mTOR activity paces human blastocyst stage developmental progression.

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.

Evolution of diapause in the African turquoise killifish by remodeling the ancient gene regulatory landscape.

Suspended animation states allow organisms to survive extreme environments. The African turquoise killifish has evolved diapause as a form of suspended development to survive a complete drought. However, the mechanisms underlying the evolution of extreme survival states are unknown. To understand diapause evolution, we performed integrative multi-omics (gene expression, chromatin accessibility, and lipidomics) in the embryos of multiple killifish species. We find that diapause evolved by a recent remodeling of regulatory elements at very ancient gene duplicates (paralogs) present in all vertebrates. CRISPR-Cas9-based perturbations identify the transcription factors REST/NRSF and FOXOs as critical for the diapause gene expression program, including genes involved in lipid metabolism. Indeed, diapause shows a distinct lipid profile, with an increase in triglycerides with very-long-chain fatty acids. Our work suggests a mechanism for the evolution of complex adaptations and offers strategies to promote long-term survival by activating suspended animation programs in other species.

Dormancy, Quiescence, and Diapause: Savings Accounts for Life.

Life on Earth has been through numerous challenges over eons and, one way or another, has always triumphed. From mass extinctions to more daily plights to find food, unpredictability is everywhere. The adaptability of life-forms to ever-changing environments is the key that confers life's robustness. Adaptability has become synonymous with Darwinian evolution mediated by heritable genetic changes. The extreme gene-centric view, while being of central significance, at times has clouded our appreciation of the cell as a self-regulating entity informed of, and informing, the genetic data. An essential element that powers adaptability is the ability to regulate cell growth. In this review, we provide an extensive overview of growth regulation spanning species, tissues, and regulatory mechanisms. We aim to highlight the commonalities, as well as differences, of these phenomena and their molecular regulators. Finally, we curate open questions and areas for further exploration.

Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.

Nutrient deprivation induces mouse embryonic diapause mediated by Gator1 and Tsc2.

Embryonic diapause is a special reproductive phenomenon in mammals that helps embryos to survive various harsh stresses. However, the mechanisms of embryonic diapause induced by the maternal environment is still unclear. Here, we uncovered that nutrient deficiency in uterine fluid was essential for the induction of mouse embryonic diapause, shown by a decreased concentration of arginine, leucine, isoleucine, lysine, glucose and lactate in the uterine fluid of mice suffering from maternal starvation or ovariectomy. Moreover, mouse blastocysts cultured in a medium with reduced levels of these six components could mimic diapaused blastocysts. Our mechanistic study indicated that amino acid starvation-dependent Gator1 activation and carbohydrate starvation-dependent Tsc2 activation inhibited mTORC1, leading to induction of embryonic diapause. Our study elucidates the essential environmental factors in diapause induction.

A quantitative model of temperature-dependent diapause progression.

Winter diapause in insects is commonly terminated through cold exposure, which, like vernalization in plants, prevents development before spring arrives. Currently, quantitative understanding of the temperature dependence of diapause termination is limited, likely because diapause phenotypes are generally cryptic to human eyes. We introduce a methodology to tackle this challenge. By consecutively moving butterfly pupae of the species Pieris napi from several different cold conditions to 20 °C, we show that diapause termination proceeds as a temperature-dependent rate process, with maximal rates at relatively cold temperatures and low rates at warm and extremely cold temperatures. Further, we show that the resulting thermal reaction norm can predict P. napi diapause termination timing under variable temperatures. Last, we show that once diapause is terminated in P. napi, subsequent development follows a typical thermal performance curve, with a maximal development rate at around 31 °C and a minimum at around 2 °C. The sequence of these thermally distinct processes (diapause termination and postdiapause development) facilitates synchronous spring eclosion in nature; cold microclimates where diapause progresses quickly do not promote fast postdiapause development, allowing individuals in warmer winter microclimates to catch up, and vice versa. The unveiling of diapause termination as one temperature-dependent rate process among others promotes a parsimonious, quantitative, and predictive model, wherein winter diapause functions both as an adaptation against premature development during fall and winter and for synchrony in spring.

Sensory integration of food and population density during the diapause exit decision involves insulin-like signaling in Caenorhabditis elegans.

