Open Access Research

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

Najeeb U Siddiqui12, Xiao Li1, Hua Luo1, Angelo Karaiskakis1, Huayun Hou13, Thomas Kislinger4, J Timothy Westwood5, Quaid Morris16* and Howard D Lipshitz1*

Author Affiliations

1 Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8

2 Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto Medical Discovery Tower, 101 College Street, Toronto, Ontario, Canada M5G 1L7

3 School of Life Sciences, Peking University, No.5 Yiheyuan Road, Haidian District, Beijing, China 100871

4 University Health Network and Department of Medical Biophysics, University of Toronto, Toronto Medical Discovery Tower, 101 College Street, Toronto, Ontario, Canada M5G 1L7

5 Department of Cell and Systems Biology and Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6

6 Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario, Canada M5S 3E1

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Genome Biology 2012, 13:R11  doi:10.1186/gb-2012-13-2-r11

Published: 20 February 2012

Additional files

Additional file 1:

Distribution of the MuDPIT results. An ordered plot showing the ratios of PGC:soma spectral counts for all of the proteins identified in this study (unique peptide number ≥2). The spectral counts shown in the plot are the geometric mean of the two replicates in the PGCs and somatic cells. The minimal measurement of all the spectral counts was added to each spectral count to avoid division by zero. The dashed lines indicate the thresholds used to determine PGC- or soma-specific (brown lines) and PGC- or soma-enriched proteins (black lines). PGC, primordial germ cell.

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Additional file 2:

Correlation between replicates. (a) Spearman's correlation coefficients between mass spectrometry replicates. (b) Spearman's correlation coefficients between microarray replicates.

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Additional file 3:

All proteins detected by mass spectrometry. The listed proteins had unique peptide number larger than 2, in either 1-to-3 hour GFP-positive or GFP-negative cells. GFP, green fluorescent protein.

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Additional file 4:

Supplementary text.

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Additional file 5:

Supplementary methods.

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Additional file 6:

PGC and somatic cell-enriched and -specific proteins. (a) Proteins that are specific to (fold change ≥5) and enriched in (5 ≥ fold change ≥ 2) 1-to-3 hour somatic cells relative to 1-to-3 hour PGCs. (b) Proteins that are specific (fold change ≥5) and enriched (5 ≥ fold change ≥ 2) in 1-to-3 hour PGCs relative to 1-to-3 hour somatic cells. The lists were determined by comparing MuDPIT data for 1-to-3 hour GFP-positive and GFP-negative cells, using spectral counts as the measure of protein level. Asterisks indicate that the denominators of the fold change were zero. GFP, green fluorescent protein; MuDPIT, multidimensional protein identification technology; PGC, primordial germ cell.

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Additional file 7:

The somatic cell proteome. A GeneMANIA-generated network seeded with the proteins specific to somatic cells at 1-to-3 hours and linked to the most relevant 20 proteins predicted by GeneMANIA. Soma-specific proteins according to our MuDPIT analysis are labeled in gray. In each case the 20 most relevant predicted proteins are labeled in white (if they were not detected by our MuDPIT analysis) or in orange (if they were detected by our MuDPIT analysis). The predictions of GeneMANIA were based on co-expression, co-localization, physical interaction and predicted interactions [90]. All the detected proteins had a unique peptide number larger than two in the results from mass spectrometry. MuDPIT, multidimensional protein identification technology.

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Additional file 8:

Gene Ontology (GO) term enrichment analysis of proteins enriched in PGCs or soma (listed in Additional file 6). (a) GO term results for the 1-to-3 hour PGC-specific proteins. (b) GO term results for the 1-to-3 hour PGC-enriched transcripts. (c) GO term results for the 1-to-3 hour soma-specific proteins. (d) GO term results for the 1-to-3 hour soma-enriched proteins. The proteins expressed in 1-to-3 hour somatic cells and PGCs served as the background sets. Terms with FDR < 10% were considered to be significant and are listed in the table.

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Additional file 9:

Transcripts expressed in PGCs or soma. (a) Transcripts that are significantly expressed in PGCs at the 1-to-3 hour time point. The list was determined using the expression profiles of 1-to-3 hour PGCs. (b) Transcripts that are significantly expressed in PGCs at the 3-to-5 hour time point. The list was determined using the expression profiles of 3-to-5 hour PGCs. (c) Transcripts that are significantly expressed in PGCs at the 5-to-7 hour time point. The list was determined using the expression profiles of 5-to-7 hour PGCs. (d) Transcripts that are significantly expressed in the somatic cells at the 1-to-3 hour time point. The list was determined using the expression profiles of 1-to-3 hour GFP-negative cells.

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Additional file 10:

Transcripts enriched in PGCs or soma. (a) Transcripts that are enriched in PGCs at the 1-to-3 hour time point. (b) Transcripts that are enriched in somatic cells at the 1-to-3 hour time point. The lists in (a, b) were determined by comparing the expression profiles of GFP-positive and GFP-negative cells at the 1-to-3 hour time point. All the reported transcripts have a FDR < 5% and the corresponding fold-change larger than two.

