XB-ART-48872Development. May 1, 2014; 141 (9): 1927-39.
High-resolution analysis of gene activity during the Xenopus mid-blastula transition.
The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here, we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilisation wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the seventh cleavage and extending beyond the MBT at the twelfth cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal mRNAs that are deadenylated shortly after fertilisation, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organisation of gene regulatory networks in the early Xenopus embryo.
PubMed ID: 24757007
PMC ID: PMC3994770
Article link: Development.
Grant support: A252-5RG50 Medical Research Council , A252-5RG70 Medical Research Council , MC_U117597140 Medical Research Council , MC_U117597137 Medical Research Council , MRC_MC_U117597137 Medical Research Council , MRC_MC_U117597140 Medical Research Council
Genes referenced: adcy3 aplnr bix1.1 bmp4 ccnb3 cer1 cog8 dpp8 eef1a1 ehmt1 eno1 eomes flrt2 fmn2 foxi4.2 gata5 gdf3 gtf2ird1 hmg20b hs3st1 hspa8 ints12 kif9 kifap3 klf17 lekr1 lhx5 lrr1 mapk4 mixer mixl1 mn1 msgn1 mybl2 myf5 ninl nodal3.1 nodal6 nol8 not nup155 odc1 pak4 plod2 rasl11b ric3 sox17a ssh3 tbx2 tbxa2r tbxt tfap2a ube2s vegt ventx1.2 znf574
Morpholinos referenced: eomes MO1 eomes MO2 mixer MO4 t MO2 t MO3 t2 MO2 t2 MO3 vegt MO3
Article Images: [+] show captions
|Fig. 2. Consistency and reproducibility in time series data. (A) Pairwise Spearman correlation between all samples for each series; asterisks mark replicates in Series 2 (4.5 and 6.5 hpf). We chose ranked Spearman correlations over Pearson correlations as they reveal better the biological structure in the data. (B) Pairwise Spearman correlation between Series 1 and 2 samples for genes with onset fold change ≥5 validates per gene correction to join time series (see text). Circles mark cross-correlations around join. (C) Histograms of Pearson correlation coefficients between genes in Series 1/2 joined data and Series 3. Left: all genes with median normalised read count >10; right: genes with onset fold change ≥5 in either series. Blue, compared by actual sampling times; red, compared with Series 3 development rate correction.|
|Fig. 3. Expression profiles showing variability in normalised maternal mRNA levels. Study of overlapping data points between Series 1 (squares) and Series 2 (crosses). (A) Almost clean joins (left) and small offsets between series (middle) corrected with a per gene adjustment (right) (see text). (B,C) Highly divergent expression profiles suggest real variability in underlying populations of maternal mRNAs between clutches (from different mothers).|
|Fig. 5. Gene activity profiles showing successive waves. Each plot shows the number of genes with onset times at each sampling point in the various time series, showing the data for different fold change thresholds (see text). Positive vertical axes represent onset of rising transcript levels, negative axes represent onset of falling levels. All data are plotted against actual sample times, except the 2× Series 3 in A, which is corrected for development rate (Materials and Methods). MBT (pink bar) gives consistent developmental time scale. (A) PolyA+ Series 1 and 2. Also shown is time-adjusted Series 3 data using a more sensitive end-of-series 2× threshold (Materials and Methods) to show detection of the third wave in this developmentally shorter time series. (B) PolyA+ Series 3. (C) PolyA+ Series 3 analysed at one-hour intervals. (D) Ribo-Zero Series 3. (E,F) Activity profiles using 10× threshold, organised by gene type: TF, transcription factor; SIG, signalling molecules and receptors; other, genes not classified as TF or SIG. Stage diagrams reproduced from Nieuwkoop and Faber (Nieuwkoop and Faber, 1994) with permission of Garland Science/Taylor & Francis, LLC.|
|Fig. 6. Expression profiles of split sample polyA+/Ribo-Zero RNA-seq data illustrating mechanisms underlying changes in polyA+ transcript levels. Data from Series 3: squares, Ribo-Zero; crosses, polyA+. (A) Eg family of genes: controls for detection of post-fertilisation deadenylation of maternal mRNAs; and other deadenylated genes. (B) Post-fertilisation polyadenylation of maternal mRNAs. (C) Deadenylation of maternal mRNAs at MBT. (D) Transcriptional activation of genes with onset time in the second wave.|
|Fig. 7. Comparison of polyA+ and total mRNA expression profiles confirming underlying mechanisms. Although the polyA+ mRNA and NanoString total RNA data are taken from different clutches, the two data sets are entirely consistent. (A,B) Higher and lower expression level genes with onset times before 3.0 hpf showing polyadenylation of maternal mRNAs. (C) Genes with onset times after 3.0 hpf showing zygotic transcription.|
|Fig. 8. Variable transcriptional deficit at MBT in embryos treated with cordycepin. (A) Confirmation of inhibition of polyadenylation: shortened polyA+ tail lengths in cordycepin-treated embryos for the polyadenylated maternal genes VegT, Fam46c and Ccnb3. Lanes: ‘co’, long and variable polyA+ tail in controls; ‘cordycepin’, consistently short polyA+ tail in treated embryos; ‘restriction digest’, confirmatory restriction digest of a gene-specific PCR fragment (Materials and Methods). (B-D) Transcriptional activation of genes in cordycepin-treated versus control embryos by NanoString nCounter (red/blue) or qPCR (green/black). (B) Transcription unaffected by cordycepin treatment. (C) Substantially reduced transcription levels after cordycepin treatment. (D) Loss of primary transcripts for Mix1 shown by intron qPCR confirms loss of transcription, not degradation of deadenylated transcripts.|
|Fig. 9. Targets of MBT-activated transcription factors are found activated within 1-2 h in the third wave. (A) Onset times from Series 2 data for published targets of transcription factors Mixer (Loose and Patient, 2004; Sinner et al., 2006) and Brachyury (Gentsch et al., 2013). Numbers of targets shown at each time point: light blue, onset in second wave; dark blue, onset in third wave or beyond. Onset time of transcription factor is indicated by circle on time axis. (B,C) Expression profiles of Mixer and Brachyury, and published targets selected for qPCR. Circled data points show onset times, dashed lines indicate qPCR sample points. (D,E) qPCR data relative to Odc1 for selected targets at 6.5 hpf and 9.5 hpf, comparing expression in transcription factor morpholino (MO)-injected versus control MO-injected embryos. Arrows show reduced expression in transcription factor knockdown. Data are two technical replicates from pairs of biological replicates: Mixer biological replicates are from a single clutch, Brachyury biological replicates are from different clutches. (F) Inhibition of in vitro translation of Mixer by Mixer MO showing the threshold concentration of morpholino able to block the translation of HA-tagged Mixer.|