Decisions made over long time scales, such as life cycle decisions, require coordinated interplay between sensory perception and sustained gene expression. The Caenorhabditis elegans dauer (or diapause) exit developmental decision requires sensory integration of population density and food availability to induce an all-or-nothing organismal-wide response, but the mechanism by which this occurs remains unknown. Here, we demonstrate how the Amphid Single Cilium J (ASJ) chemosensory neurons, known to be critical for dauer exit, perform sensory integration at both the levels of gene expression and calcium activity. In response to favorable conditions, dauers rapidly produce and secrete the dauer exit-promoting insulin-like peptide INS-6. Expression of ins-6 in the ASJ neurons integrates population density and food level and can reflect decision commitment since dauers committed to exiting have higher ins-6 expression levels than those of noncommitted dauers. Calcium imaging in dauers reveals that the ASJ neurons are activated by food, and this activity is suppressed by pheromone, indicating that sensory integration also occurs at the level of calcium transients. We find that ins-6 expression in the ASJ neurons depends on neuronal activity in the ASJs, cGMP signaling, and the pheromone components ascr#8 and ascr#2. We propose a model in which decision commitment to exit the dauer state involves an autoregulatory feedback loop in the ASJ neurons that promotes high INS-6 production and secretion. These results collectively demonstrate how insulin-like peptide signaling helps animals compute long-term decisions by bridging sensory perception to decision execution.

Altered circadian rhythm, sleep, and rhodopsin 7-dependent shade preference during diapause in Drosophila melanogaster.

To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various Drosophila species undergo adult reproductive diapause in response to cool temperatures and/or short day-length. While flies are less active during diapause, it is unclear how adverse environmental conditions affect circadian rhythms and sleep. Here we show that in diapause-inducing cool temperatures, Drosophila melanogaster exhibit altered circadian activity profiles, including severely reduced morning activity and an advanced evening activity peak. Consequently, the flies have a single activity peak at a time similar to when nondiapausing flies take a siesta. Temperatures ≤15 °C, rather than photoperiod, primarily drive this behavior. At cool temperatures, flies rapidly enter a deep-sleep state that lacks the sleep cycles of flies at higher temperatures and require high levels of stimulation for arousal. Furthermore, we show that at 25 °C, flies prefer to siesta in the shade, a preference that is virtually eliminated at 10 °C. Resting in the shade is driven by an aversion to blue light that is sensed by Rhodopsin 7 outside of the eyes. Flies at 10 °C show neuronal markers of elevated sleep pressure, including increased expression of Bruchpilot and elevated Ca2+ in the R5 ellipsoid body neurons. Therefore, sleep pressure might overcome blue light aversion. Thus, at the same temperatures that cause reproductive arrest, preserve germline stem cells, and extend lifespan, D. melanogaster are prone to deep sleep and exhibit dramatically altered, yet rhythmic, daily activity patterns.

The integral role of de novo lipogenesis in the preparation for seasonal dormancy.

Animals can alter their body compositions in anticipation of dormancy to endure seasons with limited food availability. Accumulation of lipid reserves, mostly in the form of triglycerides (TAGs), is observed during the preparation for dormancy in diverse animals, including insects (diapause) and mammals (hibernation). However, the mechanisms involved in the regulation of lipid accumulation and the ecological consequences of failure to accumulate adequate lipid stores in preparation for animal dormancy remain understudied. In the broadest sense, lipid reserves can be accumulated in two ways: the animal either receives lipids directly from the environment or converts the sugars and amino acids present in food to fatty acids through de novo lipogenesis and then to TAGs. Here, we show that preparation for diapause in the Colorado potato beetle (Leptinotarsa decemlineata) involves orchestrated upregulation of genes involved in lipid metabolism with a transcript peak in 8- and 10-d-old diapause-destined insects. Regulation at the transcript abundance level was associated with the accumulation of substantial fat stores. Furthermore, the knockdown of de novo lipogenesis enzymes (ACCase and FAS-1) prolonged the preparatory phase, while the knockdown of fatty acid transportation genes shortened the preparatory phase. Our findings suggest a model in which the insects dynamically decide when to transition from the preparation phase into diapause, depending on the progress in lipid accumulation through de novo lipogenesis.

Beyond energy and growth: the role of metabolism in developmental signaling, cell behavior and diapause.

Metabolism is crucial for development through supporting cell growth, energy production, establishing cell identity, developmental signaling and pattern formation. In many model systems, development occurs alongside metabolic transitions as cells differentiate and specialize in metabolism that supports new functions. Some cells exhibit metabolic flexibility to circumvent mutations or aberrant signaling, whereas other cell types require specific nutrients for developmental progress. Metabolic gradients and protein modifications enable pattern formation and cell communication. On an organism level, inadequate nutrients or stress can limit germ cell maturation, implantation and maturity through diapause, which slows metabolic activities until embryonic activation under improved environmental conditions.

Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum.

Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). In this study, we demonstrate that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. Our findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis.

Mmp-induced fat body cell dissociation promotes pupal development and moderately averts pupal diapause by activating lipid metabolism.

