Characterization of zygotic genome activation-dependent maternal mRNA clearance in mouse

Abstract

An important event of the maternal-to-zygotic transition (MZT) in animal embryos is the elimination of a subset of the maternal transcripts that accumulated during oogenesis. In both invertebrates and vertebrates, a maternally encoded mRNA decay pathway (M-decay) acts before zygotic genome activation (ZGA) while a second pathway, which requires zygotic transcription, subsequently clears additional mRNAs (Z-decay). To date the mechanisms that activate the Z-decay pathway in mammalian early embryos have not been investigated. Here, we identify murine maternal transcripts that are degraded after ZGA and show that inhibition of de novo transcription stabilizes these mRNAs in mouse embryos. We show that YAP1-TEAD4 transcription factor-mediated transcription is essential for Z-decay in mouse embryos and that TEAD4-triggered zygotic expression of terminal uridylyltransferases TUT4 and TUT7 and mRNA 3'-oligouridylation direct Z-decay. Components of the M-decay pathway, including BTG4 and the CCR4-NOT deadenylase, continue to function in Z-decay but require reinforcement from the zygotic factors for timely removal of maternal mRNAs. A long 3'-UTR and active translation confer resistance of Z-decay transcripts to M-decay during oocyte meiotic maturation. The Z-decay pathway is required for mouse embryo development beyond the four-cell stage and contributes to the developmental competence of preimplantation embryos.

Figure 1.

Dynamics of maternal mRNA clearance in mouse preimplantation embryos. (A) Illustration of the timepoints when the samples were collected for experiments in (B) and (C). (B) Expression pattern of mouse maternal transcripts at GV, zygote, and late two-cell stages. Transcripts with FPKM > 2 in GV oocytes were selected and further analyzed. Each light blue line represents the expression level of one gene, and the middle red line represents the median expression level of the cluster. Zygotes and two-cell embryos were collected from in vivo at 28 h and 43 h post-hCG injection. (C) RT-qPCR results showing the relative mRNA levels of select transcripts in mouse oocytes and embryos at the timepoints indicated in (A). Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. n = 3 biological replicates. (D) Illustration showing the treatment of mouse oocytes and embryos for RNA-seq. Zygotes were treated with or without α-amanitin (25 ng/μl), and then cultured to the two-cell stage until 43 h after hCG injection. (E) The degradation pattern of maternal transcripts in mouse embryos with or without α-amanitin treatment. Transcripts with FPKM (two-cell)/FPKM (zygote) < 1/2 were selected for analyses. Each light blue line represents the expression level of one gene. The middle red line represents the median expression level of the cluster. The green line represents the median expression level of the cluster after α-amanitin treatment. (F) RT-qPCR results showing the relative mRNA levels in mouse zygotes and two-cell embryos, which were treated and collected as illustrated in (D). Error bars, s.e.m. ***P < 0.001 and **P < 0.01 by two-tailed Student's t-test. n = 3 biological replicates.

Figure 2.

Role of maternal YAP1 and zygotic TEAD4 in the Z-decay pathway of mouse embryos. (A, B): RT-qPCR results showing the relative mRNA levels of Yap1 (A) and Tead4 (B) in GV oocytes, MII oocytes after 16 h of hCG injection, zygotes, two-cell embryos, and α-amanitin-treated embryos. The embryos were treated with α-amanitin (25 ng/μl), and then cultured to the 2-cell stage until 43 h after the hCG injection. Error bars, s.e.m. *P < 0.05 and ***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. n = 3 biological replicates. (C) Scatter plot comparing transcripts between the WT and Yap1^–/– oocytes (at the GV stage) and the four-cell embryos derived from these oocytes. Transcripts decreased or increased by >2-fold in Yap1^–/– samples were highlighted in blue or red, respectively. (D) Degradation pattern of maternal transcripts in mouse embryos with or without maternal Yap1 knockout. Transcripts with FPKM (GV) > 2; FPKM (four-cell/GV) < 1/2 were selected for the analyses. Each light blue line represents the expression level of one gene. The middle red line represents the median expression level of the cluster. The green line represents the median expression level of the cluster after maternal Yap1 knockout. (E) Venn diagrams showing the overlap of Z-decay transcripts (FPKM (GV) > 2; FPKM (zygote/GV) ≥ 1/2; FPKM (2-cell/zygote) <1/2) and the maternal transcripts that were significantly accumulated in four-cell embryos derived from Yap1^–/– oocytes (FPKM (4-cell/GV) < 1/2 in WT; FPKM (Yap1^–/–/WT) >1 at the 4-cell stage). P = 1e–308 by two-tailed Student's t-test. (F): Venn diagrams showing the overlap of ZGA-dependent Z-decay transcripts (FPKM (GV) > 2; FPKM (zygote/GV) ≥ 1/2; FPKM (two-cell/zygote) < 1/2; FPKM (two-cell/zygote) ≥ 1/2 after α-amanitin treatment) and the maternal YAP1-dependent Z-decay transcripts, i.e. the overlapping transcripts in (E). P = 1e–548 by two-tailed Student's t-test. (G–H) RT-qPCR results showing the relative mRNA levels of zygotic transcripts (G) and Z-decay transcripts (H) in two-cell embryos overexpressing a dominant negative form of TEAD4 (FLAG-TEAD4^ΔTEA) by mRNA microinjection at the zygote stage. Error bars, s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates. (I) Illustration showing a putative TEAD-binding site (M-CAT element) of the mouse Tut7 gene in mm9 genome. M-CAT element locates at 1025 bp upstream of Tut7 transcription start site, from 59 925 533 to 59 925 541 on the chromosome 13.

