Abstract

Aim

   Given its excellent capability of escaping from unavoidable harm and
   death, Turritopsissp. (T. sp.) has captured the attention and
   fascination of scientists as a less conventional tool for aging
   research. The current study introduces a method for establishment of a
   research model and comprehensive transcriptomic analysis to reveal the
   structural and functional diversity of T. sp.

Methods

   T. sp. medusae collected from the Pacific Ocean near Japan were reared
   using a common laboratory setting. Tissues of the gastrovascular cavity
   part (GP) and nerve ring part (NP) were collected, and total RNA was
   extracted. Bulk RNA-seq was performed to compare the different
   transcriptome landscapes between GP and NP.

Results

   The GP fragment could be utilized for studies related to stress
   response and systemic senescence, while the NP fragment could be used
   to explore system rejuvenation, self-repair and regeneration.

Conclusions

   As a less conventional system for aging research, by employing the most
   recently developed tools and techniques in the genomic revolution,
   comprehensive elucidation of the composition, development, and
   functions of T. sp. enabled us to explore the underlying mechanisms of
   the response to environmental stress and rejuvenation.

   Keywords: Aging, Immortal jellyfish, Regeneration, RNA-seq, Turritopsis
   sp.

Highlights

     * •
       Turritopsis sp is known as an "immortal jellyfish".
     * •
       It effectively bypass death and restart its life cycle.
     * •
       Tissue and functional diversity were comprehensively elucidated
       using RNA-seq.
     * •
       They can be utilized for study related to systemic senescence and
       regeneration.
     * •
       Turritopsis sp could be employed as a less conventional system for
       aging study.

1. Introduction

   As a representative of hydrozoans within the phylum Cnidaria,
   Turritopsis, including Turritopsis dohrnii and Turritopsis sp. (T.
   sp.), commonly known as the "immortal jellyfish", is a species of
   small, bell-shaped jellyfish. They display the greatest adaptability
   with respect to behaviors in response to environmental stress and their
   antiaging strategy. When encountering aging, injury, or other adverse
   environmental conditions, the medusa form shrinks, loses its swimming
   ability and undergoes retrograde transformation to a protective, poorly
   differentiated cyst-like structure, which eventually gives rise to a
   preceding juvenile morph, the polyp [[29]1]. These behaviors thus
   result in effectively bypassing death and allowing it to restart its
   life cycle, which is known as “rejuvenation” [[30]2].

   The basic Turritopsis life cycle proceeds from the adult medusa to the
   planula larva, and to the polyp, which is an asexual and often colonial
   stage [[31]3]. The structure of the adult medusa includes a
   gastrovascular cavity with a set of manubrium, oral lobes, radical
   canal, and gonad inside, and a velum surrounded by a nerve ring with
   tentacles [[32]4,[33]5]. The body wall of the gastrovascular cavity
   consists of an outer epidermis, outer mesoglea, endoderm, inter
   mesoglea with the formation of a gastral cavity, radial canals and ring
   canal. A layer of mononucleated, striated muscle cells and a smooth
   muscle layer are also observed in the mature medusa. A polyp could not
   regenerate from an isolated manubrium and tentacles alone, whereas
   excised manubrium and tentacles additionally containing the
   tissue/cells around the velum with nerve ring maintained the capability
   of stolon formation.

   Given its excellent capability of escaping from unavoidable harm and
   death, Turritopsis has captured the attention and fascination of
   scientists as a less conventional tool for examining factors governing
   regeneration, aging, and immortality. By employing the most recently
   developed tools and techniques in the genomic revolution, such as
   next-generation sequencing, single-cell/molecular sequencing, spatial
   transcriptomics, multi-omics integration, and CRISPR-Cas9 genome
   editing [[34]6,[35]7], it has been possible to comprehensively
   elucidate the composition, development, and functions of Turritopsis,
   enabling us to explore the underlying mechanism of the response to
   environmental stress and rejuvenation. In the current study, a basic
   method for comparative transcriptome analysis of the upper half of the
   medusa without the nerve ring and the lower half of the medusa
   including the nerve ring is introduced. The transcriptomic landscape
   revealed the structural and functional diversity of different fragments
   of T. sp., a Turritopsis medusa which was collected from the Pacific
   Ocean near Japan, in particular focusing on the study of aging.