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Additional file 11:

Gene Ontology (GO) term enrichment analysis of transcripts enriched in PGCs or soma (listed in Additional file 10). (a) GO term results for the 1-to-3 hour PGC-enriched transcripts. The transcripts expressed in 1-to-3 hour PGCs served as the background set when performing the analysis. (b) GO term results for the 1-to-3 hour somatic-cell-enriched transcripts. The transcripts expressed in 1-to-3 hour somatic cells served as the background set. Terms with a FDR < 10% were considered to be the significant and are listed in the table.

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Additional file 12:

Comparison of the PGC proteome and transcriptome. The log2 ratios of the geometric mean of the expression of PGC proteins (x-axis) and PGC mRNAs (y-axis) versus the soma. First, protein expression was normalized using the trimmed mean (see Materials and methods); second, the minimal expression level of the protein was added to avoid division by zero. Extra rules were applied to the lists shown in Additional files 6 and 10 (see Materials and methods). PGC, primordial germ cell.

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Additional file 13:

Heat maps showing the kinetics of the transcriptome during the maternal-to-zygotic transition in PGCs. (a) Expression profile of PGC transcripts that decreased in level at the 3-to-5 hour time point relative to the 1-to-3 hour time point. (b) As in (a) but showing PGC transcripts that decreased in level at 5-to-7 hours relative to the 3-to-5 hour time point. (c) The expression profile of the PGC transcripts that increased in level at the 3-to-5 hour time point. (d) As in (c) but showing the PGC transcripts that increased at the 5-to-7 hour time point. PGC, primordial germ cell.

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Additional file 14:

Transcripts degraded during the PGC MZT. (a) Transcripts that decreased in level at the 3-to-5 hour time point. The list was determined by comparing the expression profiles in PGCs at the 3-to-5 hour and 1-to-3 hour time points. (b) Transcripts that decreased in level at the 5-to-7 hour time point. The list was determined by comparing the expression profiles in PGCs at the 5-to-7 hour and 3-to-5 hour time points. All the reported transcripts have a FDR < 5% and the corresponding fold change larger than two.

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Additional file 15:

Transcripts in classes I to IX. Detailed description of the nine classes is in the legend to Figure 4. The lists were obtained by overlapping the decay lists (Additional file 14), transcription lists (Additional file 19) and stable transcript lists (FDR > 30% and fold change < 1.2; Additional file 16) at the 3-to-5 hour and 5-to-7 hour time points. All transcripts probed by the array served as the background set. Terms with a FDR < 10% were considered to be the significant and are highlighted in yellow.

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Additional file 16:

Transcripts whose expression level remained constant in PGCs. (a) Transcripts that are stable between the 1-to-3 and 3-to-5 hour time points. The list was determined by comparing the expression profiles in PGCs at 3-to-5 hour and 1-to-3 hour time points. (b) Transcripts that are stable between the 3-to-5 and 5-to-7 hour time points. The list was determined by comparing the expression profiles in PGCs at 5-to-7 hour and 3-to-5 hour time points. All the reported transcripts have a FDR > 30% and the corresponding fold change > 0.83 but < 1.2.

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Additional file 17:

GO term enrichment analysis of transcripts degraded during the PGC MZT (listed in Additional file 14). (a) GO term results for the transcripts that decrease in level at the 3-to-5 hour time point. The transcripts expressed in 1-to-3 hour PGCs served as the background set. (b) GO term results for the transcripts that decrease in level at the 5-to-7 hour time point. The transcripts expressed in 1-to-3 hour PGCs served as the background set. Terms with a FDR < 10% were considered to be the significant GO terms and are listed in the table.

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Additional file 18:

GO term enrichment analysis of transcripts in classes I to IX. Transcripts are listed in Additional file 15. The transcripts expressed in 1-to-3 hour PGCs served as the background set for classes I, II, IV and V. All transcripts probed by the array served as the background set for classes III, VI, VII, VIII and IX.

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Additional file 19:

Transcripts that are zygotically transcribed in PGCs. (a) Transcripts that increase in level at the 3-to-5 hour time point. The list was determined by comparing the expression profiles in PGCs at the 3-to-5 and 1-to-3 hour time points. (b) Transcripts that increase in level at the 5-to-7 hour time point. The list was determined by comparing the expression profiles in PGCs at the 5-to-7 and 3-to-5 hour time points. All the reported transcripts have a FDR < 5% and the corresponding fold change larger than two.

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Additional file 20:

GO term enrichment analysis of transcripts that are zygotically transcribed in PGCs (listed in Additional file 19). (a) GO term results for the transcripts that increased in level at the 3-to-5 hour time point. All transcripts probed by the array served as the background set. (b) GO term results for the transcripts that increased in level at the 5-to-7 hour time point. All transcripts probed by the array served as the background set. Terms with a FDR < 10% were considered to be the significant and are listed in the table.

