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

Edited by Bob Goldstein, University of North Carolina, Chapel Hill, NC; received September 30, 2022; accepted December 21, 2022 by Editorial Board Member Rebecca Heald
January 24, 2023
120 (5) e2216739120

Significance

Tardigrades are microscopic ubiquitous animals that are known for their extremotolerance, including exposure to space vacuum. Here, we report a gene expression system for tardigrades, which enables the expression of any protein in tardigrades, realizing live imaging. We have derived a series of promoters from the tardigrade genome to enable expression in various tissues, and observed the expression of genes contributing to their tolerance. Promoters functioned similarly across multiple species, even for species which undergo significant upregulation upon drying, suggesting that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins are also observed. Transgenic technology for tardigrades facilitates anhydrobiosis researches using tardigrades as the model organism.

Abstract

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca2+ indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.
Water is essential for life on Earth. In contrast, some organisms, e.g., yeast, Artemia and tardigrades, can enter an almost completely dehydrated state, referred to as anhydrobiosis (1). Anhydrobiotic organisms halt their metabolic activity and maintain the structures of living systems under water deficit conditions, until they resume active metabolism upon rehydration. Tardigrades in the anhydrobiotic state can tolerate various extreme conditions, e.g., temperature at almost absolute zero (2), high pressure (7.5 GPa) (3), high doses of irradiation of UV and gamma rays (411), and even exposure to space vacuum (1214).
In terrestrial tardigrades, the mechanisms underlying anhydrobiosis have been gradually revealed through analyses of genomes and transcripts (15, 16). Tardigrades have evolved lineage-specific IUP (intrinsically unstructured protein) families localized in the cytosol, nucleus, mitochondria, and extracellular space, i.e., CAHS (cytoplasmic abundant heat-soluble), Dsup (damage suppressor), MAHS (mitochondrial abundant heat-soluble), and SAHS (secretory abundant heat-soluble) (1719). Previous studies used heterologous hosts for molecular analyses of these proteins and showed that exogenous expression of Dsup and MAHS confers tolerance to human cells (18, 19) and plants (20, 21), and a deficit of CAHS transcripts by RNAi attenuated the survival rate in the dehydrated state (22); however, little is known about the actual tissue specificity or the actual workings of these tardigrade-specific IUPs within tardigrades during anhydrobiosis due to the lack of a genetic toolkit for use in these species.
Recently, several reports indicated that recombinant CAHS proteins form fibers or a gel-like lump at high concentrations in vitro, and similar fibrous condensates or aggregates were observed in human cultured cells and Escherichia coli cells (2327). These gel-like structures are assumed to sustain the structure of a whole cell or to prevent membranes and proteins from cohesion in the cytoplasm. This concept follows the research of another anhydrobiosis-related IUPs, namely, the LEA (late embryogenesis abundant) protein, one of which from Artemia is reported to form liquid–liquid phase separation (LLPS) upon dehydration in vitro (28). Interestingly, such condensation is rapidly reversible; that is, condensates formed with applied osmotic pressure rapidly dissociate upon removal of such stress (23). This highly dynamic nature demands a live imaging toolkit for in vivo study. On the other hand, reports thus far are all based on in vitro experiments or heterologous systems, such as human culture cell lines, yeast, and bacteria, since direct approaches applicable in tardigrades are currently restricted to RNAi and IHC (immunohistochemistry). Therefore, the actual subcellular localization in tardigrade cells and the dynamics during anhydrobiosis, as well as the tissue specificity of these expressions in tardigrades, remain elusive. IHC has been used widely in tardigrades to reveal cell morphology, such as neural systems, and the localization of tardigrade protein in the whole body (2936), but it nevertheless cannot be used to observe the dynamics of proteins and cells in a living tardigrade.
In the present study, we have developed an in vivo expression system, with a vector designated the TardiVec system, using a promoter that originated from an anhydrobiotic tardigrade, Ramazzottius varieornatus; it allows the introduction of exogenous genes such as GFP for live imaging in tardigrades. Tardigrade vectors show distinct expression patterns depending on the promoter sequence, and the GFP signals were retained for more than 10 d in tardigrades. The genetically encoded calcium indicator GCaMP (37) functions in the muscle and neurons of tardigrades, and its function is maintained throughout anhydrobiosis and after rehydration. We also confirmed that promoters that originated from not only housekeeping genes but also tardigrade-specific genes function widely in other tardigrade species within the order Parachela, and to some extent within the class Eutardigrada including the other order Apochela. Furthermore, expression of tardigrade-specific genes with this transgenic technology was distinct in different tissues, contrary to the previous expectations that these proteins are expressed and synergistically function within a single cell (for convenience, this is referred to as the “single-cell hypothesis” hereafter) (15, 1719, 38). CAHS genes are mainly expressed in epidermal cells, and the proteins are localized in the cytosol, while SAHS proteins are expressed exclusively in storage cells, which are tardigrade-specific free-floating cells in the body cavity. Therefore, the transgenic technology enables live imaging and transient expression in tardigrades and is especially useful in examining the details and dynamics of anhydrobiosis.

Results

Establishment of an In Vivo Expression System Based on a Plasmid Vector with a Tardigrade-Specific Promoter.

