Šimková K et al. (2023), RNA localization during early development of th...

XB-ART-60405

Front Cell Dev Biol 2023 Jan 01;11:1260795. doi: 10.3389/fcell.2023.1260795.

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RNA localization during early development of the axolotl.

Šimková K , Naraine R , Vintr J , Soukup V , Šindelka R .

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The asymmetric localization of biomolecules is critical for body plan development. One of the most popular model organisms for early embryogenesis studies is Xenopus laevis but there is a lack of information in other animal species. Here, we compared the early development of two amphibian species-the frog X. laevis and the axolotl Ambystoma mexicanum. This study aimed to identify asymmetrically localized RNAs along the animal-vegetal axis during the early development of A. mexicanum. For that purpose, we performed spatial transcriptome-wide analysis at low resolution, which revealed dynamic changes along the animal-vegetal axis classified into the following categories: profile alteration, de novo synthesis and degradation. Surprisingly, our results showed that many of the vegetally localized genes, which are important for germ cell development, are degraded during early development. Furthermore, we assessed the motif presence in UTRs of degraded mRNAs and revealed the enrichment of several motifs in RNAs of germ cell markers. Our results suggest novel reorganization of the transcriptome during embryogenesis of A. mexicanum to converge to the similar developmental pattern as the X. laevis.

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Species referenced: Xenopus laevis
Genes referenced: aco1 actb ap2b1 atp11b atp1a1 cap1 ccna1 crem csk dazl ddx5 dnd1 dvl3 dync1i2 fuca1 grip2 kif4a man2b1 mcm2 nanos1 nek9 pgc pi4ka pum2 rassf3 rbfox1 rbm24 rbpms2 rrp12 spire1 sulf1 tnf velo1 wee2 wnk2 wnt1 wnt5a wnt5b zfp36 zp4

