Sorting C. elegans
embryos expressing a stage specific GFP marker via FACS (eFACS) made it possible to obtain a large staged embryo population. As a proof of principle we used eFACS to obtain large samples of staged one-cell and two-to-four cell embryos. Due to the low abundance of one-cell zygotes in embryos extracted via standard bleaching, resorting is necessary to yield a pure one-cell population. Methanol fixation allows resorting. Our resorted one-cell sample had a purity of >98%. Methanol fixation did not alter miRNA expression (Supplementary Fig. 2
) or mRNA expression (data not shown). Nevertheless, we cannot rule out that methanol fixation or sorting does induce some artifacts when using eFACS for other purposes. We observed some differences in miRNA expression fold-changes determined by sequencing and qRT-PCRs in independently assayed embryos (), including an outstanding discrepancy for miR-58. We believe that this discrepancy can be in part explained by saturation effects in the library preparation for sequencing because miR-58 is by far the most highly expressed miRNA. This problem and biases in sequencing in general may also be responsible for other discrepancies. Moreover, we observed a shift in overall miRNA fold-changes towards increased fold-changes quantified by qRT-PCR. An inherent problem when comparing sequencing and qRT-PCR data is that both methods require normalization. Sequencing data were normalized under the assumption that net fold-changes are close to zero while qRT-PCRs were normalized to an internal standard. While both assumptions have their problems, different normalization procedures only shift the baseline of fold-changes and do not influence the relative fold-changes to each other and thus do not influence any conclusions presented in this study.
In principle, eFACS can be used to extract large samples of embryos enriched in any desired embryonic stage. Thus, eFACS opens the door to many modern high-throughput technologies to assay embryonic stage specific gene expression. Several of such investigations are already ongoing. A limitation of eFACS is that eFACS depends on the availability of a good fluorescent marker gene for the desired embryonic stage. State-of-the-art FACS allows the simultaneous usage of up to eight fluorescence channels. Strains expressing different fluorescent fusion proteins with temporally overlapping changes in gene expression could be combined and thus it should be possible to use eFACS in situations where a single optimal marker gene is not available. Moreover, it was shown that it is possible to engineer the specific degradation of a protein at a specific time and in a specific cell type25
Alternatively, we have shown that embryos can be sorted alive to obtain staged samples at a purity of ~70%. However, one technical constraint in eFACS is that the large size of the embryos forced us to sort at very low speeds of ~400/second. We cooled embryos (15°C) to delay cell divisions but were still unable to resort living embryos. We also experimented with lower temperature settings (4-10°C). However, these settings reduced viability (<60%) after sorting and still resulted in relatively low purity. It is entirely possible that more advanced FACS machines will allow to sort at higher speeds comparable to standard cell sorting (>20,000/second). In this case, one could sort and even resort to obtain samples of the same size and purity as our fixed embryo eFACS runs. Fixation could be omitted and eFACS just with the OMA-1::GFP strain could be used to obtain thousands of staged living embryos that could be allowed to develop synchronously to the embryonic stage of interest. Further improvements to this approach might also be achieved by careful animal staging11
prior to eFACS.
We applied eFACS to profile small RNA expression during embryogenesis. Previous large-scale studies of small RNA expression had to use samples composed of mixed-stage embryos. These studies could not detect the orchestrated and dynamic changes between and within different classes of small RNAs that we observed when comparing the one-cell embryo to later stages. Several examples illustrate this finding. Firstly, it is very interesting that the majority of miRNAs is already expressed in the one-cell embryo, suggesting that they are maternally deposited. This raises the question why so many miRNAs are expressed in the early embryo. Secondly, we showed that miRNAs from the miR-35 cluster are likely early embryo specific. Genetic knockouts and mutations for 95 miRNAs have been published26
. Strikingly, the miR-35 cluster is the only known miRNA cluster with an embryonic lethal knockout phenotype. Thirdly, we observed many small RNAs of uniform length mapping sense to rRNAs in one-cell embryos (living-sorted or methanol-fixed), with decreased expression in two–to-four-cell embryos, but virtually absent in samples from older stages that were in part also obtained by sorting. Thus, although we do not have independent validation, it seems unlikely that the observed rRNA expression is an experimental artifact. rRNAs, unlike mRNAs, are already transcribed in the one-cell embryo13
. One may speculate about a turnover of maternally and paternally provided rRNAs to zygotically transcribed rRNAs. Finally, we find consistent evidence for a turnover of mitochondrial components in the one-cell embryo. We observed degradation products of mitochondrial tRNAs in the early embryo as well as many siRNAs directed against mitochondrial enzymes. Thus, it is tempting to speculate about mechanisms that selectively degrade paternal mitochondria in early zygotes, as described in vertebrates27
Our data allowed us to shed light on the nature of yet virtually undescribed classes of small RNAs such as 26G-RNAs. Observations of small RNAs, in particular of length ~26 nt with a 5′ Guanine bias have been reported earlier17,20
and were recently dubbed 26G-RNAs24
. However, we found that 26G-RNAs are dynamically expressed and that they cluster in several intergenic regions. Northern blot analysis suggested that they may be generated or modified such that they appear in different sizes. Besides an increase of 26G-RNAs in older embryos we also observed an increase of 26nt endo-siRNAs mapping antisense to coding mRNAs. We failed to computationally detect a ‘ping pong’ biogenesis mechanism28,29
between 26G-RNAs and 26nt endo-siRNAs.
Our eFACS data and analyses raise many more open questions. However, altogether one is tempted to conclude that the complexity of small RNA expression dynamics in very early embryogenesis is comparable to the expression dynamics of protein encoding genes, and that eFACS will make a contribution towards a more complete understanding of gene regulatory networks during early animal development.