Figure 3.

Role of zygotic TUT4 and TUT7 in the Z-decay pathway of mouse embryos. (A) RT-qPCR results showing the relative mRNA levels of indicated transcripts in mouse oocytes (GV and MII), zygotes, and two-cell embryos (with or without 25 ng/μl α-amanitin treatment). Error bars, s.e.m. **P < 0.01 and ***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. n = 3 biological replicates. (B) Changes of relative mRNA copy numbers in WT and Tut4/7-depleted embryos at the indicated stages. **P < 0.01 by two-tailed Student's t-test. (C) Scatter plot comparing transcripts between WT and Tut4/7-depleted two-cell embryos. Transcripts decreased or increased >2-fold in Tut4/7-depleted embryos were highlighted in blue or red, respectively. (D) Venn diagrams showing the overlap of upregulated transcripts in Tut4/7-depleted embryos (FPKM (siTut4/7/WT) > 2 in two-cell embryos) and the degraded transcripts from the zygote to 2-cell embryos in WT (FPKM (two-cell/zygote) < 1/3 in WT). P = 1e–1738 by two-tailed Student's t-test. (E) Degradation pattern of maternal transcripts in mouse embryos with or without zygotic Tut4/7 depletion. Transcripts with FPKM (2-cell/zygote) < 1/3 were selected for the analyses. Each light blue line represents the expression level of one gene. The middle red line represents the median expression level of the cluster. The green line represents the median expression level of the cluster after zygotic Tut4/7 depletion. (F) Venn diagrams showing the overlap of ZGA-dependent Z-decay transcripts (FPKM (GV) > 2; FPKM (zygote/GV) ≥ 1/2; FPKM (two-cell/zygote) < 1/2; FPKM (two-cell/zygote) ≥ 1/2 after α-amanitin treatment), YAP1-dependent Z-decay transcripts, and TUT4/7-dependent Z-decay transcripts. (G) RT-qPCR results showing the relative mRNA levels of indicated Z-decay transcripts in two-cell embryos with or without zygotic Tut4/7-depletion. Error bars, s.e.m. **P < 0.01 and ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates. (H) Changes in RT-qPCR results obtained from oligo-(dA)-versus random primer-mediated RT reactions reflecting the 3′-oligouridylation levels of selected Z-decay transcripts in two-cell embryos with or without zygotic Tut4/7-depletion. Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates. (I) Venn diagrams showing the overlap of downregulated transcripts in Tut4/7-depleted embryos (FPKM (siTut4/7/WT) > 2 in two-cell embryos) and the zygotically activated transcripts in WT embryos (FPKM (2-cell/zygote) > 2 in WT). P = 1e–903 by two-tailed Student's t-test. (J, K): Developmental rates (J) and representative images (K) of preimplantation embryos after zygotic Tut4/7-depletion. Time after hCG injection (h) and numbers of analyzed embryos are indicated (n). Error bars, s.e.m. **P < 0.01; ***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. Scale bar, 100 μm. Arrows and hollow arrows indicate the normal and arrested embryos, respectively. n = 3 biological replicates.

Figure 4.