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Additional file 21:

Transcripts that are degraded in PGCs in a Smaug-dependent manner. (a) Transcripts that are Smaug dependent for decay in PGCs at 3-to-5 hour time point. The list was determined by comparing the expression profiles of the degraded transcripts (listed in Additional file 14) at the 3-to-5 hour time point (normalized to the expression level at the 1-to-3 hour time point), between wild-type and smaug-mutant PGCs. (b) Transcripts that are Smaug-dependent for decay in PGCs at the 5-to-7 hour time point. The list was determined by comparing the expression profiles of the degraded transcripts (listed in Additional file 14) at the 5-to-7 hour time point (normalized to the expression level at the 3-to-5 hour time point), between wild-type and smaug-mutant PGCs. All the reported transcripts have a FDR < 5% and the corresponding fold change larger than two.

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Additional file 22:

Smaug-dependent RNA decay in PGCs verified by in situ hybridization. Four transcripts identified in the gene expression profiling experiment as Smaug-dependent for decay at 3-to-5 hours of embryogenesis were analyzed by fluorescence in situ hybridization in wild type and smaug mutants. (a) spire, (b) orb, (c) mnt, (d) arrest. Transcripts are shown in red and nuclei (DAPI) in blue. Left panels: 2-hour-old embryos. In all cases, transcripts are present at high levels in PGCs of wild type and smaug mutants. Right panels: 3-to-5 hour old embryos in which transcript levels have decreased in wild type but persist at high levels in smaug mutants. (Note: for spire, the wild-type embryo is less than 3 hours old, by which time transcripts have already disappeared.) Since smaug mutant embryos either do not undergo germ-band extension or undergo pseudo-extension, the PGCs remain at the posterior pole or move slightly dorso-anteriorly. DAPI, 4',6-diamidino-2-phenylindole; PGC, primordial germ cell.

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Additional file 23:

GO term enrichment analysis of transcripts that are degraded in PGCs in a Smaug-dependent manner (listed in Additional file 21). (a) GO term results for the transcripts that are Smaug-dependent for decay in PGCs at the 3-to-5 hour time point. The transcripts that are degraded in wild-type PGCs at the 3-to-5 hour were used as the background set. (b) GO term results for the transcripts that are Smaug-dependent for decay in PGCs at the 5-to-7 hour time point. The transcripts that are degraded in wild-type PGCs at 5-to-7 hour were used as the background set. (c) GO term results for the transcripts that are Smaug-dependent for decay in PGCs at the 3-to-5 hour time point. The transcripts that expressed in wild-type 1-to-3 hour PGCs were used as the background set. (d) GO term results for the transcripts that are Smaug-dependent for decay in PGCs at the 5-to-7 hour time point. The transcripts that were expressed in wild-type 1-to-3 hour PGCs were used as the background set. The terms with FDR < 10% were considered to be significant and are listed in the table.

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Additional file 24:

Transcripts that are transcribed in PGCs in a Smaug-dependent manner. (a) Transcripts that are Smaug-dependent for transcription in PGCs at the 3-to-5 hour time point. The list was determined by comparing the expression profiles of the transcripts that increased in level (listed in Additional file 19) at the 3-to-5 hour time point (normalized to the expression level at the 1-to-3 hour time point), between wild-type and smaug-mutant PGCs. (b) Transcripts that are Smaug-dependent for transcription in PGCs at the 5-to-7 hour time point. The list was determined by comparing the expression profiles of the transcripts that increased in level (listed in Additional file 19) at the 5-to-7 hour time point (normalized to the expression level at the 3-to-5 hour time point), between wild-type and smaug-mutant PGCs. All the reported transcripts have a FDR < 5% and the corresponding fold change larger than two.

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Additional file 25:

GO term enrichment analysis of transcripts that are transcribed in PGCs in a Smaug-dependent manner (listed in Additional file 24). (a) GO term results for the transcripts that are Smaug-dependent for transcription in PGCs at the 3-to-5 hour time point. The transcripts that are transcribed at the 3-to-5 hour in wild-type PGCs were used as the background set. (b) GO term results for the transcripts that are Smaug-dependent for transcription in PGCs at the 5-to-7 hour time point. The transcripts that are transcribed at 5-to-7 hours in wild-type PGCs were used as the background set. (c) GO term results for the transcripts that are Smaug-dependent for transcription in PGCs at the 3-to-5 hour time point. All transcripts probed by the array were used as the background set. (d) GO term results for the transcripts that are Smaug-dependent for transcription in PGCs at the 5-to-7 hour time point. All transcripts probed by the array were used as the background set. GO terms with a FDR < 10% were considered to be significant and are listed in the table.

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Additional file 26:

microRNA target site enrichment in the different classes of PGC transcripts. Targets of miR families and miR clusters were determined using Targetscan and PicTar, respectively. The listed P-values are uncorrected hypergeometric P-values, with a FDR < 10% after multiple test correction.

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Additional file 27:

Summary of the datasets derived from the microarray analyses. This table summarizes all the datasets used in this study.

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