To establish an in vivo expression system in tardigrades, we first designed DNA plasmids based on the expression system for housekeeping genes that are highly expressed in the transcriptome of R. varieornatus: actin, ubiquitin, and tubulin. The genome size of R. varieornatus is relatively small, 56 Mbp, and the median length of intergenic regions is less than 1 kbp (SI Appendix, Fig. S1) (19, 39). Therefore, we extracted the 1-kbp sequence upstream and downstream of each target gene as the potential promoter and 3’UTR regions to activate and terminate transcription. The expression marker used was a monomeric enhanced green fluorescent protein (mEGFP) with a nuclear localization sequence (NLS) sequence tag at the C terminus (Fig. 1A), so it was expected that concentrated fluorescence would be observed in the nucleus even if the expression level was quite low. As a result of the introduction of the plasmid vector into tardigrades (Fig. 1B), we observed GFP fluorescence in tardigrades (Fig. 1C and SI Appendix, Fig. S2). Fluorescence in most cells was observed to spread throughout the cell, although it was most intensive in the nucleus; it is thought that this is due to the overflow from the nucleus. Injection of the plasmid solution was insufficient for successful in vivo expression, and electroporation or transfection reagents were necessary. The survival rate after microinjection was almost 100%, but 13% tardigrades died after electroporation, presumably depending on the amount of injected solution. In more than 80% of the tardigrades treated with the TardiVec, GFP fluorescence was observed after at least 24 h (SI Appendix, Table S1). We have so far observed GFP fluorescence in the muscle, epidermis, nervous system, storage cells, and ovary, and the fluorescence in muscle cells was more intense than that in other tissues. This is probably due to the difference in the efficiency of introduction, as well as the promoter activity in each tissue. The variation in expression among individuals possibly depends on the differing membrane damage during electroporation, where the amount of DNA injected affects electrical resistance of tardigrade individuals and the corresponding membrane damage. Injection of low-concentration plasmid solution had a low success rate, but the high-concentration mixture of the two plasmids showed equal expression even though each concentration was halved. Transfection reagents were functional in tardigrade cells, but the toxicity was not negligible, and there was a tendency for expression to occur only in the periphery of the injection site. The plasmid DNA was detected in the tardigrades that were kept for 10 d after introduction, although they were degraded gradually (SI Appendix, Fig. S3A). GFP fluorescence was observed and remained strong in the individuals on the 10th day: intensity was comparable to that at 48 h and 72 h after introduction (SI Appendix, Fig. S3B). When the mEGFP inserted downstream of the actin promoter was replaced with the calcium indicator GCaMP6s, we observed blinking of fluorescence in the muscle cells and neurons, i.e., calcium oscillation associated with muscle contraction and neural activity (Fig. 1D and Movie S1). The genetically encoded Ca2+ indicator can also function in tardigrades, and therefore, their in vivo calcium concentration is at a comparable level with other organisms (the Kd for Ca2+ of GCaMP6s is approximately 140 nM) (40). We also observed that the GCaMP signal increased during dehydration and even through after rehydration (Movie S2). In addition, the GCaMP signal was observed just before drying (at 00:21 of Movie S2), and it might indicate the increase in intracellular calcium concentration just before drying. TardiVec thus realized a direct observation of intracellular molecular changes in anhydrobiotic animals. Notably, GCaMP consists of circularly permuted GFP, with calcium-binding protein calmodulin at the C terminus and the calmodulin-interacting M13 peptide at the N terminus; therefore, its functional blinking indicates that the transcription and translation of the inserted gene with a length of 1,251 bp was completely continuous from the N-terminal domain to the C-terminal domain in the TardiVec system.
Fig. 1.
Tardigrade vector system enables in vivo expression in tardigrades. (A) Tardigrade vector named TardiVec. TardiVec consists of a promoter and terminator that originate from the genome of tardigrades. (B) Microinjection into tardigrades. To perform microinjection, a thin cover glass is affixed on the slide glass, and tardigrades are soaked in halocarbon oil with a small amount of water for fixation. (C) Tardigrade, R. varieornatus, expressing mEGFP-NLS under the actin promoter of R. varieornatus, pRvACT. White arrowheads indicate the mEGFP signal in muscle cells, and a red arrowhead indicates the signal in a neuron. (DR. varieornatus expressing GCaMP6s and mCherry with pRvACT. White arrowheads indicate the GCaMP signal in muscle cells, and a red arrowhead indicates the signal in a neuron. (E) Another anhydrobiotic tardigrade, H. exemplaris, expressing mEGFP-NLS under the actin promoter that originates from the genome of H. exemplaris. (F) R. varieornatus and H. exemplaris expressing mEGFP-NLS by interchange introduction of TardiVec. The merged images were obtained by adding transmission light to observe tardigrade body under the condition of observing GFP signal. (Scale bars, 100 μm.)
Movie S1.
GCaMP6s and mCherry. The left panel is a merged bright field displaying GCaMP6s (green) and mCherry (red). The right panel shows merged GCaMP6s (green) and mCherry (magenta) (2× speed). GCaMP6s signal appear in a neuron at the point of 00:09 of the Movie S1.
Movie S2.
A tardigrade expressing GCaMP6s was dehydrated and rehydrated (2× speed). Water was added to rehydrate the tardigrade at 00:24.
Similar to R. varieornatus we developed other DNA vectors based on the genome and transcriptome data of Hypsibius exemplaris, which is another anhydrobiotic tardigrade widely studied for development due to its transparency (41), another suitable characteristic for fluorescence observation. These two species are closely related, and both belong to the superfamily Hypsibioidea, but there is a fundamental difference in the mode of anhydrobiotic entry; R. varieornatus constitutively expresses anhydrobiosis-related proteins to cope with rapid desiccation, whereas H. exemplaris slowly prepares and expresses these proteins during a period called “preconditioning” upon the detection of water loss (42). Comparison between these two species is therefore important to understand the assembly of anhydrobiotic machinery and the evolution of anhydrobiosis. Although H. exemplaris has approximately twice the genome size of R. varieornatus, the median length of the intergenic region is only slightly longer at approximately 1,200 bp (SI Appendix, Fig. S1) (39); therefore, we adopted the same design as R. varieornatus in the in vivo expression of H. exemplaris. Actin and ef1α, which are highly expressed in H. exemplaris, were initially selected for the DNA vector. These promoters showed high expression of mEGFP ubiquitously in H. exemplaris (Fig. 1E and SI Appendix, Fig. S4). Interestingly, we observed that these vectors also functioned interchangeably in R. varieornatus, and vice versa, where the vectors of R. varieornatus also functioned seamlessly in H. exemplaris, even though the identity rate of the actin promoter between these two species is only approximately 50% (Fig. 1F and SI Appendix, Fig. S4).

Tissue-Specific Expression Patterns of Anhydrobiosis-related Genes Are Conserved in Anhydrobiotic Tardigrades.