Dynamics of 3′-terminal polyadenylation and oligouridylation in maternal transcripts undergoing M-decay and Z-decay. (A) Illustration of BTG4 Trim-Away experiments. Zygotes were collected from in vivo at 20 h after hCG injection and were co-injected with Flag-Trim21 mRNA and anti-BTG4 antibody or were only injected with Flag-Trim21 mRNA as a negative control. Microinjected zygotes were cultured for another 4 h before sample collection for western blotting. (B) Western blot results showing the levels of BTG4, CNOT7, and TRIM21. Total proteins were collected from 100 zygotes at 4 h after microinjection and were loaded in each lane. DDB1 was blotted as a loading control. (C) RT-qPCR results showing the relative mRNA levels of indicated Z-decay transcripts in two-cell embryos with or without BTG4 Trimming-away. Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates. (D and E) Immunofluorescence (D) and quantification (E) of 5-ethynyl uridine (EU) fluorescence showing RNA transcription in two-cell embryos with or without BTG4 Trimming-away, and in maternal Btg4 knockout two-cell embryos. Scale bar, 20 μm. Error bars, s.e.m. n.s.: non-significant. **P < 0.01 by two-tailed Student's t-test. (F) RT-qPCR results showing the relative mRNA levels of indicated zygotic transcripts in two-cell embryos with or without BTG4 Trimming-away. Error bars, s.e.m. n.s.: non-significant. *P < 0.05 and ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates. (G) RT-qPCR results showing the relative mRNA levels of indicated transcripts in zygote and two-cell embryos with or without BTG4 Trim-away and α-amanitin treatment. Error bars, s.e.m. n.s.: non-significant. *P < 0.05 and ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates. (H-I): Developmental rates (H) and Representative images (I) of preimplantation embryos after zygotic BTG4-depletion. Time after hCG injection (h) and numbers of analyzed embryos are indicated (n). Zygotes were microinjected as in (A), and then cultured until 96 h after hCG injection. Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t-test. Scale bar, 100 μm. Arrows and hollow arrows indicate the normal embryos and arrested embryos, respectively.

Figure 5.

Dynamics of 3′-terminal polyadenylation and oligouridylation in maternal transcripts undergoing M-decay and Z-decay. (A) Poly(A) tail assay results showing changes in the poly(A)-tail length tendency of indicated M-decay and Z-decay transcripts during MZT. Zyg, zygote. Experiments were performed three times with reproducible results; a representative result is shown. (B) Changes in RT-qPCR results obtained with oligo-(dA)- versus random primer-mediated RT reactions reflecting the 3′-oligouridylation levels of selected M-decay and Z-decay transcripts during MZT. Zyg, zygote. Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. n = 3 biological replicates.

Figure 6.

Influence of 3′-UTR length and translational activity on the timing of mRNA degradation. (A and B) Average 3′-UTR length (A) and numbers of CPEs and PASs (B) in the 3′-UTR of murine M-decay, Z-decay, and length-controlled non-Z decay transcripts. The box indicates upper and lower quantiles, the purple thick line in the box indicates the median.***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. (C) Levels of polysome-bound M-decay and Z-decay transcripts in oocytes at the GV, MI and MII stages (two biological repeats). The expression level of each transcript was normalized by the mCherry spike-in, which was in vitro transcribed and equally added to each sample before RNA extraction. The box indicates upper and lower quantiles, and the line in the box indicates the median. ***P < 0.001 by two-tailed Student's t-test. n.s.: non-significant. (D) RT-qPCR results showing the relative mRNA levels of M-decay and Z-decay transcripts in association with polysomes at the GV and MII stages. Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t-test. n = 3 biological replicates.

Figure 7.

A summary of maternal mRNA clearance during mouse oocyte maturation and MZT. Maternal mRNAs can be classified into four categories based on their degradation dynamics: maternal decay (M-decay), zygotic decay (Z-decay), continuous decay, and stable throughout the MZT process. The maternal processes are in the pink area and the zygotic events are in the blue area. M-decay transcripts usually have short 3′-UTRs and are deadenylated by the CCR4-NOT complex during oocyte maturation. In contrast, Z-decay transcripts tend to have long 3′-UTRs, maintain long poly(A) tails, bind with polysomes, and are actively translated in maturing oocytes. After ZGA, maternally provided YAP1, together with zygotically expressed TEAD4, induce expression of zygotic TUT4/7, which mediate the 3′-oligouridylation and degradation of Z-decay transcripts. In addition, maternally translated BTG4 and CNOT7 continue to be required for Z-decay. Their transcripts are among those being removed as late as the four-cell stage. Blockage of Z-decay by depleting TUT4/7 or BTG4 in zygotes caused developmental arrest of embryos at the eight-cell stage, indicating that Z-decay is an essential MZT event.