Dataset Information

Tools for gene-regulatory analyses in the marine annelid Platynereis dumerilii.

ABSTRACT: The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.

SUBMITTER: Backfisch B

PROVIDER: S-EPMC3979674 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Tools for gene-regulatory analyses in the marine annelid Platynereis dumerilii.

Backfisch Benjamin B Kozin Vitaly V VV Kirchmaier Stephan S Tessmar-Raible Kristin K Raible Florian F

PloS one 20140408 4

The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimen ...[more]

PMID: 24714200

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Project description:Background:Wnt/β-catenin (or canonical) signalling pathway activity is necessary and used independently several times for specification of vegetal fate and endoderm, gut differentiation, maintenance of epithelium in adult intestine and the development of gut-derived organs in various vertebrate and non-vertebrate organisms. However, its conservation in later stages of digestive tract development still remains questionable due to the lack of detailed data, mainly from Spiralia. Results:Here we characterize the Pdu-Tcf gene, a Tcf/LEF orthologue and a component of Wnt/β-catenin pathway from Platynereis dumerilii, a spiralian, marine annelid worm. Pdu-Tcf undergoes extensive alternative splicing in the C-terminal region of the gene generating as many as eight mRNA isoforms some of which differ in the presence or absence of a C-clamp domain which suggests a distinct DNA binding activity of individual protein variants. Pdu-Tcf is broadly expressed throughout development which is indicative of many functions. One of the most prominent domains that exhibits rather strong Pdu-Tcf expression is in the putative precursors of endodermal gut cells which are detected after 72 h post-fertilization (hpf). At day 5 post-fertilization (dpf), Pdu-Tcf is expressed in the hindgut and pharynx (foregut), whereas at 7 dpf stage, it is strongly transcribed in the now-cellularized midgut for the first time. In order to gain insight into the role of Wnt/β-catenin signalling, we disrupted its activity using pharmacological inhibitors between day 5 and 7 of development. The inhibition of Wnt/β-catenin signalling led to the loss of midgut marker genes Subtilisin-1, Subtilisin-2, α-Amylase and Otx along with a drop in β-catenin protein levels, Axin expression in the gut and nearly the complete loss of proliferative activity throughout the body of larva. At the same time, a hindgut marker gene Legumain was expanded to the midgut compartment under the same conditions. Conclusions:Our findings suggest that high Wnt/β-catenin signalling in the midgut might be necessary for proper differentiation of the endoderm to an epithelium capable of secreting digestive enzymes. Together, our data provide evidence for the role of Wnt/β-catenin signalling in gut differentiation in Platynereis.

Project description:BackgroundRestorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered.ResultsWe generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process.ConclusionsIn this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

Dataset Information

Tools for gene-regulatory analyses in the marine annelid Platynereis dumerilii.

Publications

Tools for gene-regulatory analyses in the marine annelid Platynereis dumerilii.

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