<i>in vivo</i> expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada

Tanaka, Sayoko; Aoki, Kazuhiro; Arakawa, Kazuharu

doi:10.1101/2022.09.21.508853

Cited by 5 publications

(8 citation statements)

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“… (d) Expression of He -TDR1-mNeonGreen in transient transgenic H. exemplaris tardigrades Expression plasmids of He -TDR1-mNeonGreen (mNG) and mCherry (both under control of the He- Actin promoter) were microinjected into the body fluid of H. exemplaris adults and electroporation was performed to induce delivery into cells following the protocol of Tanaka et al 2023. Confocal microscopy was carried out on live animals immobilized in carbonated water at day 8 post-microinjection after 2 days of treatment with 20µM Hoechst 33342 to stain nuclei.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Recent progress in tardigrade transgenesis (S. Tanaka, Aoki, and Arakawa 2023) and promising findings of somatic mutagenesis by CRISPR-Cas9 (Kumagai, Kondo, and Kunieda 2022) are paving the way towards germ line gene editing in H. exemplaris .…”

Section: Discussionmentioning

confidence: 99%

“…To further examine the potential interaction of He-TDR1 with DNA in vivo, we generated a tardigrade expression plasmid with He-TDR1-mNeonGreen cDNA downstream of He-Actin promoter sequences and introduced it into tardigrade cells using a recently reported protocol (S. Tanaka, Aoki, and Arakawa 2023). He-TDR1-mNeonGreen was easily detected in muscle cells, likely due to high muscle-specific activity of the He-Actin promoter, and predominantly localized to nuclei, as observed by confocal microscopy (Figure 5d-e).…”

Section: He-tdr1 Interacts Directly With Dna In Vitro and Co-localize...mentioning

confidence: 99%

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Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Anoud,

Delagoutte,

Helleu

et al. 2023

Preprint

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Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicalsin vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily.To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model speciesHypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species,H. exemplaris,Acutuncus antarcticusandParamacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, namedTardigradeDNA damageResponse protein1(TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: He-tdr1 Interacts Directly With Dna In Vitro and Co-localize...mentioning

confidence: 99%

See 2 more Smart Citations

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Anoud,

Delagoutte,

Helleu

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Work toward the latter has begun with the use of different live fluorescent dyes to temporarily label various cellular components, such as mitochondria, lysosomes, and membrane (McGreevy et al, 2018). Transgene and CRISPR techniques were recently developed for somatic cells of adult tardigrades, but they have not yet been demonstrated to work in germline cells (Kumagai et al, 2022; Tanaka et al, 2022). There has yet to be a technique established for labeling and live imaging of specific mRNA and/or proteins or for live reporting of mRNA transcription and/or translation in germline or embryos of this system.…”

Section: Discussionmentioning

confidence: 99%

The Embryonic Origin of Primordial Germ Cells in the TardigradeHypsibius exemplaris

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Game

Smith

et al. 2023

Preprint

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Primordial germ cells (PGCs) give rise to gametes – cells necessary for the propagation and fertility of diverse organisms. Current understanding of PGC development is limited to the small number of organisms whose PGCs have been identified and studied. Expanding the field to include little-studied taxa and emerging model organisms is important to understand the full breadth of the evolution of PGC development. In the phylum Tardigrada, no early cell lineages have been identified to date using molecular markers. This includes the PGC lineage. Here, we describe PGC development in the model tardigradeHypsibius exemplaris. The four earliest- internalizing cells (EICs) exhibit PGC-like behavior and nuclear morphology. The location of the EICs is enriched for mRNAs of conserved PGC markerswiwi1(water bearpiwi1) andvasa. At early stages, bothwiwi1andvasamRNAs are detectable uniformly in embryos, which suggests that these mRNAs do not serve as localized determinants for PGC specification. Only later arewiwi1andvasaenriched in the EICs. Finally, we traced the cells that give rise to the four PGCs. Our results reveal the embryonic origin of the PGCs ofH. exemplarisand provide the first molecular characterization of an early cell lineage in the tardigrade phylum. We anticipate that these observations will serve as a basis for characterizing the mechanisms of PGC development in this animal.

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Expression patterns of FGF and BMP pathway genes in the tardigradeHypsibius exemplaris

Heikes,

Goldstein

2024

Preprint

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A small number of conserved signaling pathways regulate development of most animals, yet we do not know where these pathways are deployed in most embryos. This includes tardigrades, a phylum with a unique body shape. We examined expression patterns of components of the BMP and FGF signaling pathways during embryonic segmentation and mesoderm development of the tardigrade Hypsibius exemplaris. Among the patterns examined, we found that an FGF ligand gene is expressed in ectodermal segment posteriors and an FGF receptor gene is expressed in underlying endomesodermal pouches, suggesting possible FGF signaling between these developing germ layers. We found that a BMP ligand gene is expressed in lateral ectoderm and dorsolateral bands along segment posteriors, while the BMP antagonist Sog gene is expressed in lateral ectoderm and also in a subset of endomesodermal cells, suggesting a possible role of BMP signaling in dorsal-ventral patterning of lateral ectoderm. In combination with known roles of these pathways during development of common model systems, we developed hypotheses for how the BMP and FGF pathways might regulate embryo segmentation and mesoderm formation of the tardigrade H. exemplaris. These results identify the expression patterns of genes from two conserved signaling pathways for the first time in the tardigrade phylum.

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in vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada

Cited by 5 publications

References 58 publications

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

The Embryonic Origin of Primordial Germ Cells in the TardigradeHypsibius exemplaris

Expression patterns of FGF and BMP pathway genes in the tardigradeHypsibius exemplaris

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