Identification of the zebrafish maternal and paternal transcriptomes

Abstract

Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.

Keywords: MZT; Maternal; Paternal; Transcriptome; Zebrafish.

Fig. 1.

Identification of maternal and paternal mRNAs. (A) To identify maternal and paternal mRNAs, crosses of two different strains of zebrafish were used. A female SAT was crossed with a male WIK and the reciprocal cross of female WIK and male SAT was also performed. The four adults used were fin clipped and then subjected to exon enrichment and Illumina sequencing. On average, ∼95 million (M) reads were obtained for each sample/individual. Each sample was then run through our single-nucleotide polymorphism (SNP) calling pipeline. The exon-enriched sequences for the male and female in each cross were compared to identify homozygous SNPs that distinguished male and female alleles. (B) One hundred zebrafish embryos were collected at five different developmental stages from each cross: 2-cell, 64-cell, 3.5 hpf, 6 hpf and 9 hpf. Polyadenylated RNA was then extracted from total RNA and subjected to Illumina sequencing to produce ∼70 million reads per stage per cross. Reads were mapped to the zebrafish genome (Zv9) using TopHat and quantified using Cufflinks. SNPs identified were then used to identify maternal and paternal mRNAs. FPKM, fragments per kb per million reads; m, male; f, female.

Fig. 2.

Maternal and zygotic expression. (A) The developmental stage at which paternal SNPs (green) and the corresponding transcripts (purple) are first detected. Paternal mRNAs, which are an indicator of zygotic transcription, are first detected at 3.5 hpf. The SNPs counted displayed the same expression pattern in both the SATm/WIKf and WIKm/SATf crosses. (B-G) mRNA maternal (red) and paternal (blue) SNP count for three genes displaying only maternal expression (B,C; ehd1b ENSDARG00000014793), maternal and zygotic expression (D,E; tsr2 ENSDARG00000005772) and only zygotic expression (F,G; wnt11 ENSDARG00000004256). (B,D,F) mRNA SNP counts from the SATf and WIKm cross. (C,E,G) mRNA SNP counts from the WIKf and SATm cross. M, maternal; Z, zygotic.

Fig. 3.

Polyadenylation of maternal mRNAs. (A,B) Significant increases in the maternal mRNA SNP count and FPKM levels were observed from the 2-cell to 64-cell stage. Because these changes occur in the absence of de novo transcription they represent the post-transcriptional regulation of maternal mRNAs. The SNP count of maternal and paternal mRNAs for the gene hcfc1b (ENSDARG00000012519) in the (A) SATf/WIKm cross and (B) WIKf/SATm cross is shown. (C) FPKM for hcfc1b. (D) Polyadenylation tail assay for the gene polb (ENSDARG00000003749). A primer (p1) was first ligated onto the 3′ end of mRNAs and cDNA was generated using a complementary primer (p2). A polb gene-specific primer and p2 were then used to amplify the poly(A) tail. As shown, there was an increase in the poly(A) tail for polb from the 2-cell to the 64-cell stage.

Fig. 4.

Cytoplasmic polyadenylation elements drive the post-transcriptional regulation of maternal mRNAs. (A) To map the elements controlling polyadenylation of the maternal mRNA sox19b, four different fragments of the 3′ UTR were cloned downstream of the coding sequence of the fluorescent protein Venus. The positions of a cytoplasmic polyadenylation element (CPE) and hexamer, which were contained within fragment 3, are shown. (B) Injection of fertilised zebrafish embryos with Venus-sox19b 3′ UTR RNA demonstrated that only fragment 3 is polyadenylated. The ratio of quantitative PCR levels at the 64-cell stage to 2-cell stage are shown. cDNA was generated with oligo(dT) primers. As a negative control (neg. con.), embryos were injected with Venus RNA without a 3′ UTR. Error bars indicate s.d. (C) A CPE is present within fragment 3 of the sox19b 3′ UTR, which when deleted abolishes polyadenylation. (D) To study the cell cycle-dependent post-transcriptional regulation of cyclin B1, quantitative PCR was performed on cDNA, generated with oligo(dT) primers, derived from embryos synchronised by IVF. Embryos were collected every 3 minutes, starting just after the 2-cell stage. cyclin B1 polyadenylation levels are regulated in a cell cycle-dependent manner, with levels peaking during mitosis.

Fig. 5.

Zygotic transcription initiation in the zebrafish embryo. (A) To test the transcriptional competence of the early zebrafish embryo, fertilised embryos were injected with plasmid DNA containing the EF1α promoter upstream of the coding sequence for the fluorescent protein CFP. Lane 1, size ladder; lane 2, uninjected embryos; lane 3, embryos injected with plasmid; lane 4, embryos co-injected with plasmid DNA and the RNA polymerase II inhibitor α-amanitin. RT-PCR for CFP is shown on the left, with control cyclin B1 RT-PCR on the right. (B) Experimental design to identify the first zygotic genes to be expressed. Embryos were synchronised by IVF and half of the clutch was exposed to the translation blocker cycloheximide at the 128-cell stage. Untreated and treated embryos were then collected every 15 minutes, starting just after the 128-cell stage. RNA-seq was then performed on the samples. (C) FPKM for a sample of genes that are expressed only zygotically (supplementary material Table S6) demonstrates that the sampled stages (labelled A-F) span the period of normal zygotic transcription initiation. (D,E) In embryos treated with cycloheximide (128c), some of the zygotically expressed genes, such as vox (D), are unaffected by treatment. However, some zygotic genes, such as claudin e (E), fail to be expressed in the treated embryos. WT, wild type, untreated with cycloheximide.