Several tardigrade-specific genes have been discovered and then named based on their subcellular localization in human cultured cells, e.g., cytoplasm-localized CAHS1-3 and secretory SAHS1. They are IUPs highly expressed in tardigrades; therefore, they have been assumed to act as a “molecular shield” that has been hypothesized to be the main protective mode of anhydrobiotic protectants such as LEA proteins (43, 44). These proteins were previously assumed to function in concert; it is supposed that CAHS proteins protect the cytoplasmic components, while SAHS proteins protect the extracellular components, in coordination with other subcellular protectant proteins, MAHS and LEAM in mitochondria, and Dsup in the nucleus, altogether protecting all parts of the cells during anhydrobiosis (15, 1719, 38). To test this single-cell hypothesis, we developed vectors with promoters of CAHS3 (pRvCAHS3), which is most highly expressed among CAHS1-3, and SAHS1 (pRvSAHS1). As a result, tardigrades transfected with pRvCAHS3 plasmids showed intensive GFP signals in the epidermal tissue (Fig. 2A). Even more unexpectedly, tardigrades injected with the pRvSAHS1 plasmid showed intensive expression most exclusively in the storage cells (Fig. 2B) although some epidermal cells sometimes showed weak expression (SI Appendix, Fig. S5). Most cells expressing the mEGFP-NLS protein under the RvSAHS1 promoter leaked out of the body when pressed, confirming that these are free-floating cells (Movie S3, Upper). The CAHS gene family consists of the CAHS1/2 and CAHS3 subfamilies; to examine the expression pattern of another CAHS gene subfamily, CAHS1/2, plasmid vectors of CAHS1 were introduced into R. varieornatus. As a result, pRvCAHS1 showed the same expression pattern as pRvCAHS3 in epidermal cells (SI Appendix, Fig. S6A), suggesting that CAHS subfamilies possibly work together in the same cells rather than in different tissues. Tissue-specific expression of SAHS in storage cells was further confirmed, and this expression was validated directly through RNA sequencing of these storage cells because these cells could be collected by incision. The transcripts of the SAHS gene family were detected in much higher abundance compared with the whole body (Fig. 2C), and the fold change (×5 to 20) roughly corresponded to the ratio of the number of cells in the storage cells to the whole body (45), indicating predominant expression of SAHS in storage cells of tardigrades in their natural settings (i.e., without TardiVec transfection). It should also be noted that unlike CAHS where 11 out of 12 paralogs in H. exemplaris is significantly induced at least twofold after preconditioning, 6 out of 10 SAHS paralogs have extremely high expression (TPM > 1000) without preconditioning.
Fig. 2.
The tissue-specific expression of the tardigrade-specific genes CAHS and SAHS in R. varieornatus and H. exemplaris. (A) R. varieornatus expressing mEGFP-NLS under pRvCAHS3. (B) R. varieornatus expressing mEGFP-NLS under pRvSAHS1. (C) Comparison of transcripts of CAHS (red circle) and SAHS (purple circle) gene families between storage cells and the whole body of R. varieornatus (D) H. exemplaris expressing mEGFP-NLS under pRvCAHS3. (E) H. exemplaris expressing mEGFP-NLS under pRvSAHS1. (F) Comparison of transcripts of CAHS (red circle) and SAHS (purple circle) gene families between storage cells and the whole body of H. exemplaris without preconditioning. (G) T. ruffoi expressing mEGFP-NLS under pRvCAHS3. (E) T. ruffoi expressing mEGFP-NLS under pRvSAHS1. (I) M. inceptum expressing mEGFP-NLS under pRvCAHS3. D, E, G, H, and I are the results in heterologous expression experiment. The merged images were obtained by adding transmission light to observe tardigrade body under the condition of observing GFP signal. (Scale bars, 100 μm.)
Movie S3.
R. varieornatus and H. exemplaris expressing mEGFP-NLS under pRvSAHS1 (2× speed). In R. varieornatus, storage cells expressing mEGFP-NLS were ejected by pressure.
Surprisingly, in heterologous expression experiment, these two plasmids that consist of promoters of R. varieornatus also showed similar intense expression patterns in H. exemplaris in active state without any preconditioning, contrary to the premise that H. exemplaris requires a stimulation and preconditioning period to enhance the expression of anhydrobiosis-related genes (Fig. 2 DF and Movie S3, Lower). Furthermore, the aquatic tardigrade Thulinius ruffoi also expressed the pRvCAHS3 and pRvSAHS1 plasmids with similar tissue specificity (Fig. 2 G and H). T. ruffoi belongs to the order Parachela with R. varieornatus and H. exemplaris but is highly sensitive to desiccation, and therefore was expected to lack the capacity to express CAHS and SAHS. Additionally, the other order Apochela, Milnesium inceptum, which were collected from the field, also expressed mEGFP-NLS under pRvCAHS3 promoter (Fig. 2I). Moreover, it was found that pHeCAHS (BV898_02951, CAHS 86272), which is composed of the 5′ and 3′ regions of the most highly expressed CAHS gene in the anhydrobiotic state of H. exemplaris, functions not only in H. exemplaris but also in R. varieornatus in heterologous expression experiment (SI Appendix, Fig. S6B). Taken together, these results indicate that the 1-kbp sequence of the 5′ region of anhydrobiosis-related genes contains the conserved elements required for its transcription and tissue specificity, but not for the regulation of transcription, at least in terms of anhydrobiotic response or suppression thereof.

Subcellular Localization and Dynamics of Anhydrobiotic Proteins in Tardigrade Cells.

Next, to confirm the subcellular localization and dynamics of CAHS and SAHS proteins in actual tardigrade cells, we introduced plasmids in which mEGFP-NLS was replaced with intact proteins fused with mEGFP without NLS, such as CAHS3-mEGFP or SAHS1-mEGFP, into pRvCAHS3 or pRvSAHS1 vectors. The CAHS3-mEGFP is localized in the cytoplasm, and fewer signals were observed in the nucleus, which is the same outcome as that reported in human cultured HEK293T cells, while the mEGFP without NLS control localized throughout the cytosol and in the nucleus (Fig. 3A). To investigate the dynamics of these anhydrobiotic proteins when entering anhydrobiosis, we next observed tardigrades with pRvCAHS3-CAHS3-mEGFP without NLS introduced under desiccation. As a control, we prepared a vector to express only mEGFP without NLS and mCherry without NLS. Contrary to the report in cultured cells (27), we did not observe filament formation of CAHS3-mEGFP in tardigrade cells (Movie S4). Since dehydration of the specimen made the fluorescence observation challenging due to cell compression, we then tested osmotic stress by soaking the tardigrades in 0.1 M NaCl. Under this osmotic stress condition, CAHS3-mEGFP without NLS seemed to accumulate along membranes, sometimes forming a fiber-like assembly, while mCherry without NLS coexpressed in the same cells remained uniform (SI Appendix, Fig. S7), and mEGFP without NLS also did not show any distinct change. This result thus suggests that CAHS3-mEGFP is possibly associated with the membrane in achieving anhydrobiosis. In addition, both of CAHS3-mEGFP without NLS and the mEGFP without NLS showed inhomogeneous flow during observation of hydrated tardigrades (Movie S5), suggesting that the intracellular condition of tardigrades might be quite different from that of other experimental model organisms.
Fig. 3.
Subcellular localization of tardigrade-specific proteins fused with mEGFP in tardigrade cells. (A) Overexpression of CAHS3-mEGFP and mEGFP-only in R. varieornatus. (B) Overexpression of SAHS1-mEGFP and mCherry-NLS by cotransfection in R. varieornatus. (C) Observation of a punctured tardigrade expressing SAHS1-mEGFP and mCherry-NLS. White arrowheads indicate cells in which mEGFP and mCherry signals were merged. (Scale bars, 50 μm.)
Movie S4.
Dehydrated tardigrades expressing (A) CAHS3-mEGFP and (B) mEGFP were rehydrated (16× speed).
Movie S5.
Dynamics of (A) CAHS3-mEGFP without NLS and (B) mEGFP without NLS in tardigrade cytosol (4× speed). GFP signal flow inhomogeneously at 00:04 to 00:10 in A, the left, and at 00:14 to 00:20 in B the right.
On the other hand, SAHS1-mEGFPs without NLS were observed in the vesicle-like structure in the storage cells, while in some individuals, GFP signals were observed to spread throughout the body cavity (Fig. 3B and SI Appendix, Fig. S8A). This pattern is similar to the previous heterologous expression observed in HEK293T cells, in which weak signals in secretory pathways such as the endoplasmic reticulum or the Golgi apparatus were observed and most SAHS1-mEGFP was detected from the culture media. The fact that most SAHS1-mEGFPs were retained in the storage cells in tardigrades suggests differences in the modes of secretion and storage regulation between tardigrades and human cells, especially since the cell type is unique to tardigrades. Localization of SAHS1-mEGFP in the storage cell was again confirmed by leaking the cell contents out by puncture (Fig. 3C). These results demonstrated that the CAHS proteins localized in the cytoplasm of the epidermal tissue, and the SAHS proteins were stored in vesicles of the body cavity cells in tardigrades. Moreover, CAHS3-mEGFP and SAHS1-mEGFP that are originated from R. varieornatus also drive similar subcellular localization in H. exemplaris (SI Appendix, Fig. S8 B and C).

