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Dev Biol
2022 Sep 01;489:146-160. doi: 10.1016/j.ydbio.2022.06.013.
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Evolutionary conservation of maternal RNA localization in fishes and amphibians revealed by TOMO-Seq.
Naraine R
,
Iegorova V
,
Abaffy P
,
Franek R
,
Soukup V
,
Psenicka M
,
Sindelka R
.
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Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized transcripts is still limited to a few models. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four vertebrate models, two amphibians (Xenopus laevis, Ambystoma mexicanum) and two fishes (Acipenser ruthenus, Danio rerio) using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important transcripts such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. It was also observed that there was a poor correlation between the vegetally localized transcripts but a relatively good correlation between the animally localized transcripts. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.
Fig. 1. Relationship between the studied taxa and their developmental features. A) Taxonomic tree of select animal models based on Timetree (v. 4.0) (Kumar et al., 2017). Highlighted are the model organisms used in this research. B) Images of the egg, 4-cell stages, and cell fate maps for the analysed models. Images have been cropped and brightness adjusted for clarity. C) Genomic and egg developmental properties for each model.
Fig. 2. The three datasets created from this research and their usages.
Fig. 3. Localization of maternal differentially localized transcripts (DLTs). A) Schematic of the five main localization profiles detected for the maternal transcripts within the egg. The darker colored regions within the egg represent higher transcript count saturation. The line graphs represent the median expression of the transcripts within all models that showed the strongest distributions for each profile (dataset2). B) Distribution of DLTs with a localization profile for each model (dataset1 â differentially localized (padj <0.1), overall oocyte transcript count >20). Venn diagram shows the overlap in the number of unique gene symbols or orthologous units. C) Dendogram showing the similarity between the shared asymmetry amongst the analysed models. The list of transcripts used comprised of only those with an assigned orthologous unit number that did not have a paralog in a contrasting localization profile.
Fig. 4. Comparative analysis between the maternal asymmetry observed within each studied model and with data from previous publications. Overlap of asymmetrical maternal transcripts shared between this dataset and A) Three published datasets on the Xenopus laevis egg, B) One published dataset on the Danio rerio egg. Highlighted in red are the subset of transcripts that are shared across all datasets. C) The differentially localized presence within all four analysed models for the top most significant transcripts derived from the overlap between all datasets/all RNA-Seq datasets for the X. laevis model from panel A. The full reference for the published datasets can be found in the methodology.
Fig. 5. Summary of the conservation of the differentially localized transcripts amongst the different models as derived from dataset3. A) Conservation of extreme vegetal or vegetal localized transcripts. B) Conservation of central localized transcripts. C) Conservation of extreme animal or animal localized transcripts. The Upset plots show the number of transcripts shared between the different models. The tables show the number of conserved localized transcripts for the specific models, examples of these conserved transcripts, the median expression of the transcripts within this group for all models and an example of a significantly enriched motif within the 3â²UTR (top) and 5â²UTR (bottom) sequences. 1Danio rerio transcript nomenclature.
Fig. 6. Summary of number of significantly (padj < 0.05) enrichment Gene Ontology (GO) terms associated with the transcripts from the localization profiles. Venn diagrams show the overlap of the enriched GO (Biological Process) terms between each model while heatmaps shows summarized GO terms for the, A) The extreme vegetal and vegetal transcripts, and B) Extreme animal and animal transcripts. The left most heatmap represents the padj values for the enriched GO terms. Second heatmap shows the similarity matrix of the GO terms.
Fig. 7. RNA localization of some essential transcripts within the egg of several species. Localization profiles for some key transcripts belonging to A) PGC markers, B) Endodermal and mesodermal determinants, C) wnt ligands, other D) Known Xenopus laevis vegetal transcripts. Graphs represent the average relative expression of the transcripts of interest from the RNA-Seq data. Transcripts in brackets and graphs with dashed lines indicate that the RNA-Seq data for the transcript was not differentially localized, failed the statistical analysis using DESeq or did not meet the threshold criteria. Transcripts listed with âOtherRT-qPCRâ indicate that the RT-qPCR profiles were variable between the replicates. Danio rerio GDF3 transcript is an ortholog of GDF1 transcript. velo1 (amphibians and A. ruthenus) and buc2 (D. rerio) are orthologous transcripts. DDX19B in A. ruthenus was closest blast hit to DDX25 while DDX19A used as ortholog in D. rerio. 1transcript nomenclature for X. laevis; 2transcript nomenclature for D. rerio; âprimers worked on later stages; ¬ detected even at later stages.