Integrating ATAC-Seq and RNA-Seq Reveals the Signal Regulation Involved in the Artemia Embryonic Reactivation Process.

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia. In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause.

Genomic and single-cell analyses reveal genetic signatures of swimming pattern and diapause strategy in jellyfish.

Jellyfish exhibit innovative swimming patterns that contribute to exploring the origins of animal locomotion. However, the genetic and cellular basis of these patterns remains unclear. Herein, we generated chromosome-level genome assemblies of two jellyfish species, Turritopsis rubra and Aurelia coerulea, which exhibit straight and free-swimming patterns, respectively. We observe positive selection of numerous genes involved in statolith formation, hair cell ciliogenesis, ciliary motility, and motor neuron function. The lineage-specific absence of otolith morphogenesis- and ciliary movement-related genes in T. rubra may be associated with homeostatic structural statocyst loss and straight swimming pattern. Notably, single-cell transcriptomic analyses covering key developmental stages reveal the enrichment of diapause-related genes in the cyst during reverse development, suggesting that the sustained diapause state favours the development of new polyps under favourable conditions. This study highlights the complex relationship between genetics, locomotion patterns and survival strategies in jellyfish, thereby providing valuable insights into the evolutionary lineages of movement and adaptation in the animal kingdom.

New insights into how to induce and maintain embryonic diapause in the blastocyst.

Embryonic diapause in mammals is a period of developmental pause of the embryo at the blastocyst stage. During diapause, the blastocyst has minimal cell proliferation, metabolic activity and gene expression. At reactivation, blastocyst development resumes, characterised by increases in cell number, biosynthesis and metabolism. Until recently, it has been unknown how diapause is maintained without any loss of blastocyst viability. This review focuses on recent progress in the identification of molecular pathways occurring in the blastocyst that can both cause and maintain the diapause state. A switch to lipid metabolism now appears essential to maintaining the diapause state and is induced by forkhead box protein O1. The forkhead box protein O transcription family is important for diapause in insects, nematodes and fish, but this is the first time a conclusive role has been established in mammals. Multiple epigenetic modifications are also essential to inducing and maintaining the diapause state, including both DNA and RNA methylation mechanisms. Finally, it now appears that diapause embryos, dormant stem cells and chemotherapeutic-resistant cancer cells may all share a universal system of quiescence.

Diapause-like Drug-Tolerant Persister State: The Key to Nirvana Rebirth.

Cancer is one of the leading causes of death in the world. Various drugs have been developed to eliminate it but to no avail because a tumor can go into dormancy to avoid therapy. In the past few decades, tumor dormancy has become a popular topic in cancer therapy. Recently, there has been an important breakthrough in the study of tumor dormancy. That is, cancer cells can enter a reversible drug-tolerant persister (DTP) state to avoid therapy, but no exact mechanism has been found. The study of the link between the DTP state and diapause seems to provide an opportunity for a correct understanding of the mechanism of the DTP state. Completely treating cancer and avoiding dormancy by targeting the expression of key genes in diapause are possible. This review delves into the characteristics of the DTP state and its connection with embryonic diapause, and possible treatment strategies are summarized. The authors believe that this review will promote the development of cancer therapy.

Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

Histone acetylation is associated with pupal diapause in cotton bollworm, Helicoverpa armigera.

BACKGROUND: Diapause is an environmentally preprogrammed period of arrested development that is important to insect survival and population growth. Histone acetylation, an epigenetic modification, has several biological functions, but its role in agricultural pest diapause is unknown. In this study, we investigated the role of histone H3 acetylation in the diapause of Helicoverpa armigera. RESULTS: The histone H3 gene of H. armigera was cloned, and multiple sequence alignment of amino acids revealed that the potential lysine acetylation sites were highly conserved across species. Investigation of histone H3 acetylation levels in diapause- and nondiapause-type pupae showed that acetylation levels were down-regulated in diapause-type pupae and were lower in diapausing pupae compared to nondiapause pupae. By screening the genome, six histone acetyltransferase (HAT) and eight histone deacetylase (HDAC) genes responsible for antagonizing catalytic histone acetylation modifications were identified in H. armigera, and most of them exhibited different expression patterns between diapause- and nondiapause-type pupae. To elucidate the effect of histone H3 acetylation on diapause in H. armigera, the diapause pupae were injected with the histone acetylation activator trichostatin A (TSA). The results indicated that TSA injection increased the levels of histone H3 acetylation, causing the diapausing pupae to revert to development. Furthermore, transcriptome analysis revealed that 259 genes were affected by TSA injection, including genes associated with metabolism, resistance, and immunological responses. CONCLUSION: These results suggest that histone acetylation is inseparably related to the pupal diapause of H. armigera, which promises to be a potential target for pest control. © 2023 Society of Chemical Industry.