Discussion

Inception of Live Imaging in Anhydrobiotic Tardigrades.

Tardigrada is one of the phyla in Ecdysozoa. There are model organisms with modern genetic toolkits around the phylum Tardigrada in the ecdysozoan tree of life, such as C. elegans and D. melanogaster. Such an experimental model species was lacking in Tardigrada, even though the phenomenon of anhydrobiosis in tardigrades has been enthralling researchers for decades. Following the establishment of the stable culture system, complete genome sequences have been published for R. varieornatus and H. exemplaris over the past decade, and as a result, several tardigrade-specific genes have been discovered to contribute to anhydrobiotic machinery. Our transient transgenic technology with TardiVec achieves prolonged expression in various tissues across different orders within Tardigrada, with the capability to cotransfect multiple vectors at the same time. It is therefore expected to contribute not only to the study of these anhydrobiosis-related proteins, but also to various studies of tardigrades, including development, physiology, and ethology. Transgenic technology for tardigrades facilitates anhydrobiosis researches using tardigrades as the model organism.
Our transgenic approach easily enables the introduction of live imaging with tags (e.g., GFP) or indicators (e.g., GCaMP). Fluorescent proteins fused with target proteins possibly enable us to reveal not only the subcellular localization but also the protein dynamics during dehydration and rehydration without sample loss. For example, GCaMP will reveal neural activity and networks, building on the tardigrade anatomy that was revealed through IHC and electron microscopy imaging (2936, 46, 47).
Of note, the TardiVec can work beyond the genus, the family, and the order, indicating that it can be readily applied to field-collected samples that have no precise genome data and adequate rearing systems. Indeed, we succeeded in the introduction of our vector plasmids that originated from R. varieornatus to anhydrobiotic tardigrades of the other order, Apochela, M. inceptum, which were collected from the field. The class Heterotardigrada, in which there is no isolated rearing system established yet for any of the species, has only a draft genome sequence of Echiniscus testudo (48). The same vector system based on this species should also work across orders within heterotardigrades, opening up new frontiers in Tardigradology.
As shown in SI Appendix, Fig. S3, vector plasmids and GFP fluorescence were maintained for 10 d, suggesting that there is no active degradation mechanism by nucleases. Since cell division rarely occurs in tardigrades (49), plasmids may not be lost by cell division as they are in cultured cells. Moreover, the half-life of GFP as a protein is approximately 26 h (50); therefore, it is reasonable to assume that the plasmid vector is functional up to 10 d after introduction. Although the length of the open reading frame is relatively short in R. varieornatus, the vector system could transcribe more than 1.6 kbp of product as reported here (CAHS3-mEGFP). We conducted cotransfection using two distinct vectors (e.g., pRvCAHS3-CAHS3-mEGFP and pRvCAHS3-mCherry), and these two fluorescence signals were observed in almost entirely the same cells. This is possibly because plasmid DNA is imported selectively into cells with sufficient membrane damage by electroporation.
Recently, a CRISPR/Cas9 system functioning with gRNA and Cas9 proteins was reported in tardigrade somatic cells (51). This system unfortunately did not seem to function in the germline, which is necessary to obtain a mutant strain by gene editing. Introduction of constructs to germlines is also a rare event in our method, but the TardiVec system will possibly provide another approach to deliver the gene-editing system to the germline, paving the way to produce gene-edited strains in tardigrades.

Conservation and Diversity of the Expression Mechanism of Genes Constituting Anhydrobiotic Machinery.

Our present study showed that vectors derived from tardigrade-specific genes, i.e., CAHS and SAHS, successfully expressed inserted genes in R. varieornatus, H. exemplaris, and T. ruffoi. These results indicate that the 1-kb sequence upstream and downstream of each gene contains functional promoter and terminator sequences for constitutively expressing them with tissue specificity. The RvCAHS genes are constitutively expressed, while the expression of HeCAHS genes is induced only after dehydration cues; e.g., the fold change of HeCAHS is ×20 to ×500 compared with the normal condition. Therefore, the pHeCAHS vector was expected to be nonfunctional under normal conditions of H. exemplaris, but such suppression was not observed and preconditioning treatment did not enhance the expression (SI Appendix, Fig. S9); therefore, this result suggests that the regulation of the expression of these genes might be suppressed in trans by repressor binding on distal regions and/or chromosomal regulation, partially suggested by the fact that these anhydrobiotic genes often colocalize within the genome in tandem. Moreover, this kind of global or metaregulation of anhydrobiosis-related genes is in line with the fact that the shift from rapid anhydrobiosis to preconditioning or the loss of anhydrobiotic ability in Tardigrada seems to occur in multiple phylogenetic lineages and is a recurring adaptive strategy to cope with xeric, mesic, and aquatic environments (16). It is obviously more energetically cost effective to only require the change in global regulation rather than evolving different transcription factor-promoter network rewiring for all related genes. Why is CAHS or SAHS expression conserved in T. ruffoi, which seemingly lost its anhydrobiotic capability? One possibility is another dormant state of tardigrades, which is known to be present in T. ruffoi, to form a cyst, an adaptive state to withstand unfavorable environmental conditions, although this corresponds with loss of anhydrobiotic ability (52). The remaining promoter activity for anhydrobiosis-related genes in T. ruffoi allowed us to speculate regarding the possibility that the two dormant states of tardigrades, anhydrobiosis and encystment, are in fact somewhat related although there could be other possibilities such as promoter evolution in T. ruffoi.

