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Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, Austrofundulus limnaeus

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Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, Austrofundulus limnaeus

Amie L Romney et al. Evodevo.

Abstract

Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifish Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to "escape" from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species.

Results: Using high-throughput RNA sequencing, we generated transcriptomic profiles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1-2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafish and other fishes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle.

Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by differential expression of diapause-specific genes. Rather, evidence suggests a role for trajectory-specific splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafish, the A. limnaeus 1-2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development.

Keywords: Alternative splicing; Diapause; Maternal effect; Maternal-to-zygotic transition; RNA-seq; Transcriptome.

Figures

Fig. 1
Fig. 1
Alternative splicing of poly-A RNA in embryos of A. limnaeus that will develop along two alternative developmental trajectories. a Differential exon usage in mRNA gene transcripts that are packaged into diapause- and escape-destined 1–2 cell stage embryos of A. limnaeus. Of the 57 exons that were significantly different between trajectories (red symbols, FDR < 0.1, t test) 49 are upregulated in diapause-bound embryos, while only 8 are upregulated in escape-bound embryos. b GO term analysis for transcript variants between diapause- and escape-bound embryos of A. limnaeus (P < 0.05) suggests enrichment for exons expressed in genes for a variety of molecular and metabolic pathways including glycolysis and the insulin/IGF signaling pathway
Fig. 2
Fig. 2
Top 10 genes with developmental trajectory-specific splice variants based on statistical significance. Each biological replicate is graphed separately in the exon usage graphs with orange lines indicating escape-bound embryos and blue lines indicating diapause-bound embryos. The x-axis on the plots indicates the exon number and the mapping location of the exon on the appropriate contig from the A. limnaeus genome file. Note that the y-axis is a log scale which tends to mask the differential expression of the exons, and thus we have provided a bar graph on a linear scale to better illustrate the mean (±SD) levels of expression for the differentially expressed exon within each gene. Blue bars indicate diapause-bound embryos, while orange bars represent escape-bound embryos
Fig. 3
Fig. 3
Comparative analysis of poly-A transcriptomes in 1–2 cell stage embryos of D. rerio and A. limnaeus. a The 20 most abundant transcripts and their FPKM values that are unique to the transcriptome of either A. limnaeus or D. rerio. b Venn diagram depicting the number of shared (orthologous) and non-shared transcripts in 1–2 cell stage embryos of A. limnaeus (turquoise) and D. rerio (gray). Frequency histograms show the distribution of expression values of shared (orthologous) and non-shared genes and indicate similar patterns of transcript abundance between the two species
Fig. 4
Fig. 4
The relationship of the maternally packaged transcriptome of A. limnaeus to other teleosts. a Venn diagram showing shared numbers of the 20 most abundant transcripts in 3 species of fish with 1–2 cell stage Illumina-sequenced transcriptomes: A. limnaeus (this study), D. rerio [17] and H. hippoglossus [44]. bd Gene ontology analysis comparing the top 100 transcripts in the 1–2 cell stage transcriptomes of A. limnaeus and D. rerio. Pie charts represent the quantities of GO categories in (b) D. rerio and c A. limnaeus for biological process classification (top) and protein class (bottom). d Go terms enriched in the A. limnaeus compared to D. rerio (P < 0.05) transcriptome. For more details see the text and Additional file 5
Fig. 5
Fig. 5
Maternally packaged sncRNA transcriptome of A. limnaeus. Frequency distribution of a normalized sequence reads and b unique sequences as a function of sequence length in the sncRNA libraries (n = 12). Each library is a different color. c There is a high diversity of sncRNA sequences with lengths between 15 and 23 nucleotides that are unknown (blue line) compared to those that could be annotated (red line) by sequence similarity to those cataloged in RNA databases
Fig. 6
Fig. 6
Rfam database annotation of the maternally packaged sncRNA transcriptome of A. limnaeus. a The most abundant sncRNAs are 16–17 nucleotides in length and annotate as antisense RNAs, while the second most abundant group are sequences that are 26 nucleotides in length that annotate as ribosomal RNA (see panel c for a color key to annotation category). b The highest diversity of unique sncRNA sequences is in the 15 and 17 nucleotide length categories. Note the enrichment of miRNA sequences in the 20–22 nucleotide range as expected, even though miRNAs are not a dominant part of the sncRNA transcriptome. c Some annotation categories have distinct size ranges, while others span the entire range of sizes explored in this study. d Putative micro-RNA precursor structures and consensus mature sequences (highlighted in yellow) annotated as mir-181a (Alim-mir-181a1-3) and mir-10b (Alim-mir-10b1-4)

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