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. 2012 Feb 20;13(2):R11.
doi: 10.1186/gb-2012-13-2-r11.

Genome-wide analysis of the maternal-to-zygotic transition in Drosophila primordial germ cells

Najeeb U Siddiqui  1 , Xiao Li, Hua Luo, Angelo Karaiskakis, Huayun Hou, Thomas Kislinger, J Timothy Westwood, Quaid Morris, Howard D Lipshitz
Affiliations

Genome-wide analysis of the maternal-to-zygotic transition in Drosophila primordial germ cells

Najeeb U Siddiqui et al. Genome Biol. .

Abstract

Background: During the maternal-to-zygotic transition (MZT) vast changes in the embryonic transcriptome are produced by a combination of two processes: elimination of maternally provided mRNAs and synthesis of new transcripts from the zygotic genome. Previous genome-wide analyses of the MZT have been restricted to whole embryos. Here we report the first such analysis for primordial germ cells (PGCs), the progenitors of the germ-line stem cells.

Results: We purified PGCs from Drosophila embryos, defined their proteome and transcriptome, and assessed the content, scale and dynamics of their MZT. Transcripts encoding proteins that implement particular types of biological functions group into nine distinct expression profiles, reflecting coordinate control at the transcriptional and posttranscriptional levels. mRNAs encoding germ-plasm components and cell-cell signaling molecules are rapidly degraded while new transcription produces mRNAs encoding the core transcriptional and protein synthetic machineries. The RNA-binding protein Smaug is essential for the PGC MZT, clearing transcripts encoding proteins that regulate stem cell behavior, transcriptional and posttranscriptional processes. Computational analyses suggest that Smaug and AU-rich element binding proteins function independently to control transcript elimination.

Conclusions: The scale of the MZT is similar in the soma and PGCs. However, the timing and content of their MZTs differ, reflecting the distinct developmental imperatives of these cell types. The PGC MZT is delayed relative to that in the soma, likely because relief of PGC-specific transcriptional silencing is required for zygotic genome activation as well as for efficient maternal transcript clearance.