Tissue-Specific Expression Pattern of Tardigrade-Specific Proteins Suggests Systematic Organismal Anhydrobiosis Machinery.

Our study revealed that the expression of tardigrade-specific genes varied in specific tissues, although it had been assumed that all cells express and produce equally the full set of anhydrobiotic genes to protect their own in previous idea (15, 1719, 38). This single-cell hypothesis is based on the experimental results on human cultured cells; CAHS proteins localized in the cytosol and SAHS proteins were secreted into the medium (17, 18). As our results showed, these characteristics of subcellular localization in human cells are coincident with those in tardigrade cells, as was also reported for the subcellular localizations of RvLEAM and Dsup proteins by IHC of tardigrade embryos. The tissue-specific expression of these genes was revealed by whole-body observation with our transgenic approach, suggesting a more complex and systematic organismal anhydrobiosis machinery with a specialized set of proteins functioning in each of the specific tissue types. Of course, this does not rule out the fact that some secretory proteins are produced in a specific cell type but are spread throughout the body. In sleeping chironomids, trehalose, the main protectant molecule, is synthesized in the fat body, secreted to body fluid, and eventually spread throughout the whole body. Similarly, it is reasonable to assume that SAHS proteins are synthesized in storage cells and are secreted into the body cavity. Moreover, it is interesting that in most tardigrade samples, SAHS proteins seemed to remain in storage cells in our observation, whereas in rare cases, SAHS proteins were also observed to be present in the body cavity (SI Appendix, Fig. S8A). This suggests a regulatory mechanism governing secretion from cells in addition to transcription and translation. Additionally, since not all cells are in contact with the body cavity, it is possible that other mechanisms exist to protect the outside of the cells.
Although the single-cell hypothesis is attractive for establishing technologies for dry preservation of cells or other biological materials, the actual anhydrobiotic mechanism of tardigrades appears to be more complex; CAHS proteins mainly function in the epidermis, and SAHS proteins are produced in storage cells and possibly transported to other cells via the body cavity. Tissue-specific expression of each anhydrobiotic gene allows us to speculate with respect to the existence of other candidate proteins for accomplishing anhydrobiosis in various cell types. Tardigrade genomes are found to code multiple IUPs, and dozens of IUPs are highly expressed in R. varieornatus and induced in H. exemplaris, similar to CAHS and SAHS genes (15); it is reasonable to assume that they are candidates as protectants of other cell types. In addition, the tissue specificity forces us to reconsider the sufficient amounts of protectants for understanding anhydrobiosis correctly.
Expression of the CAHS3 gene via TardiVec was most often observed in epidermal cells. Since there are more than 10 paralogs of CAHS genes, there is still a possibility that the other paralogs of the CAHS family are expressed in other tissue types, or that all CAHS proteins may have a specific role in epidermal cells. Since the variety of CAHS paralogs makes us speculate that CAHS proteins are used differently based on their characteristics, future studies should analyze in more detail the differences in CAHS subfamilies and their tissue specificity. In addition, difficulty was encountered in observing the fluorescence of dehydrated samples due to insufficient light penetration and possible change in refractive index, but nevertheless, fluorescence observation is possible in the process of dehydration and rehydration. It has also been observed that GFP expressed in tardigrade cells exhibits LLPS-like dynamics during the dehydration process, suggesting that the condition of the cytoplasm might differ from that of other organisms. The fact that it became possible to observe the live cells of anhydrobiotic animals in vivo has enabled us to examine various possibilities to understand life without water.
To conclude, we developed a vector-based in vivo expression system consisting of tardigrade-specific promoters, with a vector named the TardiVec. This technology enabled live imaging in several tardigrades in the class Eutardigrada, and the basic concept should be directly applicable for heterotardigrades as well. With this system, the dynamics of proteins and cells were revealed through observation using fluorescent tags and indicators in anhydrobiotic tardigrades. Live imaging would open new doors in the study of anhydrobiosis and make it possible to utilize tardigrades as a model system for anhydrobiology, morphology, physiology, and other various fields of biology.

Materials and Methods

Animals.

The YOKOZUNA-1 strain of R. varieornatus, the Z151 strain of H. exemplaris, previously described as Hypsibius dujardini (53), and the PL.014 strain of T. ruffoi were used. These strains were maintained on water-layered agar plates by feeding algae Chlorella vulgaris (Chlorella Industry) (4, 41). M. inceptum were collected from the field (Tsuruoka City, Yamagata, Japan, 38.74615685138436, 139.82520286521836) and kept on an agar gel plate likewise R. varieornatus and H. exemplaris, but rotifer Lecane inermis was added as food.

Design of the Vector System and Preparation of the Injection Solution.

TardiVec was designed to consist of a promoter, mEGFP, 3′UTR, selection marker, and a replication origin for amplification in E. coli. The promoter and 3′UTR regions correspond to 1 kbp DNA sequences located before and after an open reading frame, respectively. The expression marker is a mEGFP fused with the NLS (PKKKRKV), and thus it facilitates the observation of fluorescence signals by concentrating them in the nucleus. The selection marker was ampicillin. Promoter, mEGFP and 3′UTR fragments were amplified by PrimeSTAR Max DNA polymerase (Takara). The replication origin and selection marker were derived from a mammalian expression vector, pCAGGS. pCAGGS was digested by SpeI and BamHI and assembled with the PCR products by an In-Fusion HD cloning system (Clontech). After transformation into DH5α, the amplified plasmids were extracted by a Plasmid Maxi Kit (QIAGEN) to a concentration of approximately 2 μg/μL. The resulting pellets were eluted with pure water.

Introduction of Plasmids to Tardigrades via Microinjection.

