Rolling back to BOULE.

T presence of a dedicated germline, a population of cells that give rise exclusively to gametes, is a unique feature of animal development. Despite variation in the timing of germ cell determination (1, 2) and in the details of lineage and migration by which they populate the gonad, numerous germline genes are conserved across the bilaterian phyla. It is therefore likely that biologists of today are studying variations on a theme first played by a common ancestor of the protostomes and deuterostomes, roughly 600 million years ago (3). In this issue of PNAS , Xu et al. (4) provide further support for deep conservation of germline function with their discovery in mammals of a homologue of another invertebrate gene encoding the RNA-binding protein Boule (5, 6). For some time, however, researchers had believed that they already had the mammalian boule homologues in the Y-linked gene DAZ (deleted in Azoospermia), only in humans and old world monkeys (7–9), and its autosomal ancestor, DAZL (DAZlike, also known as DAZH; refs. 10–12). The work of Xu et al. suggests that DAZL and DAZ are in fact the divergent products of two nested gene-duplication events, and that the slower evolving progenitor, BOULE, remains a distinct gene in mammals. A phylogenetic perspective reveals that each duplication event generated one paralog retaining the ancestral function and one that is divergent. The solitary DAZ relative in Drosophila, boule, is expressed only during and is required for male meiosis (5, 13), and the expression data of Xu et al. (4) are consistent with conservation of this role for mouse and human BOULE. Thus, it is likely that the ancestral Boule gene functioned only in male meiosis (Fig. 1). DAZL was generated by a duplication of BOULE in an ancestor of the vertebrates but was expressed in both male and female primordial germ cells, where it was required for their development in Xenopus and mice (14, 15). BOULE itself has not been isolated from fish or amphibians but is predicted to exist (see ref. 4 for discussion). Finally, DAZ was born in primates as a Y-translocated duplicate of the autosomal DAZL but is expressed only in male germ cells (like BOULE). DAZL is expressed before meiosis and is partially redundant with other family members, as judged by the variable penetrance of DAZ deletions in men (7). The phylogenetic view also indicates that in nematodes, BOULE has changed from an ancestral male role to be oocyte-specific, an anomaly that coincides with the invention in nematodes of amoeboid sperm. It is tempting to think that these two may be related. The bursts of functional divergence among DAZ family members also have been accompanied by variable rates of sequence evolution, which can be seen readily in the nucleotide sequence divergence among mammalian DAZ family members (Table 1). For example, the mouse DAZL gene is separated equally in time from each of the human and cynomolgus monkey DAZyDAZL sequences. Nevertheless, the DAZ genes are 50% more divergent relative to the mouse standard than their autosomal counterparts (Table 1 mDAZL). This discrepancy is even more apparent when comparing the degree of sequence change in DAZ and DAZL since the separation of the human lineage from that of the cynomolgus monkey. Although their DAZL orthologues are a mere 1.5% different at the nucleotide level, the DAZ genes are 10.9% diverged (Table 1 hDAZL), a difference in substitution rate of nearly 7-fold. As relatively conservative as DAZL seems to be, BOULE is even more so (Table 1 mBOULE vs. hBOULE). The higher rates of sequence change seen in DAZ genes also are accompanied by modest increases in the inferred proportion of amino acidaltering nucleotide substitutions (Ka:Ks) in pairwise comparisons involving them (Table 1 decimal number in each row). This phenomenon is consistent with some selection acting to change the DAZ sequence, although a lack of constraint on amino acid sequence or an inability to repair Y-linked mutations also may explain this trend.