CG13617 and inversion 2j evolutionary history

Inversion 2j is widespread in Drosophila natural populations (HASSON et al. 1995) and can be considered evolutionary successful. In this work we have described a position effect associated with the breakpoints of this inversion. The expression change of gene CG13617 provides a molecular mechanism able to link this chromosomal rearrangement with the phenotypical differences in adult body size and developmental time observed between 2st and 2j arrangements (BETRÁN et al. 1998) and, consequently, with the adaptive value of inversion 2j.

The reduction in CG13617 expression level is caused by the presence of an antisense transcript that has been associated to 2j chromosomes. Two main events have contributed to the generation of an antisense RNA overlapping gene CG13617, and therefore, to the detected expression change: the GalileoK insertion and inversion 2j itself. If the insertion of this particular TE copy happened first, then the inversion of a portion of chromosome 2 by ectopic recombination in the adjacent GalileoG fragment situated GalileoK in the current position and orientation, which allowed the antisense transcription of CG13617. Alternatively, GalileoK could have inserted in an already inverted chromosome. In this case, the antisense transcription, with the consequent silencing of CG13617, might have resulted in a favorable mutation that swept all other 2j chromosomes in the population, causing also an increase in inversion frequency. This last possibility would explain that the different 2j alleles at the breakpoints are younger than the inversion itself (CÁCERES et al. 2001). Consistent with this, LAAYOUNI et al. (2003) state that their nucleotide diversity analysis is compatible with a scenario where historical frequencies of inversion 2j have remained quite low during most of the time, and that its rise in frequency, likely due to selection, has only occurred recently.

Thus, the selected favorable change could be CG13617 silencing in embryos caused by the insertion of GalileoK inside the initial GalileoG copy that originated inversion 2j. However, for such an escenario to be possible, the reduction of CG13617 expression level in 2j embryos should cause a phenotypical change in traits affecting fitness.

Developmental time and adult body size are two traits of adaptive importance. Faster development can increase fitness in many insects like Drosophila either by an increase in larval survival in wild conditions or a demographic advantage for early reproduction (LEWONTIN

1965). A positive correlation has been reported in D. buzzatii between body size and longevity, mating success and fecundity, three major fitness components (SANTOS et al. 1992). In Drosophila, large flies have also been shown to present lower metabolic rates, higher desiccation tolerance and greater dispersal ability (SANTOS et al. 1992). Based on their effects on fitness, developmental time and body size are connected by a trade-off because, everything else being equal, it takes a longer time to grow to a larger size (ROFF 2000). Since a larger body size requires a longer development, a certain individual will experience either the advantages of having a short development or the ones derived from a large body size. In D. buzzatii, the phenotypic effects of inversion 2j are related to these two traits. Inversion 2j carriers have a

larger adult body size (RUIZ et al. 1991) but also a longer development when compared to 2st individuals (BETRÁN et al. 1998). This means that the carriers of the 2st arrangement are smaller as adults but they benefit from a shorter development. Therefore, the two chromosomal arrangements are thought to be maintained as a balanced polymorphism in D.

buzzatii natural populations because of a trade-off between developmental time and body size.

We have shown in this work that as a consequence of CG13617 natural (in D. buzzatii 2j embryos) or experimental (in D. melanogaster) silencing, the expression levels of several genes involved in DNA replication and regulation of cell cycle are decreased as well. This indicates that CG13617 protein participates directly or indirectly in the regulation of these processes (additional pieces of evidence linking gene CG13617 to these activities come from its putative promoter sequences, which might contain a TFBS for Rfx, a transcription factor also known to bind the promoter of other genes that take part in DNA replication, and maybe a DRE element). The importance of some of the affected genes in both biological processes suggests that it is possible that their lower expression leads to a reduced DNA replication rate. Studies in yeast, plants, mammalian cells or Drosophila (WEIGMANN et al. 1997, NEUFELD et al. 1998) have shown that cells forced to divide more slowly become much larger than controls because cell growth continues for a longer period of time (SU and O'FARRELL 1998). For example, D.

melanogaster giant (gt) mutants, which exhibit an extended period of growth during the third larval instar that allows them to reach twice the size of wild type larvae and generate giant adults, have a reduced rate of DNA synthesis measured as the incorporation of DNA precursors (NARACHI and BOYD 1985). In addition, gt flies also show many single-strand and double-strand breaks, further indicating an altered DNA metabolism (NARACHI and BOYD

1985). Larvae carrying another mutation with similar phenotypical effects, l(2)gl, also exhibit an slowed DNA synthesis (but less so than in gt). In both cases, these observations correlate with a slowed growth and development. Besides, in gt larvae, the increased body size appears to be caused by an increase in cell size and not in cell number (SIMPSON and MORATA 1980).

Therefore, the consequences of slowing down DNA replication and cell division could be: (1) larger cells, which could result in larger animals, and (2) longer developmental times, since it would take longer to complete the cell divisions required to generate a fully developed individual. Interestingly, a larger adult body size and longer developmental times are precisely the two phenotypical characteristics that distinguish inversion 2j carriers from 2st individuals.

Nonetheless, this hypothesis has some drawbacks. In the first place, CG13617 expression change in 2j lines takes place in embryos, a developmental stage where cell proliferation occurs without growth (the ~50000-cell larvae that hatches from the egg has roughly the same size that the initial single-cell embryo) (O'FARRELL 2004). So, most of the embryonic cells decrease in size with each division, rather than doubling their mass, although it is also true that some cell types of the embryo clearly grow, like neuroblasts, which enlarge considerably in the early embryo (EDGAR and NIJHOUT 2004). And in second place, adult body size in Drosophila is largely determined by the size of the larva at the time of pupation.

Thus, if body size is effectively regulated by the mechanism that controls the timing of the onset of metamorphosis in third instar larvae, how could an expression change that takes place in embryos affect this process? In larvae, if development is delayed, pupation takes place later, giving rise to larger individuals. For example, Drosophila giant (gt) mutants have reduced hormonal signals for pupation, slow-growing imaginal discs (groups of cells from which adult structures like wings or legs will develop) and delayed metamorphosis (SCHWARTZ et al. 1984).

Therefore, these larvae grow to a giant size before undergoing metamorphosis because it takes longer for the discs to achieve their final size. Also, in studies on imaginal disc regeneration in Drosophila larvae, it has been shown that regenerating discs are able to delay pupation until regeneration is complete (SIMPSON et al. 1980). So, while imaginal discs are growing, larvae can not initiate metamorphosis but they keep growing nonetheless (EDGAR and NIJHOUT 2004). If for some reason imaginal discs growth takes longer to complete, the resulting animal is larger.

Since no significant expression differences have been detected so far between 2st and 2j lines in larvae, the detected CG13617 expression change is probably not affecting imaginal disc development. However, similar mechanisms could be operating during embryonic development, so that that slowing down the growth of a certain tissue or group of cells could cause a delay in the development of the whole animal. As a result of a longer developmental time, a total larger size could be reached later on. In the case of inversion 2j, gene CG13617 reduced expression in 2j embryos could be the primary cause of a delay in development starting at the embryonic stage. Also, we need to take into account that the earlier a change occurs in development the more unpredictable and major the consequences can be, since they can be affecting a larger number of cells and in the critical moment of formation of the organism.