Glass capillaries (GD-1, NARISHIGE) were pulled by a puller (PC-100, NARISHIGE); the temperatures were set at 66.2 and 62.0 °C. Our microinjection system consists of an inverted microscope (AXIO Vert. A1, Zeiss) equipped with an injector (IM-31, NARISHIGE) and manipulators (MMN-1 and MHW-103, NARISHIGE). Tardigrades were mounted as described previously by Tenlen et al. for RNAi experiments without anesthesia (Fig. 1B) (54, 55). After the microinjections, individuals were collected and transferred to a cuvette (CUY505P5, NEPA GENE) for electroporation using a super electroporator (NEPA21 type 2, NEPA GENE). The poring pulse was emitted twice at 250 V for 5 ms with a 50-ms interval, and the transfer pulse was emitted five times at 30 V for 50 ms with a 50-ms interval. Tardigrades were maintained with algal food on an agar plate until observation at 17 °C.

Observation of Fluorescence in Tardigrades.

Tardigrades were sandwiched between cover glasses for observation. Polystyrene particles were sometimes used to maintain the distance between the glasses. The bright field and fluorescence images were obtained using an IX73 inverted microscope with a DP74 camera adapter (Evident (Olympus)). LUCPLFLN 20X PH and LUCPLFLN 40X PH lenses (Evident (Olympus)) were used. For observation of GCaMP6s and subcellular localization, a TCS SP8 confocal laser scanning microscope (Leica Microsystems) equipped with HC PL APO CS2 20×/0.75, HC PL APO CS2 63×/1.20, and HC PL APO CS2 100×/1.40 objectives (Leica Microsystems) was used. Images were analyzed with ImageJ2 and Fiji.

Detection of Plasmid DNA from Transfected Tardigrade Samples.

Tardigrades transfected with pRvACT-mEGFP-NLS were kept for 10 d, and 10 individuals were collected at each time point as samples. Tardigrade samples were stored at −80 °C until extraction treatment. An RNeasy mini kit (Qiagen) was used for extraction of the total nucleic acid. Amplification of target fragments was performed by PrimeSTAR Max DNA Polymerase (Takara).

RNA Sequencing.

Storage cells were obtained by dissecting tardigrades in 0.1× PBS under a stereomicroscope with a sterile knife and quickly aspirating the cells that were released. Collected cells, as well as active individuals as controls for the whole body, were used to prepare RNA-Seq libraries using the SMART-Seq v.4 Kit (Clontech) as previously described (56). Single-end sequencing was performed with a NextSeq 500 with a High Output Mode 75 Cycles Kit (Illumina). Expression levels were computed as transcripts per million (TPM) using kallisto software (v.0.46.1) (57). The RNA-Seq data as well as computed abundances for each gene obtained were deposited into NCBI GEO under the accession ID GSE212632.

q-PCR.

H. exemplaris were introduced pHeCAHS-mEGFP-NLS, and incubated on an ager plate with algae solution at 17 °C for 24 h. Then, 200 μL pure water containing live tardigrades was put on a nylon net filter (Millipore, USA; pore size 11 μm, 25 mm diameter) placed on a filter paper (GE Healthcare, UK; 25 mm diameter) in a 35-mm plastic dish. The dish was transferred to 95% RH container that contained saturated solution of potassium nitrate. After 24 h’ incubation, tardigrades with preconditioning were harvested. Control samples were live tardigrades that kept on an ager plate with pure water at 17 °C for 24 h. Each batch contained 7 individuals and all samples were kept at −80 °C just after sampling. RNA was extracted from each batch of tardigrades using the Direct-zol RNA MicroPrep Kit (Zymo Research) after homogenizing manually using BioMasher II (Nippi), and were reverse transcribed with the PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio). qPCR reaction was performed on LightCycler 96 System (Roche) with the KAPA SYBR FAST qPCR Master Mix Kit (KAPA BioSystems) according to the manufacturer’s protocol, including amplification cycle conditions. Primer used for mEGFP was 5′-AGAACGGCATCAAGGTGAAC-3′ and 5′-TGCTCAGGTAGTGGTTGTCG-3′, and that for actin was 5′-CAGGGAAAAGATGACCCAGA-3′ and 5′-GAGGCAGAGCATAACCTTCG
-3′. Relative abundance of actin and mEGFP was calculated by delta CT method.

Data, Materials, and Software Availability

Gene expression data have been deposited in NCBI GEO (GSE212632).

Acknowledgments

We are grateful to Esraa Hassan Ahmed Youssef for thoroughly conducting experiments associated with plasmid vectors. Naoko Ishii, Ayako Shirahata, Yuki Takai, and Takahiro Bino provided technical assistance. The C. vulgaris used to feed the tardigrades was provided courtesy of Chlorella Industry. This work is supported by KAKENHI Grant-in-Aid for Transformative Research Areas (A), Grant-in-Aid for Early-Career Scientists, and Grant-in-Aid for Challenging Research (Exploratory) from the Japan Society for the Promotion of Science (JSPS, grant Numbers 21H05279, 20K15781, and 22K19302), Joint Research by Exploratory Research Center on Life and Living Systems (ExCELLS program Nos. 19-208, 19-501, and 22EXC601) and partly by research funds from the Yamagata Prefectural Government and Tsuruoka City, Japan.

Author contributions

S.T. and K. Arakawa designed research; S.T. and K. Arakawa performed research; S.T., K. Aoki, and K. Arakawa contributed new reagents/analytic tools; S.T. and K. Arakawa analyzed data; S.T., K. Aoki, and K. Arakawa writing - Review & Editing; and S.T. and K. Arakawa wrote the paper.

Competing interest

The authors declare no competing interest.

Supporting Information

Appendix 01 (PDF)
Movie S1.
GCaMP6s and mCherry. The left panel is a merged bright field displaying GCaMP6s (green) and mCherry (red). The right panel shows merged GCaMP6s (green) and mCherry (magenta) (2× speed). GCaMP6s signal appear in a neuron at the point of 00:09 of the Movie S1.
Movie S2.
A tardigrade expressing GCaMP6s was dehydrated and rehydrated (2× speed). Water was added to rehydrate the tardigrade at 00:24.
Movie S3.
R. varieornatus and H. exemplaris expressing mEGFP-NLS under pRvSAHS1 (2× speed). In R. varieornatus, storage cells expressing mEGFP-NLS were ejected by pressure.
Movie S4.
Dehydrated tardigrades expressing (A) CAHS3-mEGFP and (B) mEGFP were rehydrated (16× speed).
Movie S5.
Dynamics of (A) CAHS3-mEGFP without NLS and (B) mEGFP without NLS in tardigrade cytosol (4× speed). GFP signal flow inhomogeneously at 00:04 to 00:10 in A, the left, and at 00:14 to 00:20 in B the right.