Temperature dependence of gas exchange patterns shift as diapause progresses in the butterfly Pieris napi.

Insects have the capacity to significantly modify their metabolic rate according to environmental conditions and physiological requirement. Consequently, the respiratory patterns can range from continuous gas exchange (CGE) to discontinuous gas exchange (DGE). In the latter, spiracles are kept closed during much of the time, and gas exchange occurs only during short periods when spiracles are opened. While ultimate causes and benefits of DGE remain debated, it is often seen during insect diapause, a deep resting stage that insects induce to survive unfavourable environmental conditions, such as winter. The present study explores the shifts between CGE and DGE during diapause by performing long continuous respirometry measurements at multiple temperatures during key diapause stages in the green-veined white butterfly Pieris napi. The primary goal is to explore respiratory pattern as a non-invasive method to assess whether pupae are in diapause or have transitioned to post-diapause. Respiratory pattern can also provide insight into endogenous processes taking place during diapause, and the prolonged duration of diapause allows for the detailed study of the thermal dependence of the DGE pattern. Pupae change from CGE to DGE a few days after pupation, and this shift coincides with metabolic rate suppression during diapause initiation. Once in diapause, pupae maintain DGE even at elevated temperatures that significantly increase CO2 production. Instead of shifting respiratory pattern to CGE, pupae increase the frequency of DGE cycles. Since total CO2 released during a single open phase remains unchanged, our results suggest that P. napi pupae defend a maximum internal ρCO2 set point, even in their heavily suppressed diapause state. During post-diapause development, CO2 production increases as a function of development and changes to CGE during temperature conditions permissive for development. Taken together, the results show that respiratory patterns are highly regulated during diapause in P. napi and change predictably as diapause progresses.

The molecular mechanisms of diapause and diapause-like reversible arrest.

Diapause is a protective mechanism that many organisms deploy to overcome environmental adversities. Diapause extends lifespan and fertility to enhance the reproductive success and survival of the species. Although diapause states have been known and employed for commercial purposes, for example in the silk industry, detailed molecular and cell biological studies are an exciting frontier. Understanding diapause-like protective mechanisms will shed light on pathways that steer organisms through adverse conditions. One hope is that an understanding of the mechanisms that support diapause might be leveraged to extend the lifespan and/or health span of humans as well as species threatened by climate change. In addition, recent findings suggest that cancer cells that persist after treatment mimic diapause-like states, implying that these programs may facilitate cancer cell survival from chemotherapy and cause relapse. Here, we review the molecular mechanisms underlying diapause programs in a variety of organisms, and we discuss pathways supporting diapause-like states in tumor persister cells.

RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

The diapause-like colorectal cancer cells induced by SMC4 attenuation are characterized by low proliferation and chemotherapy insensitivity.

In response to adverse environmental conditions, embryonic development may reversibly cease, a process termed diapause. Recent reports connect this phenomenon with the non-genetic responses of tumors to chemotherapy, but the mechanisms involved are poorly understood. Here, we establish a multifarious role for SMC4 in the switching of colorectal cancer cells to a diapause-like state. SMC4 attenuation promotes the expression of three investment phase glycolysis enzymes increasing lactate production while also suppressing PGAM1. Resultant high lactate levels increase ABC transporter expression via histone lactylation, rendering tumor cells insensitive to chemotherapy. SMC4 acts as co-activator of PGAM1 transcription, and the coordinate loss of SMC4 and PGAM1 affects F-actin assembly, inducing cytokinesis failure and polyploidy, thereby inhibiting cell proliferation. These insights into the mechanisms underlying non-genetic chemotherapy resistance may have significant implications for the field, advancing our understanding of aerobic glycolysis functions in tumor and potentially informing future therapeutic strategies.

