Retrotransposition of R2 elements in somatic nuclei during the early development of Drosophila
© Eickbush and Eickbush; licensee BioMed Central Ltd. 2011
Received: 8 July 2011
Accepted: 29 September 2011
Published: 29 September 2011
R2 retrotransposable elements exclusively insert in the 28S rRNA genes of their host. Their RNA transcripts are produced by self-processing from a 28S R2 cotranscript. Because full-length R2 transcripts are found in most tissues of R2-active animals, we tested whether new R2 insertions occurred in somatic tissues even though such events would be an evolutionary dead end.
PCR assays were used to identify somatic R2 insertions in isolated adult tissues and larval imaginal discs of Drosophila simulans. R2 somatic mosaics were detected encompassing cells from individual tissues as well as tissues from multiple body segments. The somatic insertions had 5' junction sequences characteristic of germline insertions suggesting they represented authentic retrotransposition events.
Body segments are specified early in Drosophila development, thus the detection of the same somatic insertion in cells from multiple tissues suggested that the R2 retrotransposition events had occurred before the blastoderm stage of Drosophila development. R2 activity at this stage, when embryonic nuclei are rapidly dividing in a common cytoplasm, suggests that some retrotransposition events appearing as germline events may correspond to germline mosaicism.
Mobile element insertions during the development of somatic tissues provide no benefit to the element, as these insertions are not transferred to subsequent generations. Thus in animals, where the separation of somatic and germline tissues is established early, the ability of a mobile element to generate new insertions in somatic tissues would most likely be selected against. Consistent with this prediction, early studies in Drosophila melanogaster showed that P element transpositions were dependent upon a germline-specific RNA splicing component , and I elements were only transcribed in ovaries . However, counter to this model, mobile elements in other animals have been shown to generate new insertions in somatic tissues (for example, Tc1 elements in Caenorhabditis elegans , L1 elements in mammals [4, 5]).
Several explanations can be put forward for the somatic activity of mobile elements. First, somatic events are inconsequential to the host and thus there is little selective pressure for a mobile element to evolve specificity to the germline. Second, somatic events are harmful, however it is risky for a mobile element to become dependent on a germline-specific mechanism, as it provides another opportunity for the host to control the element. Third, on occasions somatic events provide a benefit to the host. This last fascinating possibility has been suggested to explain the ability of L1 to retrotranspose in nerve tissues .
Somatic mosaics were defined as the presence of unique PCR bands in only a subset of the tissues tested from a single animal. To be scored as a somatic insertion, each new PCR band also had to be reproducibly detected using two different PCR primer combinations. Examples of an R2 insertion in one tissue of the four tested from a third instar larva, and of another insertion detected in three of seven adult tissues are shown in Figure 1C, D. The PCR bands representing potential somatic events were less intense than the bands derived from the R2 elements inherited from the mother or father, as expected if not all cells of a tissue type contained the insertion. Generally new bands could be reproducibly observed if they corresponded to at least one-tenth the intensity of those bands derived from inherited R2 copies. In total, tissues from 29 individuals (14 females, 15 males) were scored. A total of 15 potential somatic insertions were detected in 7 animals (2 females with 4 total events, and 5 males with 11 total events). The detection of greater numbers of new insertions in males compared to females was likely due to the greater sensitivity of the PCR assay in males. The rRNA genes in D. simulans are located on the × chromosome : thus males contain a single rDNA locus, compared to two copies of the rDNA locus in females. The somatic events were detected in essentially all tissues examined, although the numbers of events were not sufficient to make conclusions about relative frequencies.
Because the development of Drosophila has been intensively investigated, the timing of the retrotransposition events that generated the observed somatic mosaics can be estimated. By mid-embryogenesis (10-12 h), small clusters of cells (10-40 cells) are specified to become individual imaginal discs [21, 22]. Each imaginal disc primordium divides during the 3 larval instars to form from 10,000 to 60,000 cells by late larval development . Because the observed somatic events were present in a significant fraction of the cells present in a third instar larval disc or an adult tissue, the retrotransposition events probably occurred before or early in imaginal disc development. Those retrotransposition events detected in more than one disc or adult appendage probably occurred even earlier in development, before determination of body segments at the blastoderm stage (2-3 h). Of the 15 events we observed, 5 were detected in cells derived from more than 1 body segment. Because we surveyed only a fraction of all body segments in either the larvae or adult, it is likely that a larger fraction of the somatic R2 insertion events we observed occurred before the blastoderm stage. This developmental period corresponds to rapid nuclear division in a common cytoplasm. During this period there is little RNA synthesis but active protein synthesis using the RNA synthesized by the nurse cells and deposited in the oocyte during oogenesis . Because rRNA synthesis also does not occur in these first hours of development , R2 retrotransposition events occurring during this time probably use RNA templates synthesized by the nurse cells during oogenesis.
It should be noted that the observed somatic retrotransposition events likely occurred at a time when embryonic nuclei had not yet entered the pole plasma of the egg to become the germline. Thus in addition to somatic mosiacism there is also likely to be germline mosiacism of R2 elements in Drosophila. As a result, re-evaluation of a previous study of retrotransposition in the germline of males and females appears warranted . We have previously suggested that the rate of R2 insertion inherited through the male germline was one-third to one-quarter the rate of insertions through the female germline. Based on the findings in this report, it is possible that all of the insertions scored as inherited through the male germline (that is, during spermatogenesis), actually occurred during early embryogenesis.
Because preblastoderm development in male and female embryos are similar, we suggest the higher rate of R2 insertions observed through the female germline represents this germline mosiacism as well as authentic germline events during oogenesis. Two separate periods of R2 activity in females was also consistent with experiments to monitor a large fraction of the offspring from individual females. In the most comprehensive study, new insertions were assayed in 213 progeny of a single female . Of the 32 different R2 insertions detected in these progeny, 27 were found in only 1 individual and 4 were detected in 2 individuals. These insertions appeared to have occurred late in the development of the germline (that is, during oogenesis). The final R2 insertion was detected in 13 progeny, and could correspond to an insertion during early development. Additional evidence for germline mosiacism was found in the analysis of progeny from another female in which 6 of 17 individuals contained the same new R2 insertion.
In conclusion, we suggest that R2 elements are active early in Drosophila development, and as in the case with L1 elements in mouse and humans [4, 5], can lead to both somatic and germline mosiacism. To determine if R2 elements are also active in somatic tissues later in development will require assaying many smaller samples from individual tissues or more sensitive approaches to detect insertions in smaller percentages of cells. Finally, R2 should serve as a reminder in the study of other mobile elements that events early in development can give rise to insertion mosaics that could be misinterpreted as germline events in the subsequent generation.
The research was support by funds from the National Institutes of Health grant GM42790. The authors would like to thank B Burke for help with the DNA sequencing, D Eickbush for comments on the manuscript, and M Welte for discussions of early Drosophila development.
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