References

1
J. H. Crowe, F. A. Hoekstra, L. M. Crowe, Anhydrobiosis. Annu. Rev. Physiol. 54, 579–599 (1992).
2
P. Becquerel, La suspension de la vie au dessous de 1/20 K absolu par demagnetization adiabatique de l’alun de fer dans le vide les plus eléve. C. R. Hebd. Seance Acad. Sci. 231, 261–263 (1950).
3
F. Ono et al., Effect of ultra-high pressure on small animals, tardigrades and Artemia. Cogent Physics 3, 1167575 (2016).
4
D. D. Horikawa et al., Establishment of a rearing system of the extremotolerant tardigrade Ramazzottius varieornatus: A new model animal for astrobiology. Astrobiology 8, 549–556 (2008).
5
T. Altiero, R. Guidetti, V. Caselli, M. Cesari, L. Rebecchi, Ultraviolet radiation tolerance in hydrated and desiccated eutardigrades. J. Zoolog. Syst. Evol. Res. 49, 104–110 (2011).
6
R. M. May, M. Maria, J. Gumard, Action différentielle des rayons x et ultraviolets sur le tardigrade Macrobiotus areolatus, a l’état actif et desséché. Bull. Biol. Fr. Belg. 98, 349–367 (1964).
7
E. Beltrán-Pardo et al., Effects of ionizing radiation on embryos of the tardigrade Milnesium cf. tardigradum at different stages of development. PLoS One 8, e72098 (2013).
8
E. Beltrán-Pardo, K. I. Jönsson, M. Harms-Ringdahl, S. Haghdoost, A. Wojcik, Tolerance to gamma radiation in the tardigrade Hypsibius dujardini from embryo to adult correlate inversely with cellular proliferation. PLoS One 10, e0133658 (2015).
9
K. I. Jönsson, A. Wojcik, Tolerance to X-rays and heavy ions (Fe, He) in the tardigrade Richtersius coronifer and the bdelloid rotifer Mniobia russeola. Astrobiology 17, 163–167 (2017).
10
K. Ingemar Jönsson, M. Harms-Ringdahl, J. Torudd, Radiation tolerance in the eutardigrade Richtersius coronifer. Int. J. Radiat. Biol. 81, 649–656 (2005).
11
E. J. Charlotta Nilsson, K. Ingemar Jönsson, J. Pallon, Tolerance to proton irradiation in the eutardigrade Richtersius coronifer–A nuclear microprobe study. Int. J. Radiat. Biol. 86, 420–427 (2010).
12
D. Persson et al., Extreme stress tolerance in tardigrades: Surviving space conditions in low earth orbit. J. Zoolog. Syst. Evol. Res. 49, 90–97 (2011).
13
K. I. Jönsson, E. Rabbow, R. O. Schill, M. Harms-Ringdahl, P. Rettberg, Tardigrades survive exposure to space in low Earth orbit. Curr. Biol. 18, R729–R731 (2008).
14
L. Rebecchi et al., Tardigrade resistance to space effects: First results of experiments on the LIFE-TARSE mission on FOTON-M3 (September 2007). Astrobiology 9, 581–591 (2009).
15
K. Arakawa, Examples of extreme survival: Tardigrade genomics and molecular anhydrobiology. Annu. Rev. Anim. Biosci. 10, 17–37 (2022).
16
Y. Yoshida, S. Tanaka, Deciphering the biological enigma—Genomic evolution underlying anhydrobiosis in the phylum Tardigrada and the chironomid Polypedilum vanderplanki. Insects 13, 557 (2022).
17
A. Yamaguchi et al., Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. PLoS One 7, e44209 (2012).
18
S. Tanaka et al., Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells. PLoS One 10, e0118272 (2015).
19
T. Hashimoto et al., Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein. Nat. Commun. 7, 12808 (2016).
20
J. Kirke Analysis of tardigrade damage suppressor protein (dsup) expressed in tobacco, search.proquest.com. Master thesis, Florida Atlantic University, Boca Raton, FL (2019).
21
J. Kirke, X.-L. Jin, X.-H. Zhang, Expression of a tardigrade Dsup gene enhances genome protection in plants. Mol. Biotechnol. 62, 563–571 (2020).
22
T. C. Boothby et al., Tardigrades use intrinsically disordered proteins to survive desiccation. Mol. Cell 65, 975–984.e5 (2017).
23
M. Yagi-Utsumi et al., Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade. Sci. Rep. 11, 1–9 (2021).
24
A. Malki et al., Intrinsically disordered tardigrade proteins self-assemble into fibrous gels in response to environmental stress. Angew. Chem. Int. Ed Engl. 61, e202109961 (2021), https://doi.org/10.1002/anie.202109961.
25
C. S. Hesgrove, Tardigrade CAHS proteins act as molecular swiss army knives to mediate desiccation tolerance through multiple mechanisms. bioRxiv [Preprint] (2021), 2021.08.16.456555 (Accessed 16 April 2022).
26
M. T. Veling et al., Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells. ACS Synth. Biol. 11, 1292–1302 (2022).
27
A. Tanaka et al., Stress-dependent cell stiffening by tardigrade tolerance proteins that reversibly form a filamentous network and gel. PLoS Biol. 20, e3001780 (2022).
28
C. Belott, B. Janis, M. A. Menze, Liquid-liquid phase separation promotes animal desiccation tolerance. Proc. Natl. Acad. Sci. U.S.A. 117, 27676–27684 (2020).
29
W. N. Gabriel, B. Goldstein, Segmental expression of Pax3/7 and engrailed homologs in tardigrade development. Dev. Genes Evol. 217, 421–433 (2007).
30
V. Gross, L. Epple, G. Mayer, Organization of the central nervous system and innervation of cephalic sensory structures in the water bear Echiniscus testudo (Tardigrada: Heterotardigrada) revisited. J. Morphol. 282, 1298–1312 (2021).
31
L. Hering, J.-E. Bouameur, J. Reichelt, T. M. Magin, G. Mayer, Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades. Elife 5, e11117 (2016).
32
C. Schulze, A. Schmidt-Rhaesa, The architecture of the nervous system of Echiniscus testudo (Echiniscoidea, Heterotardigrada). J. Limnol. 72, e6 (2013).
33
C. Schulze, A. Schmidt-Rhaesa, Organisation of the musculature of Batillipes pennaki (Arthrotardigrada, Tardigrada). Meiofauna Marina 19, 195–207 (2011).
34
F. W. Smith, B. Goldstein, Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda. Arthropod Struct. Dev. 46, 328–340 (2017).
35
F. W. Smith, P. J. Bartels, B. Goldstein, A hypothesis for the composition of the tardigrade brain and its implications for panarthropod brain evolution. Integr. Comp. Biol. 57, 546–559 (2017).
36
K. A. Halberg, D. Persson, N. Møbjerg, A. Wanninger, R. M. Kristensen, Myoanatomy of the marine tardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae). J. Morphol. 270, 996–1013 (2009).
37
J. Nakai, M. Ohkura, K. Imoto, A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).
38
J. D. Hibshman, J. S. Clegg, B. Goldstein, Mechanisms of desiccation tolerance: Themes and variations in brine shrimp, roundworms, and tardigrades. Front. Physiol. 11, 592016 (2020).
39
Y. Yoshida et al., Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus. PLoS Biol. 15, e2002266 (2017).
40
T.-W. Chen et al., Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
41
W. N. Gabriel et al., The tardigrade Hypsibius dujardini, a new model for studying the evolution of development. Dev. Biol. 312, 545–559 (2007).
42
K. Kondo, T. Kubo, T. Kunieda, Suggested involvement of PP1/PP2A activity and de novo gene expression in anhydrobiotic survival in a tardigrade, Hypsibius dujardini, by chemical genetic approach. PLoS One 10, e0144803 (2015).
43
A. Tunnacliffe, M. J. Wise, The continuing conundrum of the LEA proteins. Naturwissenschaften 94, 791–812 (2007).
44
S. Chakrabortee et al., Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc. Natl. Acad. Sci. U.S.A. 104, 18073–18078 (2007).
45
I. Giovannini et al., Production of reactive oxygen species and involvement of bioprotectants during anhydrobiosis in the tardigrade Paramacrobiotus spatialis. Sci. Rep. 12, 1938 (2022).
46
M. Richaud et al., Ultrastructural analysis of the dehydrated tardigrade Hypsibius exemplaris unveils an anhydrobiotic-specific architecture. Sci. Rep. 10, 4324 (2020).
47
V. Gross, I. Minich, G. Mayer, External morphogenesis of the tardigrade Hypsibius dujardini as revealed by scanning electron microscopy. J. Morphol. 278, 563–573 (2017).
48
Y. Murai et al., Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada. BMC Genomics 22, 813 (2021).
49
V. Gross, R. Bährle, G. Mayer, Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog. Tissue Cell 51, 77–83 (2018).
50
P. Corish, C. Tyler-Smith, Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12, 1035–1040 (1999).
51
H. Kumagai, K. Kondo, T. Kunieda, Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades. Biochem. Biophys. Res. Commun. 623, 196–201 (2022).
52
K. Janelt, I. Poprawa, Analysis of encystment, excystment, and cyst structure in freshwater Eutardigrade Thulinius ruffoi (Tardigrada, Isohypsibioidea: Doryphoribiidae). Diversity 12, 62 (2020).
53
P. Gasiorek, D. Stec, W. Morek, Ł Michalczyk, An integrative redescription of Hypsibius dujardini (Doyère, 1840), the nominal taxon for Hypsibioidea (Tardigrada: Eutardigrada). Zootaxa 4415, 45–75 (2018).
54
J. R. Tenlen, S. McCaskill, B. Goldstein, RNA interference can be used to disrupt gene function in tardigrades. Dev. Genes Evol. 223, 171–181 (2013).
55
J. R. Tenlen, Microinjection of dsRNA in Tardigrades. Cold Spring Harb. Protoc. 2018, https://doi.org/10.1101/pdb.prot102368 (2018).
56
K. Arakawa, Y. Yoshida, M. Tomita, Genome sequencing of a single tardigrade Hypsibius dujardini individual. Sci. Data 3, 160063 (2016).
57
N. L. Bray, H. Pimentel, P. Melsted, L. Pachter, Erratum: Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 888 (2016).