Fat enough for the winter? Does nutritional status affect diapause?

Many insects enter a dormant state termed diapause in anticipation of seasonal inhospitable conditions. Insects drastically reduce their feeding during diapause. Their reduced nutrient intake is paired with substantial nutrient costs: maintaining basal metabolism during diapause, repairing tissues damaged by adverse conditions, and resuming development after diapause. Many investigators have asked "Does nutrition affect diapause?" In this review, we survey the studies that have attempted to address this question. We propose the term nutritional status, a holistic view of nutrition that explicitly includes the perception, intake, and storage of the great breadth of nutrients. We examine the studies that have sought to test if nutrition affects diapause, trying to identify specific facets of nutritional status that affect diapause phenotypes. Curiously, low quality host plants during the diapause induction phase generally induce diapause, but food deprivation during the same phase generally averts diapause. Using the geometric framework of nutrition to identify specific dietary components that affect diapause may reconcile these contrasting findings. This framework can establish nutritionally permissive space, distinguishing nutrient changes that affect diapause from changes that induce other dormancies. Refeeding is another important experimental technique that distinguishes between diapause and quiescence, a non-diapause dormancy. We also find insufficient evidence for the hypothesis that nutrient stores regulate diapause length and suggest manipulations to investigate the role of nutrient stores in diapause termination. Finally, we propose mechanisms that could interface nutritional status with the diapause program, focusing on combined action of the nutritional axis between the gut, fat body, and brain.

Activation of protein arginine methyltransferase 1 and subsequent extension of moth lifespan is effected by the ROS/JNK/CREB signaling axis.

Previous studies have demonstrated that high physiological levels of reactive oxygen species induce pupal diapause and extend lifespan in the moth Helicoverpa armigera. This has been shown to occur via protein arginine methyltransferase 1 (PRMT1) blockade of Akt-mediated phosphorylation of the transcription factor FoxO, after which activated FoxO promotes the initiation of diapause. However, it is unclear how PRMT1 is activated upstream of FoxO activity. Here, we show that high reactive oxygen species levels in the brains of H. armigera diapause-destined pupae activate the expression of c-Jun N-terminal kinase, which subsequently activates the transcription factor cAMP-response element binding protein. We show that cAMP-response element binding protein then directly binds to the PRMT1 promoter and upregulates its expression to prevent Akt-mediated FoxO phosphorylation and downstream FoxO nuclear localization. This novel finding that c-Jun N-terminal kinase promotes FoxO nuclear localization in a PRMT1-dependent manner to regulate pupal diapause reveals a complex regulatory mechanism in extending the healthspan of H. armigera.

Endometrial extracellular matrix components do not change over the course of embryonic diapause and reactivation in the roe deer (Capreolus capreolus).

The modification of the endometrial extracellular matrix (ECM) is a crucial step for embryo implantation in many mammalian species. The embryo of the European roe deer (Capreolus capreolus) displays a 4-5 months long temporary reduction of developmental pace termed embryonic diapause. A reduction of epithelial cell height during diapause has previously been described. Co-occurring ECM modifications may contribute to the changes of the intra-uterine milieu during reactivation at which the embryo regains developmental velocity. We assessed the localization of five ECM proteins (collagen I and IV, fibronectin, laminin, and extracellular matrix protein 1) using immunohistochemistry in animals with early, late, and post-diapause (elongating) embryos. While our results confirmed the reduction of epithelial height during diapause, we only detected marginal differences in localization and staining intensities of the selected ECM proteins. Major ECM remodelling events in the roe deer endometrium are thus likely to occur only at implantation.

Molecular Regulators of Embryonic Diapause and Cancer Diapause-like State.