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Figures

Figure 1
Figure 1
Flow cytometry and flowchart of experiments. (a) GFP-positive (GFP(+)) and GFP-negative (GFP(-)) cells were obtained by flow cytometric sorting (see Materials and methods). GFP-positive cells are ones with high GFP fluorescence while the GFP-negative cells have low GFP fluorescence. GFP fluorescence is shown on the x-axis (log scale) and FL2 (575 nm band pass sensor; to discriminate dead cells, which autofluoresce) on the y-axis (log scale). The GFP-negative (100 to 102) and GFP-positive (2 × 102 to 4 × 104) cells are boxed in blue. (b) GFP fluorescence is shown on the x-axis (log scale) and the number of cells in each fluorescence class on the y-axis (linear scale). The GFP-negative cells comprise 96% of the total while the GFP-positive cells comprise 2.23% (horizontal bars indicate the cells that fall into each class). A representative fluorescence activated cell sorting (FACS) run is shown. (c) Flowchart of the experiments presented in this study. Protein and RNA levels in the cells were measured by MuDPIT and microarray gene expression profiling, respectively.
Figure 2
Figure 2
Venn diagram comparing our lists of proteins and RNAs to previous studies. (a) Venn diagram showing proteins detected in 1-to-3 hour soma and PGCs that have previously been reported to be present in 0-to-1.5 and 3-to-4.5 hour old fly embryos [27]. (b) Venn diagram showing overlap between our PGC-enriched and soma-enriched transcripts relative to ones previously reported as 'pole cell localized at stages 4 to 6' in the BDGP in situ database [36] (105 genes in total). (c) As in (b), but comparing our transcripts with those annotated as 'pole cell enriched at stages 4 to 5' in the Fly-FISH database [37] (230 genes in total). The PGC-enriched and soma-enriched transcripts were determined by comparing the expression profiles of 1-to-3 hour GFP-positive and GFP-negative cells (Additional file 9).
Figure 3
Figure 3
The PGC proteome. (a) A GeneMANIA-generated network seeded with the proteins specific to PGCs at 1-to-3 hours and linked to the most relevant 20 proteins predicted by GeneMANIA. (b) A GeneMANIA-generated network seeded with the proteins enriched in PGCs at 1-to-3 hours and linked to the most relevant 20 proteins predicted by GeneMANIA. The PGC-specific/enriched proteins are gray-filled circles. Proteins that function in germ plasm and/or PGC development are labeled in red. In each case the 20 most relevant predicted proteins are white-filled circles (if they were not detected by our MuDPIT analysis) or orange-filled (if they were detected by our MuDPIT analysis). The predictions of GeneMANIA were based on co-expression, co-localization, genetic interactions, physical interactions, predicted interactions, and shared protein domains [90]. All the detected proteins had a unique peptide number larger than two in the results from mass spectrometry.
Figure 4
Figure 4
Classes of transcript profiles during the MZT in PGCs. (a) Decision tree defining the different transcript classes. Expression profiles of transcripts in wild-type PGCs. Class I: transcripts that decrease in level at both the 3-to-5 hour and the 5-to-7 hour time points. Class II: transcripts that decrease in level at 3-to-5 hour but then do not change in abundance at the 5-to-7 hour time point. Class III: transcripts that decrease in level at 3-to-5 hour but then increase in level at the 5-to-7 hour time point. Class IV: transcripts that are present at the same level at the 1-to-3 and 3-to-5 hour time points, but then decrease at the 5-to-7 hour time point. Class V: transcripts that are present at the same level throughout the time course. Class VI: transcripts that are present at the same level at 1-to-3 and 3-to-5 hours, but increase in level at the 5-to-7 hour time point. Class VII: transcripts that increase in level at 3-to-5 hours but then decrease in level at the 5-to-7 hour time point. Class VIII: transcripts that increase in level at 3-to-5 hours, and then remain at the same level at the 5-to-7 hour time point. Class XI: transcripts that increase in level at both the 3-to-5 and 5-to-7 hour time points. (b) Expression profiles in wild-type PGCs of the transcripts in these nine classes across the 1-to-3, 3-to-5 and 5-to-7 hour time points. (c) Expression profiles of transcripts in Classes I to IX in smaug-mutant PGCs. In (b, c) each line represents the average expression profile of a transcript from all the replicates.
Figure 5
Figure 5
Smaug protein persists in PGCs. Double immunostains of Vasa, a PGC marker, and Smaug in PGCs. (a-c) Smaug is enriched in PGCs when they form during developmental stage 4, 1.5 hours after fertilization. (d-f) Smaug persists in the PGCs at stages 9 and 10, as they sit in the midgut pocket 3-to-5 hours after fertilization. (g-i) Smaug persists as the PGCs migrate through the midgut epithelium at stage 10. (j-l) Smaug is still detectable at stage 11, 5-to-7 hours after fertilization, at which time the PGCs lie dorsally between the midgut epithelium and the overlying mesoderm. SMG, Smaug protein.
Figure 6
Figure 6
Smaug-dependent RNA decay and/or transcription of class I to IX transcripts. (a) Smaug-dependent RNA decay. Class I: transcripts stabilized at either 3-to-5 or 5-to-7 hours. Class II: transcripts stabilized at 3-to-5 hours. Class III: transcripts stabilized at 3-to-5 hours. Class IV: transcripts stabilized at 5-to-7 hours. Class VII: transcripts stabilized at 5-to-7 hours. (b) Smaug-dependent zygotic transcription. Class III: transcripts that fail to increase at 5-to-7 hours. Class VI: transcripts that fail to increase at 5-to-7 hours. Class VII: transcripts that fail to increase at 3-to-5 hours. Class VIII: transcripts that fail to increase at 3-to-5 hours. Class XI: transcripts that fail to increase either at 3-to-5 or 5-to-7 hours. Each line represents the average expression profile of a transcript from all the replicates. Each pair of plots represents the expression profiles of the same group of Smaug-dependent transcripts in wild-type (left panel) and smaug-mutant PGCs (right panel).
Figure 7
Figure 7
Enrichment of RBP binding sites in PGC transcripts. (a) Enrichment of SREs evaluated by comparing the percentage of the transcripts having at least one SRE (that is, CNGGN(0-3) loop sequence with a four base pair stem) in the specific transcript category, relative to the background set (that is, all transcripts expressed at the same time point). Significance of enrichment was assessed by Bonferroni-corrected hypergeometric P-values. (b) Enrichment of SREs tested by comparing accessibility and the presence of CNGG in the specific category (that is, the positive set) versus the control category (that is, the negative set, which contained the transcripts expressed at the same time point but without any change in the expression level). Area under the receiver operator characteristic (AUROC) and Bonferroni-corrected Wilcoxon-Mann-Whitney rank sum P-values were used to represent the enrichment results and the significance level. AUROC equal to 0.5 (the dashed line), larger than 0.5 or smaller than 0.5 separately indicate that the binding site is represented equal to, more, or less in the positive set versus the negative set. (c, d) Graphs similar to (b), but representing the enrichment results for the Pumilio-binding-site (c) and AU-rich element (ARE) (d). Results that pass the significance test are shown with filled bars (multiple-test-corrected P-value ≤0.05) and the non-significant results as unfilled empty bars (multiple-test-corrected P-value > 0.05). Decay or transcription ('trans.') at the 3-to-5 hour time point relative to 1-to-3 (I) or at the 5-to-7 hour time point relative to 3-to-5 (II). smg, smaug mutant.
Figure 8
Figure 8
Model for delayed MZT in the PGCs relative to the soma. (a-c) Wild type, (d-f) smaug mutant. (a, d) The model. (b, c, e, f) The dynamics of maternal decay and zygotic genome activation with the model diagrammed above the curves. (a, b) In wild-type soma, maternal mRNAs are targeted for decay by a maternally encoded decay machinery ('M') that includes Smaug. Among the targeted transcripts are ones encoding transcriptional repressors that keep the zygotic genome silent. As these are eliminated so zygotic genome activation (ZGA) initiates. Among the zygotic transcripts are components of the zygotically encoded decay machinery ('Z') that feed back to further destabilize maternal mRNAs. ZGA also produces transcriptional activators that feed back to upregulate transcription. (a, c) In wild-type PGCs additional layers of regulation occur (black boxes): protection of a subset of maternal mRNAs from decay factors such as Smaug; and PGC-specific transcriptional repressors that keep the zygotic genome silent (for example, Polar granule component). Only after these are eliminated does the delayed MZT commence in the PGCs. (d, e) In smaug-mutant soma, a subset of maternal mRNAs is stabilized (60%) while a subset of ZGA fails (40%). (d, f) In the smaug-mutant PGCs, there is a similar effect but on a different scale: 34% of unstable maternal mRNAs fail to be cleared and 36% of ZGA fails.
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