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Transcriptome analysis of regeneration in Xenopus laevis twin embryos

ABSTRACT: Animal embryos have the remarkable property of self-organization. Over 125 years ago Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundational experiment of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals by diverse methods. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study we investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (Stage 12) and analyzed the transcriptome of each half-embryo by RNAseq. Because many genes are activated by wound healing, stringent analyses were used to identify genes upregulated in identical twins but not in either dorsal or ventral fragments. At early gastrula cell division-related genes such as histones were identified, whereas at late gastrula pluripotency genes (such as sox2) and germ layer determining genes (such as eomesodermin, ripply2 and activing receptor ACVRI) and a number of secretory pathway components (serpinH1, fucoleptin and sialyl transferase). These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to permit formation of the organizer with a displacement of 900 from its original site. In addition, the extensive transcriptomic data presented here (30 RNA-seq libraries of individual whole or regenerating half-embryos) provides a useful resource for data mining gene expression during early vertebrate development.

ORGANISM(S): Xenopus laevis

PROVIDER: GSE124563 | GEO | 2019/02/01

REPOSITORIES: GEO

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Project description:Studies of the Xenopus organizer have laid the foundation for our understanding of the conserved signaling pathways that pattern vertebrate embryos during gastrulation. Here, we use this wealth of knowledge as leverage in the design and analysis of a genomic visualization of organizer-related gene transcription. Using ectopic expression of the two major activities of the organizer, BMP and Wnt inhibition, as well as endogenous tissues, we generate a focused set of samples that represent different aspects of organizer signaling. The genomic expression values of each sample are then measured with oligonucleotide arrays. From this data, genes regulated by organizer signaling are selected and then clustered by their patterns of regulation. A new GO biological process annotation of the Xenopus genome allows us to rapidly identify clusters that are highly enriched for known gastrula patterning genes. Within these clusters, we can predict the expression patterns of unknown genes with remarkable accuracy, leading to the discovery of new organizer-related gastrula stage expression patterns for 19 genes. Moreover, the patterns of gene response observed within these clusters allow us to parse apart the contributions of BMP and Wnt inhibition in organizer function. We find that the majority of gastrula patterning genes respond transcriptionally to these activities according to only a few stereotyped patterns, allowing us to describe suites of genes that are likely to share similar regulatory mechanisms. These suites of genes demonstrate a mechanism where BMP inhibition initiates the organizer program before gastrulation, and Wnt inhibition maintains and drives the organizer program during gastrulation. Experiment Overall Design: In order to describe and separate the genomic expression changes induced by the two main organizing activities, BMP inhibition and Wnt inhibition, we created a panel of gastrula stage ventral tissues that ectopically overexpressed one or both of these activities, and compared these samples to endogenous dorsal and ventral gastrula stage tissue. Two well-studied organizer secreted factors, Noggin and Dickkopf-1 (Dkk-1), were used to ectopically inhibit BMP and Wnt signaling, respectively. Four different overexpression mixtures were injected ventrally into 4-cell embryos: noggin and eGFP (anti-BMP); noggin, dkk-1, and eGFP (anti-BMP and anti-Wnt); dkk-1 and eGFP (anti-Wnt); and eGFP alone. eGFP mRNA was used to trace targeting. Plasmid DNA was used for noggin and dkk-1 overexpression, instead of mRNA, in order to limit ectopic expression of these genes to post-MBT stages, more closely mimicking the endogenous regulation of these genes. The ventrally injected embryos were grown to early stage 10, sorted for appropriately targeted eGFP florescence, and then bisected between the dorsal and ventral halves at either stage 10 (early gastrula) or stage 11.5 (late gastrula). For the noggin and/or dkk-1 injected embryos, only the ventral halves of the embryos were saved, eliminating endogenous organizer tissues. For the embryos injected with only eGFP, both the ventral and dorsal halves were saved, creating separate ventral and dorsal control conditions. These five conditions were each generated twice at both stage 10 and 11.5, with each set of five samples coming from a single clutch of embryos, creating twenty total tissue samples from 4 different clutches. For each batch of injections some sorted embryos were allowed to develop through tailbud stages in order to validate the phenotypes induced by our constructs. Total RNA was then isolated from the twenty tissue samples and applied to Affymetrix oligonucleotide arrays.