Embryonic diapause is an enigmatic state of dormancy that interrupts the normally tight connection between developmental stages and time. This reproductive strategy and state of suspended development occurs in mice, bears, roe deer, and over 130 other mammals and favors the survival of newborns. Diapause arrests the embryo at the blastocyst stage, delaying the post-implantation development of the embryo. This months-long quiescence is reversible, in contrast to senescence that occurs in aging stem cells. Recent studies have revealed critical regulators of diapause. These findings are important since defects in the diapause state can cause a lack of regeneration and control of normal growth. Controlling this state may also have therapeutic applications since recent findings suggest that radiation and chemotherapy may lead some cancer cells to a protective diapause-like, reversible state. Interestingly, recent studies have shown the metabolic regulation of epigenetic modifications and the role of microRNAs in embryonic diapause. In this review, we discuss the molecular mechanism of diapause induction.

EGFR signal transduction is downregulated in C. elegans vulval precursor cells during dauer diapause.

Caenorhabditis elegans larvae display developmental plasticity in response to environmental conditions: in adverse conditions, second-stage larvae enter a reversible, long-lived dauer stage instead of proceeding to reproductive adulthood. Dauer entry interrupts vulval induction and is associated with a reprogramming-like event that preserves the multipotency of vulval precursor cells (VPCs), allowing vulval development to reinitiate if conditions improve. Vulval induction requires the LIN-3/EGF-like signal from the gonad, which activates EGFR-Ras-ERK signal transduction in the nearest VPC, P6.p. Here, using a biosensor and live imaging we show that EGFR-Ras-ERK activity is downregulated in P6.p in dauers. We investigated this process using gene mutations or transgenes to manipulate different steps of the pathway, and by analyzing LET-23/EGFR subcellular localization during dauer life history. We found that the response to EGF is attenuated at or upstream of Ras activation, and discuss potential membrane-associated mechanisms that could achieve this. We also describe other findings pertaining to the maintenance of VPC competence and quiescence in dauer larvae. Our analysis indicates that VPCs have L2-like and unique dauer stage features rather than features of L3 VPCs in continuous development.

Targeted depletion of uterine glandular Foxa2 induces embryonic diapause in mice.

Embryonic diapause is a reproductive strategy in which embryo development and growth is temporarily arrested within the uterus to ensure the survival of neonates and mothers during unfavorable conditions. Pregnancy is reinitiated when conditions become favorable for neonatal survival. The mechanism of how the uterus enters diapause in various species remains unclear. Mice with uterine depletion of Foxa2, a transcription factor, are infertile. In this study, we show that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent expression of uterine Msx1, a gene critical to maintaining the uterine quiescent state, suggesting that these mice enter embryonic diapause. Leukemia inhibitory factor (LIF) can resume implantation in these mice. Although estrogen is critical for implantation in progesterone-primed uterus, our current model reveals that FOXA2-independent estrogenic effects are detrimental to sustaining uterine quiescence. Interestingly, progesterone and anti-estrogen can prolong uterine quiescence in the absence of FOXA2. Although we find that Msx1 expression persists in the uterus deficient in Foxa2, the complex relationship of FOXA2 with Msx genes and estrogen receptors remains to be explored.

Cathepsin L Contributes to Reproductive Diapause by Regulating Lipid Storage and Survival of Coccinella septempunctata (Linnaeus).

Cathepsin L protease, which belongs to the papain-like cysteine proteases family, is an important player in many physiological and pathological processes. However, little was known about the role of cathepsin L in ladybird beetles (Coccinella septempuctata Linnaeus) during diapause. Here, we analyzed the characteristics of cathepsin L (CsCatL) in the females of C. septempunctata and its role during the diapause of the ladybeetle. CsCatL was cloned and identified from beetle specimens by rapid amplification of cDNA-ends (RACE). The cDNA sequence of CsCatL was 971 bp in length, including an 843 bp open reading frame encoding a protein of 280 amino acids. It was identified as the cathepsin L group by phylogenetic analysis. Knockdown of CsCatL by RNA interference led to decreased expression levels of fatty acid synthase 2 (fas 2) genes and suppressed lipid accumulation. Furthermore, silencing the CsCatL gene distinctly reduced diapause-related features and the survival of female C. spetempunctata under diapause-inducing conditions. The results suggested that the CsCatL gene was involved in fatty acid biosynthesis and played a crucial role in the survival of adult C. septempunctata during the diapause preparation stage.