2. Methods

2.1. Rearing

   About 100 T. sp. medusae were collected on August 2022 in Tanabe Bay,
   Japan (33.68’E, 135.36’N) and reared in the laboratory at 25 °C in a
   500 ml beaker and fed every other day with newly hatched Artemia
   salina. One hour after feeding, half of the seawater was removed and
   replaced with freshly prepared seawater.

   Long-term rearing for more than 1 month can be challenging and requires
   additional specialized water circulation equipment. A gentle circular
   flow pattern is required to prevent damage to the T. sp. and is able to
   create a gentle current and promote water exchange in a small tank. A
   combination of sponge filters, protein skimmers and activated carbon
   filter was used for the rearing systems. Proper lighting is crucial for
   the development of T. sp. and rejuvenates. An LED light with blue
   spectrum predominance was adjusted to mimic natural sunlight, and the
   lighting cycle simulated day and night periods with regular intervals.

2.2. Total RNA extraction and library preparation

   Whole medusae were anesthetized in 3.5 % MgCl[2] (w/v in seawater) and
   dissected into the upper half containing the manubrium, oral lobes, and
   radical canal, and the lower half containing the velum with nerve ring
   and tentacles, under a stereomicroscope. The tissues were frozen on dry
   ice and stored at −80 °C for subsequent RNA extraction.

   Total RNA was isolated using a NucleoSpin RNA Plus XS kit (TaKaRa,
   Tokyo, Japan) and quantified on a Qubit 4 fluorometer (ThermoFisher
   Scientific, Tokyo, Japan). After RNA quality assessment on a
   Bioanalyzer 2100 system (Agilent Technologies, Tokyo, Japan), samples
   with RIN value of 7.0 or higher were chosen for subsequent library
   preparation. Nanopore sequencing, which utilizes a next generation
   portable long-reading MiNION sequencer which runs off la laptop
   computer with conventional laboratory settings, was carried out using a
   ligation-based cDNA-PCR sequencing kit (Oxford Nanopore Technologies
   (ONT), Didcot, UK) according to the manufacturer’s protocol. Briefly,
   using the strand-switching protocol, full-length cDNA from 50 ng total
   RNA was prepared using Maxima H Minus Reverse Transcriptase
   (ThermoFisher Scientific). LongAmp Taq 2x Master Mix (New England
   Laboratories, Tokyo, Japan) was used for amplifying full-length
   transcripts, and the PCR products were purified using Agencourt AMPure
   XP magnetic beads (Beckman Coulter, USA). The adaptor-ligated libraries
   were sequenced using a MinION sequencer with FLO-MIN 106 flow cells and
   R9.4 chemistry (ONT).

2.3. Data acquisition and bioinformatics analysis

   Basecalling was performed using a Guppy basecaller (version
   6.4.6+ae70e8f) by translating ionic signals into a nucleotide sequence.
   Subsequently, Minimap2 software (version 2.24-r1122) was employed to
   align the FASTQ files to SAM files with the reference genome generated
   from Genome assembly TUR_r1.0 (WGS project No: BQMF01) [[36]8]. The SAM
   files were then converted to BAM files, sorted, and indexed using
   Samtools software (version 1.15.1). Finally, FeatureCounts (version
   2.0.4) was utilized to perform read counting, and functional annotation
   was performed using Diamond Blast function equipped in OmicsBox 3.0
   (Biobam Bioinformatics, Cambridge, MA, USA). The bioinformatic
   procedures were computed utilizing SHIROKANE, a high-performance
   computing apparatus situated at the Human Genome Center.

   Annotated raw bead counts were obtained and processed using a web
   portal for pathway analysis (iDEP.95/iDEP1.1;
   [37]http://bioinformatics.sdstate.edu/, accessed on 7 June 2023)
   [[38]9]. Identification of differentially expressed genes (DEGs) was
   performed by extraction with an FDR cutoff of 0.1 and min-fold change
   of 2 as the default setting. A hierarchical clustering was performed as
   input the Euclidean distance computed between genes and samples. A
   heatmap of all DEGs was subsequently generated in which numerical
   values of points were represented by a range of colors. All DEGs
   Pathway analysis was performed using Parametric Gene Set Enrichment
   Analysis (PGSEA) with gene sets from the Gene Ontology Molecular
   Function and all other available databases. The cutoff for significance
   was set at 0.2.