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	FIGURE 1. Asymmetric localization of maternal transcript in A. mexicanum early embryos. (A) Schematic representation of the workflow. (B) PCA of 500 most variable transcripts show high variability among developmental stages and embryo sections. (C) The diagram of shared DLTs among developmental stages. (D) Number of DLTs in each localization category. DLTs in the unclassified category represent those that did not fit into any of the five defined profiles.
	FIGURE 2. Vegetal sectional profile alteration. During the early development of A. mexicanum, 4 groups of vegetal DLTs altering profiles were observable. (A) Homogenous or slightly vegetal localization from the 1-cell stage until the 64-cell stage and vegetal localization at the 1K-cell stage. (B) Homogenous or slightly vegetal localization at the 1-cell stage and vegetal localization from the 4-cell stage and later. (C) Vegetal localization at the 1-cell stage and from the 4-cell stage until the 1K-cell stage the gradual decrease of transcript amount. (D) Vegetal localization at the 1-cell stage and from the 4-cell stage the localization is homogenous or slightly vegetal. Line plots represent the averaged z-score expression for the genes with shared localization profiles. Heatmap shows the z-score of the averaged relative expression across the replicates. DLTs represent genes that had a padj < 0.01 and greater than 20 transcripts per stage. DLTs were further filtered to show those that were 2x greater in either amplitude or relative to another section across the stages. 3 biological replicates were used. Embryos sections: A - extremely animal, B - animal, C - central, D - vegetal, E - extremely vegetal.
	FIGURE 3. Animal sectional profile alteration. During the early development of A. mexicanum, 3 groups of animal DLTs altering profiles were observable. (A) Homogeneous or slightly animal from 1-cell until the 64-cell stage and the creation of animal profile at the 1K-cell stage. (B) Homogeneous or slightly animal at the 1-cell stage and animal at the 4-cell stage and later. (C) Animal localization at the 1-cell stage and homogenous or slightly animal localization from 4-cell until the 1K-cell stage. Line plots represent the averaged z-score expression for the genes with shared localization profiles. Heatmap shows the z-score of the averaged relative expression across the replicates. DLTs represent genes that had a padj < 0.01 and greater than 20 transcripts per stage. DLTs were further filtered to show those that were 2x greater in either amplitude or relative to another section across the stages. 3 biological replicates were used. Embryos sections: A – extremely animal, B – animal, C – central, D – vegetal, E – extremely vegetal.
	FIGURE 4. De novo transcription during early embryogenesis of A. mexicanum. (A) De novo transcription at the 4-cell stage and biological role of DLTs proposed using gene ontology analysis. (B) De novo transcription at the 64-cell stage and RNA-seq gene examples. (C) De novo transcription at the 1K-cell and RNA-seq gene examples. Box plots in the first column represent the averaged z-score expression for the averaged total transcript across the stage replicates. Line plots in the second column represent the normalized counts for each replicate for a specific gene across the stages. DLTs represent genes that had a padj < 0.01 and greater than 20 transcripts per stage. DLTs were further filtered to show those that were 3x greater in either amplitude or relative to another section across the stages. 3 biological replicates were used. Embryos sections: A - extremely animal, B - animal, C - central, D - vegetal, E - extremely vegetal.
	FIGURE 5. Transcription degradation during early embryogenesis of A. mexicanum. (A) The massive degradation was observed only after the 1-cell stage. Box plot in the first column represents the averaged z-score expression for the averaged total transcript across the stage replicates. Line plot in the second column represents the normalized counts for each replicate for a specific gene across the stages. DLTs represent genes that had a padj < 0.01 and greater than 20 transcripts per stage. DLTs were further filtered to show those that were 3x greater in either amplitude or relative to another section across the stages. 3 biological replicates were used. (B) Gene ontology analysis of degraded DLTs. (C) Degradation of PGC markers. Line plots show the localization profile and total amount of selected PGC markers change during the development. Whole-mount in situ hybridization shows the gene expression of 3 known PGC markers and 1 unknown gene within presumptive germ cells (arrow). Detection of PGC in A. mexicanum embryos at around stage 33 using in situ hybridization. Lateral view, with A-Anterior, B-Posterior. Scale bar = 2 mm.
	FIGURE 6. Motif and RBP enrichment in PGC. (A) De novo motif analysis within 3′UTR of A. mexicanum. Enriched motifs were also scanned in other model organisms. Sequences of motifs 5 and 6 probably can be bound by 2 known RBPS—PUM2 and RBFOX1. (B) De novo motifs analysis within 5′UTR of A. mexicanum. Enriched motifs were also scanned in other model organisms. (C) Identification of RBP binding motifs using BRIO within 3′ and 5′ UTR of A.mexicanum. The RBP presence was also assessed in other model organisms.
	Fig. S1: RNA-seq data validation using RT-qPCR. A – C) RNA-seq represents the averaged z-score expression for the averaged total transcript across the stage replicates. qPCR represents mean proportion of mRNAs for individual genes in particular section. Mean ± .s.d. D) RNA-seq - localization profiles generated from RNA-seq data. Line plot represent the averaged z-score expression for the averaged total transcript across the stage replicates. RNA-seq vs. qPCR - the comparison of relative expression using qPCR and RNA-seq. Geometric mean ± g.s.d. The independent samples t-test. N.s . p>0.05, p<0.05, p<0.01, **p<0.001. 3 biological replicates were used.
	Fig. S2: Primordial germ cell markers in A. mexicanum. A) Complete list of primordial germ cell markers.
	Fig. S2: Primordial germ cell markers in A. mexicanum. B) Expression patterns of dnd1, grip2, rbpms2 and AMEXTC_0340000004005 at around embryonic stage 33 (note that the tailbud was lost in some embryos during the in situ hybridization protocol). H – heart; P – pronephros; PGC – primordial germ cells. A - anterior, P - posterior. Scale bars = 1 mm.
	Figure S3: The enrichment of motifs from previous paper (Naraine et al., 2022) within 3‘ and 5‘UTR of A. mexicanum embryos.
	Fig. S4: [panel a] The position map of de novo motifs, de novo motifs from previous publication (Naraine et al., 2022) and motifs binding miRNAs unique to the given gene and RNA-binding proteins (RBPs) unique to the given gene. The map was created using the 3’UTR for two PGC genes, dnd1 and grip2, of A. mexicanum and X. laevis.
	Fig. S4: [panel b] The position map of de novo motifs, de novo motifs from previous publication (Naraine et al., 2022) and motifs binding miRNAs unique to the given gene and RNA-binding proteins (RBPs) unique to the given gene. The map was created using the 3’UTR for two PGC genes, dnd1 and grip2, of A. mexicanum and X. laevis.

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