conservation of synteny of orthologous genes allows us to reconstruct ancestral karyotypes and to delineate homology relationships between linkage groups of different species. In ricefishes of the genus Oryzias, five different linkage groups have evolved to XY sex chromosomes. For three of these the different male sex-determining genes have been identified (11, 12), providing clear evidence for nonhomologous turnover. Similarly, within the frog family Ranidae at least five different candidates for male sex-determiners on different chromosomes have evolved (13).

The emergence of a new master sex-determining gene on a different chromosome can also lead to a shift of the heterogametic sex, [e.g., transforming a XY system into ZW (nonhomologous transition)]. Such a situation is again represented in the ricefishes. Two species have ZW sex determination on two linkage groups, which are different from those that became the X and the Y in the other five species (11). However, the same linkage group can also change its identity from X and Y to W and Z (homologous transition): for example, when a locus on the Y, whose sex determining function is dominant to that of the X or alone is sufficient to determine male, becomes recessive to a female-determining locus or haploinsufficient. A well-known case is that of the Japanese wrinkled frogs, Rana rugosa, where separate XY and ZW populations exist. All four sex chromosomes are descendants of the same linkage group (13). They have diverged independently already carrying different sets of lethal genes. It is unknown, however, what the ancestral situation was and what the sex-determining genes are in Rana rugosa. In the platyfish, males can be either XY or YY and three kinds of females are found (WX, XX, WY). The three sex chromosomes segregate at the same linkage group (14). The fact that all types of sex chromosomes coexist in the same population points to a recent emergence of the third sex chromosome and an ongoing process of homologous transition.

Now Roco et al. (8) provide convincing evidence that in X. tropicalis, a widely used laboratory organism, at least three sex chromosomes exist as well. Previous work has suggested a simple ZZ/ZW system as in Xenopus laevis, where a W-linked sex determination gene, DM-W, was identified (15). However, it soon became clear that this gene is not present in X. tropicalis.

Sex Chromosomes of Xenopus tropicalis: In Transition?

Previous work using the most modern, highly sensitive restriction site-associated DNA (RAD)-tag approach, well-suited to find sex-linked DNA sequence polymorphism and through this define sex chromosomes (16, 17), failed to uncover any region in the X. tropicalis genome that shows sex-specific heterozygosity (18) and was unable to provide further information about the genetics of sex determination. Thus, Roco et al. (8) had to come back to the more classic tools of genetics. On the basis of sex ratio analyses and anonymous DNA markers of crosses between several laboratory strains, sex ratios of polyploid and gynogenetic individuals, and breeding of hormonally sex-reversed frogs, a more complex situation was found. All results could be convincingly explained by the presence of three different sex chromosomes, namely that in addition to the originally assumed W and Z another male-determining chromosome, a Y, is present. How can these two male-determining sex chromosomes be distinguished? In the Roco et al. studies triploid frogs with the genotype ZZW developed as females, but YWW frogs were males. Thus, the Y is a much stronger male determiner than Z. A single Y is sufficient to override the feminization effect of even two Ws, whereas the Z of X. tropicalis can determine maleness only in the absence of W. This finding is consistent with our view on the “strength” of sex chromosomes (W > X, Y > Z), but also shows that this hierarchy in multiple sex-chromosome systems is context-dependent, meaning that it can vary in different organisms. In the platyfish, where two female-determining chromosomes coexist with a single male-determining chromosome, XY fish are male, but WY is female. In X. tropicalis WY frogs are consistently male. The triploids also show that sex-chromosome dosage (and consequently the action of any possible molecular determinant on them) is not relevant for sex determination.

Somewhat surprisingly, WW “superfemale” frogs that were repeatedly produced in the breeding experiments showed full viability and fertility. This result can be explained only by the fact that the W has not lost essential genes or accumulated deleterious recessive mutations. It is consistent with the inability to detect differences between the sex chromosomes of X. tropicalis on the molecular level and a large pseudoautosomal region, and indicates a young age of the W chromosome. YY frogs were not produced, but it will be interesting to see if they develop unaffected as well.

One question is whether an X-chromosome also exists. In their study, Roco et al. (8) did not obtain evidence for its presence. Certainly it is difficult in the current limited availability of molecular markers to differentiate between two female-determining chromosomes.

As a bona fide case of homologous transition, all three sex chromosomes belong to linkage group 7. From the gynogenesis experiments, the sex-determining locus on the ZW pair was mapped to the short arm at a distance of 65 cM from the centromere to the distal portion of the chromosome. At present, this region in the X. tropicalis reference genome (from an inbred Nigerian frog female) presents difficulties for obtaining high-quality genome representation. Fine-mapping of the sex-determining locus and improved assemblies of the corresponding part of LG 7 should be attempted as a basis to identify a master sex-determining gene. This will then provide the basis to isolate the corresponding loci from the other two sex chromosomes and will be a unique opportunity to understand on a molecular level how homologous transition of the sex-determining mechanism can occur during evolution.

Important insights can also be expected from taking these exciting studies further and going back to the laboratory models' African roots. It will be fascinating to learn about the geographic distribution of the three sex chromosomes and infer a possible origin of the Y. The simultaneous presence of three types of males (WY, ZZ, ZY) and two types of females (WZ, WW), and given that all types of possible matings occur, should lead to sex bias in the offspring. It would be interesting to see whether such deviations from the 1:1 sex ratio exist and what conditions in the wild would favor them. Selection for skewed sex ratios have indeed been proposed as an evolutionary force responsible for the presence of differing or mutant sex chromosomes. For example, in the wood lemming Myopus schisticolor, a mutant X is present besides the regular one, and this new X suppresses the male-determining action of the Y. The resulting female bias in the population has been proposed to be beneficial under the condition of recurrent inbreeding of this species (19).

The finding of (at least?) three different chromosomes that are involved in sex determination of X. tropicalis provides more than the opportunity to learn about the complexity of how sex can be determined by the genotype and the genetics of a multiple sex-chromosome system. With this finding, we might even have the chance to witness the emergence of new sex-determining genes and the transition of one sex-determining system to another in an experimentally tractable laboratory organism.