3. Results

3.1. Diversity of structure and function of T sp.

   Understanding the complexity and organization of T. sp. requires
   uncovering the tissue and functional diversity, which now can be
   predicted using bulk sequencing, in addition to traditional
   histological and ultrastructural observation. The medusae were
   dissected into the upper part and lower part as shown in [39]Fig. 1a.
   The manubrium, oral lobes, and radical canal are included in the upper
   part (gastrovascular cavity part, GP), while the tentacles and velum
   with the nerve ring and ring cannel are included in the lower part
   (nerve ring part, NP). Within these organs, the tissue surrounding the
   nerve ring has been considered to be an important contributor to the
   rejuvenation process. By employing comprehensive transcriptome
   sequencing, heatmap analysis extracted the top 1000 up-regulated and
   down-regulated genes in comparison between GP and NP ([40]Fig. 1b). By
   exploring the database of
   Co.Expression.Tissue.Protein.Expression.from.Human.Proteome.Map, the
   results of PGSEA predicatively revealed that heart-, adrenal-,
   prostate- and kidney-like tissues are contained in the GP fraction,
   while retina-like and placenta-like tissues are contained in the NP
   fraction ([41]Fig. 1c). These genes were functionally identified as
   belonging to four clusters. Functional enrichment revealed that the
   genes in cluster 2 (green circle) are related to metabolic processes,
   while the genes in cluster 3 are initially related to negative
   regulation of metabolic processes and transferase activity, cell
   projection assembly, rhythmic processes and phagocytosis. The network
   of these enriched pathways is shown in [42]Fig. 1d. These results
   systemically indicate the potential structural and functional diversity
   of T. sp. medusae.

Fig. 1.

   [43]Fig. 1
   [44]Open in a new tab

   Predicated structural and functional diversity of T. sp. (a) The
   medusae were dissected into gastrovascular cavity part (GP) and nerve
   ring part (NP). Left panel: Mature medusa under anesthetic condition.
   Right panel: The medusa was dissected into GP and NP fragments. (b)
   Heatmap analysis extracted the top 1000 up-regulated and down-regulated
   genes in comparison between GP and NP. Downregulated genes are
   highlighted in green; upregulated genes are highlighted in red. (c)
   Functional predication of tissues by PGSEA analysis with exploration of
   the database of
   Co.Expression.Tissue.Protein.Expression.from.Human.Proteome.Map.
   Downregulated pathways are highlighted in blue; upregulated pathways
   are highlighted in red. (d) GO enrichment networks of four clusters. GO
   enrichment used padj <0.05 as the threshold criterion for significant
   enrichment. Enriched pathways in cluster 2 (green circle) and cluster 3
   (red circle) are highlighted. No enrichment was extracted from cluster
   1 and cluster 4. The size of circles indicates the number of included
   genes.

3.2. GP fragment dominates process and response to external and internal
stress

   GO pathway enrichment analysis of differentially expressed genes (DEGs)
   revealed that pathways including cellular responses to external or
   internal stress, such as DNA damage, abiotic stimulus, radiation, and
   reactive oxygen species, are dominant in the GP fragment compared to
   the NP fragment. Functions related to regulation of sensory function,
   central neuronal system development and response to hypoxia are
   significantly activated in the NP fragment ([45]Fig. 2a). DEGs related
   to the response to the oxidative stress pathway were extracted and
   shown in [46]Fig. 2b. Overexpression of genes related to the stress
   response, including TPO, GSS, BTK, SLOX5, PKD2, and PLA2R1, was
   observed in the GP fragment. In DEGs collected from the hypoxia
   response pathway, which has been considered a major initialization
   factor of activation of stem cells, higher activation was confirmed in
   the GP fragment compared to the NP fragment. Genes encoding proteins
   involved in transcription-related genes (TGFB1, ERCC3, and FAXO3),
   fertilization and development-related genes (SRC, EP300, ADORA1, and
   ADAM17), cell cycle-related genes (ATM and NPEPPS), and structure
   formation-related genes (ANGPT4, OPRD1, MMP2, THBS1, and HSP90B1) were
   extracted.

Fig. 2.