Project description:Studies on the early embryonic development of Xenopus laevis contributed much to the understanding of vertebrate patterning. Gastrula stages are of particular interest because establishment of the axis and germ layer formation take place during these stages. While many genes belonging to several signaling pathways including FGF, Wnt and TGF-beta, have been implicated in patterning the gastrula embryo, the hierarchical interactions between these factors are incompletely known. To study this question, we took advantage of microarray technology to create a regional gene expression profile for the Xenopus gastrula. Stage 10 Xenopus embryos were dissected into four portions. The dorsal marginal zone including the blastopore and some ectoderm and dorsal yolk plug, composed mostly of endomesoderm; the ventral marginal zone, also containing a portion of the yolk plug; the animal cap, dissected just above the floor of the blastocoel; and the vegetal region, composed of the central part of the yolk plug. To avoid possible cross contamination that might blur the microrarray data, thin junctional regions between the explants were removed. The dissected explants were homogenized in Stat 60 (TEL TEST), RNA was precipitated by isopropanol, treated with DNase I, and purified using the RNeasy kit (Qiagen). Biotinylated probe was prepared from 100 ng total RNA using the OVATION RNA amplification system (Nugen Technologies, Inc). The probes were hybridized to Affymetrix Xenopus Chips containing features that represent about 15,000 genes according to the manufactureâs instructions. Hybridized arrays were further processed by the GeneChip Fluidics system (Affymetrix), and signals were detected by the GeneChip Scanner (Affymetrix). Gene expression profiles were analyzed by the GCOS software (Affymetrix). The analysis showed that 100 transcripts were enriched in the dorsal explant (dorsal vs. ventral, signal log2 ratio>1.5), including the known dorsal markers Chordin, gsc, Admp; 90 transcripts were enriched in the ventral explant (ventral vs. dorsal, signal log2 ratio>1.5) including Sizzled, bambi, PV.1; 449 transcripts were enriched in the vegetal explant (vegetal vs. dorsal, vegetal vs. ventral, vegetal vs. animal cap, all signal log2 ratio>1.5), including Mixer, Sox17ï¡.; 70 transcripts were enriched in the animal cap (animal cap vs. vegetal, signal log2 ratio>1.5; animal cap vs. dorsal, signal log2 ratio>1; animal cap vs. ventral, signal log2 ratio>1) including Epidermal type I cytokeratin and forkhead-2. RT-PCR was used to check the enrichment of some of the unknown genes; the enrichment of 8 of 9 ventral genes, and 9 of 12 dorsal genes was confirmed in these experiments. Experiment Overall Design: Ceate a regional gene expression profile in Xenopus gastrula, and predict gene expression pattern by comparing gene expression in different explant.

Project description:Studies on the early embryonic development of Xenopus laevis contributed much to the understanding of vertebrate patterning. Gastrula stages are of particular interest because establishment of the axis and germ layer formation take place during these stages. While many genes belonging to several signaling pathways including FGF, Wnt and TGF-beta, have been implicated in patterning the gastrula embryo, the hierarchical interactions between these factors are incompletely known. To study this question, we took advantage of microarray technology to create a regional gene expression profile for the Xenopus gastrula. Stage 10 Xenopus embryos were dissected into four portions. The dorsal marginal zone including the blastopore and some ectoderm and dorsal yolk plug, composed mostly of endomesoderm; the ventral marginal zone, also containing a portion of the yolk plug; the animal cap, dissected just above the floor of the blastocoel; and the vegetal region, composed of the central part of the yolk plug. To avoid possible cross contamination that might blur the microrarray data, thin junctional regions between the explants were removed. The dissected explants were homogenized in Stat 60 (TEL TEST), RNA was precipitated by isopropanol, treated with DNase I, and purified using the RNeasy kit (Qiagen). Biotinylated probe was prepared from 100 ng total RNA using the OVATION RNA amplification system (Nugen Technologies, Inc). The probes were hybridized to Affymetrix Xenopus Chips containing features that represent about 15,000 genes according to the manufacture’s instructions. Hybridized arrays were further processed by the GeneChip Fluidics system (Affymetrix), and signals were detected by the GeneChip Scanner (Affymetrix). Gene expression profiles were analyzed by the GCOS software (Affymetrix). The analysis showed that 100 transcripts were enriched in the dorsal explant (dorsal vs. ventral, signal log2 ratio>1.5), including the known dorsal markers Chordin, gsc, Admp; 90 transcripts were enriched in the ventral explant (ventral vs. dorsal, signal log2 ratio>1.5) including Sizzled, bambi, PV.1; 449 transcripts were enriched in the vegetal explant (vegetal vs. dorsal, vegetal vs. ventral, vegetal vs. animal cap, all signal log2 ratio>1.5), including Mixer, Sox17; 70 transcripts were enriched in the animal cap (animal cap vs. vegetal, signal log2 ratio>1.5; animal cap vs. dorsal, signal log2 ratio>1; animal cap vs. ventral, signal log2 ratio>1) including Epidermal type I cytokeratin and forkhead-2. RT-PCR was used to check the enrichment of some of the unknown genes; the enrichment of 8 of 9 ventral genes, and 9 of 12 dorsal genes was confirmed in these experiments. Keywords: Xenopus, gastrula, explant, gene expression, embryonic, gene regulation

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Transcriptome analysis of regeneration in Xenopus laevis twin embryos

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