Divergent diapause life history timing drives both allochronic speciation and reticulate hybridization in an adaptive radiation of Rhagoletis flies.

Divergent adaptation to new ecological opportunities can be an important factor initiating speciation. However, as niches are filled during adaptive radiations, trait divergence driving reproductive isolation between sister taxa may also result in trait convergence with more distantly related taxa, increasing the potential for reticulated gene flow across the radiation. Here, we demonstrate such a scenario in a recent adaptive radiation of Rhagoletis fruit flies, specialized on different host plants. Throughout this radiation, shifts to novel hosts are associated with changes in diapause life history timing, which act as "magic traits" generating allochronic reproductive isolation and facilitating speciation-with-gene-flow. Evidence from laboratory rearing experiments measuring adult emergence timing and genome-wide DNA-sequencing surveys supported allochronic speciation between summer-fruiting Vaccinium spp.-infesting Rhagoletis mendax and its hypothesized and undescribed sister taxon infesting autumn-fruiting sparkleberries. The sparkleberry fly and R. mendax were shown to be genetically discrete sister taxa, exhibiting no detectable gene flow and allochronically isolated by a 2-month average difference in emergence time corresponding to host availability. At sympatric sites across the southern USA, the later fruiting phenology of sparkleberries overlaps with that of flowering dogwood, the host of another more distantly related and undescribed Rhagoletis taxon. Laboratory emergence data confirmed broadly overlapping life history timing and genomic evidence supported on-going gene flow between sparkleberry and flowering dogwood flies. Thus, divergent phenological adaptation can drive the initiation of reproductive isolation, while also enhancing genetic exchange across broader adaptive radiations, potentially serving as a source of novel genotypic variation and accentuating further diversification.

Progress in the characterization of insulin-like peptides in aphids: Immunohistochemical mapping of ILP4.

Aphids were the first animals described as photoperiodic due to their seasonal switch from viviparous parthenogenesis to sexual reproduction (cyclical parthenogenesis) caused by the shortening of the photoperiod in autumn. This switch produces a single sexual generation of oviparous females and males that mate and lay diapausing cold-resistant eggs that can overcome the unfavourable environmental conditions typical of winter in temperate regions. Previous studies have hinted at a possible implication of two insulin-like peptides (ILP1 and ILP4) in the aphid seasonal response, changing their expression levels between different photoperiodic conditions. Moreover, in situ localization of their transcripts in particular neurosecretory cells (NSCs) in the aphid brain supported the idea that these neuropeptides could correspond to the formerly called virginoparin, an uncharacterized factor originally proposed to be transported directly to the aphid embryos to promote their development as parthenogenetic individuals. To further investigate the fate of these ILPs, we raised a specific antiserum against one of them (ILP4) and mapped this neuropeptide by immunohistochemistry (IHC) in Acyrthosiphon pisum and Megoura viciae aphids. Coincident with in situ localization, our results show that ILP4 is synthesized in two groups (one in each brain hemisphere) of four neurosecretory cells in the pars intercerebralis (NSC group I) and then it is transported outside the brain to the corpora cardiaca. From there, three nerves (two laterals and one medial) transport it to the abdomen. Although no precise site of release has been found, the terminations of these nerves near the germaria would be compatible with the proposal of a direct connection between group I of NSCs and the reproductive system by localized release. In addition, we detected some collateral arborizations originating from the eight NSCs going to the pars lateralis, where clock neurons and some photoreceptors have been previously localized, suggesting a possible communication between the circadian and photoperiodic systems.

A Malignant Case of Arrested Development: Cancer Cell Dormancy Mimics Embryonic Diapause.

Writing in Cancer Cell and Cell, two groups investigate the nature of dormant cancer cells that persist after chemotherapy. These cells adopt a state that resembles diapause, an evolutionarily conserved adaptation used by embryos to survive inhospitable conditions. Understanding cancer diapause could uncover therapeutic strategies that reduce cancer relapse.