   [47]Fig. 2
   [48]Open in a new tab

   Results of GO biological process pathway analysis in GP and NP
   fragments. (a) Functional prediction of differentially expressed genes
   was performed by PGSEA analysis by exploring the database of GO
   biological process. A default value of FDR < 0.2 served as the
   threshold criterion for significant cutoff. The 30 GO pathways with the
   most significant enrichment are shown in the heatmap. Downregulated
   pathways are highlighted in blue; upregulated pathways are highlighted
   in red. Normalized expression of genes included in representative
   pathways, including (b) response to oxidative stress and (c) response
   to hypoxia, was visualized in both the GP and NP samples with
   green-black-red heatmaps.

   Taken together, these results suggest that the GP fragment is
   functional dominant in the regulation of cellular responses to external
   or internal stress, whereas the NP fragment may contribute to
   developmental initialization processes in T. sp.

3.3. NP fragment may decide fate of rejuvenation

   We then explored all available databases equipped in the PGSEA module,
   and the top 30-hit pathways were summarized ([49]Fig. 3a). With
   particular interest in exploring contributors to the response to
   rejuvenation, partial DEGs involved in HMGIY target genes aroused our
   attention. The expression of genes encoding regulating factors involved
   in the initialization process of development and proliferation, such as
   PPP1R15B, ATF5, HEATR4, ROR1, FUT8, and EIF4H, was significantly higher
   in the NP fragment compared to the GP fragment ([50]Fig. 3b). Also,
   enhanced expression of PEF1 and TM7SF3, which encode factors related to
   programmed cell death, was observed. Parallel to the observation of
   previous studies, genes encoding proteins that are expected to be
   contributors involved in the neuronal network, including AOC3, ZFYVE26,
   RAB39B, CLCN3, and GABRD, were enriched in the NP fragment.

Fig. 3.

   [51]Fig. 3
   [52]Open in a new tab

   Expression of HMGIY-targeted genes in GP and NP fragments. (a)
   Functional prediction of differentially expressed genes was performed
   by PGSEA analysis by exploring all available databases currently
   equipped in iDEP1.1. A default value of FDR < 0.2 served as the
   threshold criterion for significant cutoff. The top 30 pathways with
   the most significant enrichment are shown in the heatmap. Downregulated
   pathways are highlighted in blue; upregulated pathways are highlighted
   in red. (b) Normalized expression of genes included in the HMGIY
   pathway was visualized in both GP and NP samples with a green-black-red
   heatmap.

   Together, these findings show that the NP fragment indeed serves as a
   central nervous-like system in T. sp. In view of the possibility of
   stem cell existence as a placenta-like tissue contained in NP
   fragments, the enrichment of factors involved in the initialization
   process of development and programmed cell death strongly suggests that
   the NP fragment may play a crucial role in the fate of the rejuvenative
   process in T. sp.

4. Discussion

   The answers to fundamental questions about the evolutionary and
   mechanistic response of age-related morphological and functional
   deterioration leading to death have been mainly explored in a limited
   number of experimental systems, including mammals (Mus musculus, Homo
   sapiens) and invertebrates (Saccharomyces cerevisiae, Caenorhabditis
   elegans, Drosophila melanogaster) [[53]1]. In fact, the spectrum of
   valuable systems for studying aging is much more extensive, which
   allows us to look at this phenomenon using a holistic rather than a
   reductionist approach. In the current study, with a particular focus on
   one of the aging permutations, immortality (neverending existence), the
   contribution of tissue diversity to the response to environmental
   stress and rejuvenation was dissected in a less conventional hydrozoan
   system, T. sp., by comparison of tissue fragments with and without the
   nerve ring.