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 120 | No. 5
January 31, 2023
PubMed: 36693101

Classifications

Data, Materials, and Software Availability

Gene expression data have been deposited in NCBI GEO (GSE212632).

Submission history

Received: September 30, 2022
Accepted: December 21, 2022
Published online: January 24, 2023
Published in issue: January 31, 2023

Keywords

  1. anhydrobiosis
  2. tardigrades
  3. in vivo expression
  4. live imaging

Acknowledgments

We are grateful to Esraa Hassan Ahmed Youssef for thoroughly conducting experiments associated with plasmid vectors. Naoko Ishii, Ayako Shirahata, Yuki Takai, and Takahiro Bino provided technical assistance. The C. vulgaris used to feed the tardigrades was provided courtesy of Chlorella Industry. This work is supported by KAKENHI Grant-in-Aid for Transformative Research Areas (A), Grant-in-Aid for Early-Career Scientists, and Grant-in-Aid for Challenging Research (Exploratory) from the Japan Society for the Promotion of Science (JSPS, grant Numbers 21H05279, 20K15781, and 22K19302), Joint Research by Exploratory Research Center on Life and Living Systems (ExCELLS program Nos. 19-208, 19-501, and 22EXC601) and partly by research funds from the Yamagata Prefectural Government and Tsuruoka City, Japan.
Author Contributions
S.T. and K. Arakawa designed research; S.T. and K. Arakawa performed research; S.T., K. Aoki, and K. Arakawa contributed new reagents/analytic tools; S.T. and K. Arakawa analyzed data; S.T., K. Aoki, and K. Arakawa writing - Review & Editing; and S.T. and K. Arakawa wrote the paper.
Competing Interest
The authors declare no competing interest.

Notes

This article is a PNAS Direct Submission. B.G. is a guest editor invited by the Editorial Board.

Authors

Affiliations

Institute for Advanced Biosciences, Keio University, Tsuruoka, 997-0017, Japan
Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, 444-8787, Japan
Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, 444-8787, Japan
National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, 444-8787, Japan
Faculty of Life Science, Graduate University for Advanced Studies, Okazaki, 444-8787, Japan
Institute for Advanced Biosciences, Keio University, Tsuruoka, 997-0017, Japan
Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, 444-8787, Japan
Graduate School of Media and Governance, Keio University, Fujisawa, 252-0882, Japan
Faculty of Environment and Information Studies, Keio University, Fujisawa, 252-0882, Japan

Notes

1
To whom correspondence may be addressed. Email: [email protected] or [email protected].

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    In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada
    Proceedings of the National Academy of Sciences
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