   Compared to sessile polyps, the prominent nerve ring in free-swimming
   medusae has been considered a much higher level of centralized nervous
   system, containing a well-developed optical system that can be
   integrated into sophisticated light and gravity-sensing organs
   [[54]10,[55]11]. The structure of the nerve ring contributes to the
   control of swimming behaviors such as the avoidance of obstacles or
   escape from predators, by generating Ca^2+-dependent or Na^+-dependent
   depolarization potentials [[56]12]. In current study, a retina-like
   structure was also confirmed in the NP fragment. Beyond that, an
   unexpected potential association between the NP structure and system
   development was identified. Compared to the GP fragment, the NP
   fragment is more dominant in the regulation of the systemic response to
   hypoxia, regulation of organelle assembly, control of developmental
   initialization and programmed cell death. As a featured signaling
   pathway, but not limited to this, up-regulated HMGIY genes could be one
   of the initialization factors of transcriptional activation of stem
   cells in the NP fragment as the existence of breakpoints outside the
   gene. These transcriptome characteristics are closely related to
   functional activity, such as proliferation and differentiation of stem
   cells. On the other hand, the GP fragment is more responsible for the
   systemic response to different kinds of stimuli, such as DNA damage,
   oxidative stress and radiation. Less response to harmful external
   stimuli in NP could be protective for the retrograde cyst and
   contribute to a high propensity to tolerate manipulations within
   environmental variations. Therefore, we assume that the GP fragment
   could be mainly responsible for systemic senescence, whereas the NP
   fragment may be fundamental for the rejuvenation process. The report of
   Piraino et al. supports our assumption [[57]3]. They dissected
   Turritopsis nutricula, another hydrozoan which was assumed not to
   undergo organismic death, into small fragments. Under an in vitro
   culture condition, the transdifferentiating rates of fragments were
   evaluated. The results indicated that the structure surrounding the
   nerve ring, including exumbrella epidermic, velum, tentacle bulbs, and
   partial tentacle, which was defined as the NP fragment in the current
   study, was critical for destabilization and stolon formation in the
   polyp. For aging studies using the T. sp. system, the GP fragment could
   be utilized for study related to stress response and systemic
   senescence, while the NP fragment could be explored for self-repair and
   regeneration approaches. Identification of specific genes or pathways
   associated with stress response and rejuvenation in T. sp. provides
   potential targets for therapeutic interventions. This knowledge may
   contribute to the development of pharmaceutical interventions aimed at
   modulating stress response and promoting regenerative processes in
   human tissues. Moreover, the study sheds light on the mechanisms
   underlying the response to environmental stress. Such knowledge has
   broader implications for understanding how organisms, including humans,
   adapt to environmental challenges. Insights into stress response
   mechanisms can have applications in developing strategies to mitigate
   the impact of environmental stressors on human health.

   While T. sp. possesses unique biological features that make it an
   intriguing research subject, there are certain limitations and
   challenges associated with using it as a research tool. First of all,
   the mature medusae of immortal T. sp. are notably sensitive to
   alterations in water quality, temperature and other environmental
   factors. Maintaining stable and optimal conditions for their survival
   and growth can be demanding, requiring precise control of parameters
   such as water chemical composition, temperature and feeding regimen.
   Secondly, while T. sp. has fascinating regenerative abilities, its
   biological mechanisms may not directly translate to other organisms,
   including humans. Its genomic resources are also relatively limited
   compared to other well-established model organisms. Extending broad
   conclusions or generalized findings from T. sp. to other species
   requires caution and further comparative studies across different
   organisms. Finally, the collection and manipulation of T. sp. specimens
   need to be conducted responsibly and in accordance with ethical
   guidelines. Ensuring the welfare and humane treatment of T. sp. during
   research is of utmost importance.

   Is there any possibility of applying the rejuvenation process, an
   exceptional ability to revert back to an earlier developmental stage
   through transdifferentiation, to humans? Or are homo sapiens equipped
   with this fantastic ability but it has not yet been noticed? Is it
   possible that aging-related tumor development or death could just be a
   precursor for the transdiffentiation process, similar to what is
   observed in T. sp.? Maybe extraordinary advances in the technological
   challenges of storing and reviving a deceased human body while
   preserving its structural integrity and stem cell viability could help
   us to answer these questions. In any case, achieving true rejuvenation
   and reversing the effects of aging in humans, as seen in T. sp.,
   remains firmly in the realm of science fiction and further exploration
   is required for essential elucidation of the fundamental biological
   differences between T. sp. and humans.

Source of funding

   S.L. and M.M. were funded by a Grant-In-Aid for Scientific Research
   from the Ministry of Education, Science, and Culture, Japan (No.
   22K08564). Y.S. was funded by a Grant-In-Aid for Scientific Research
   from the Ministry of Education, Science, and Culture, Japan (No.
   21K16549).

CRediT authorship contribution statement

   Shuang Liu: Writing – original draft, Visualization, Project
   administration, Methodology, Investigation, Formal analysis, Data
   curation, Conceptualization. Erika Takemasa: Methodology,
   Investigation, Formal analysis. Yasuyuki Suzuki: Methodology, Formal
   analysis. Masaki Mogi: Writing – review & editing, Supervision, Funding
   acquisition.

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Acknowledgments