In Lepidoptera and Diptera, the fat body dissociates into single cells in nondiapause pupae, but it does not dissociate in diapause pupae until diapause termination. Using the cotton bollworm, Helicoverpa armigera, as a model of pupal diapause insects, we illustrated the catalytic mechanism and physiological importance of fat body cell dissociation in regulating pupal development and diapause. In nondiapause pupae, cathepsin L (CatL) activates matrix metalloproteinases (Mmps) that degrade extracellular matrix proteins and cause fat body cell dissociation. Mmp-induced fat body cell dissociation activates lipid metabolism through transcriptional regulation, and the resulting energetic supplies increase brain metabolic activity (i.e., mitochondria respiration and insulin signaling) and thus promote pupal development. In diapause pupae, low activities of CatL and Mmps prevent fat body cell dissociation and lipid metabolism from occurring, maintaining pupal diapause. Importantly, as demonstrated by chemical inhibitor treatments and CRISPR-mediated gene knockouts, Mmp inhibition delayed pupal development and moderately increased the incidence of pupal diapause, while Mmp stimulation promoted pupal development and moderately averted pupal diapause. This study advances our recent understanding of fat body biology and insect diapause regulation.

Both entry to and exit from diapause arrest in Caenorhabditis elegans are regulated by a steroid hormone pathway.

Diapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. Although a steroid hormone pathway regulates the entry decision into C. elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions and establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit process. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling.

FoxO induces pupal diapause by decreasing TGFß signaling.

Diapause is a form of dormancy used widely by insects to survive adverse seasons. Previous studies have demonstrated that forkhead box O (FoxO) is activated during pupal diapause initiation in the moth Helicoverpa armigera. However, it is unclear how FoxO induces diapause. Here, we show that knockout of FoxO causes H. armigera diapause-destined pupae to channel into nondiapause, indicating that FoxO is a master regulator that induces insect diapause. FoxO activates the ubiquitin-proteasome system (UPS) by promoting ubiquitin c (Ubc) expression via directly binding to the Ubc promoter. Activated UPS decreases transforming growth factor beta (TGFß) receptor signaling via ubiquitination to block developmental signaling to induce diapause. This study significantly advances the understanding of insect diapause by uncovering the detailed molecular mechanism of FoxO.

Genome-wide analysis reveals transcriptional and translational changes during diapause of the Asian corn borer (Ostrinia furnacalis).

BACKGROUND: Diapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB). RESULTS: Our results unveil genes classified as "forwarded", "exclusive", "intensified", or "buffered" during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process. CONCLUSIONS: In summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.

Developmental ecology in embryos of an estuarine pupfish endemic of the Yucatan peninsula: Survival out of water, metabolic depression, and asynchronous hatching.

BACKGROUND: Theory predicts that drought-resistant embryos with extended incubations are evolutionarily favored in environments with high mortality of larvae but safe for eggs. Here, we experimentally test, under common garden conditions, the effect of three incubation temperatures and media on embryonic developmental length, extended incubation out of the water, survival, metabolic rate, and hatching dynamics in the estuarine pupfish Garmanella pulchra. We also described the morphological changes of embryonic cortical structures related to air exposure. RESULTS: We found that embryos incubated out of water in low and medium temperatures present an extended incubation period beyond their hatching capability with a deep metabolic depression. Also, these embryos exhibited a hatching asynchrony not related to water availability. Embryos incubated at high temperatures did not show extended incubation, with decreased probability of survival out of water. Our morphological observations of the embryonic cortical structures reveal that the perivitelline space and hair-like filaments buffer the deleterious drought effects. CONCLUSIONS: Our results reveal that G. pulchra possesses life-history traits typical of two separate phenomena: delay hatching and diapause; supporting a true continuum between them, rather than a dichotomy. The evolution of these traits may respond to aerial exposure during low tides in the estuaries of Yucatán they inhabit.

Analysis of the transcriptional pathways associated with the induction of quiescent embryonic arrest in the calanoid copepod Acartia tonsa.

The copepod species Acartia tonsa (Dana)(Crustacea) have the unique ability to induce quiescent embryonic dormancy if adverse environmental conditions occur; a characteristic shared by 41 other species belonging to the superfamily Centropagoida in the Calanoida order. However, the transcriptional changes characterizing this process are not known. Here, we compare the transcriptome of embryos in arrested quiescence with the normal development to identify pathways and differentially regulated transcripts involved in quiescent embryogenesis. Quiescence was induced by incubating eggs at 4 °C with anoxia for 26 h(hr), while eggs undergoing normal immediate development were incubated at 16.9 °C in normoxia for 7 h (where gastrulation occurs) or 14 h (where organogenesis occurs) before collecting for RNA extraction and analysis by RNA-sequencing. Results indicate that the expression profile of the quiescent embryo is not as different from the normal embryonic gastrulation as initially expected: None of the mapped transcripts is uniquely expressed in quiescence. Moreover, in quiescence a large proportion of the annotated transcripts display expression values halfway in-between the normal, immediate developmental stages of gastrulation and organogenesis. In depth comparison between the organogenesis stage and quiescent samples, reveal a high degree of divergence, confirming that a developmental arrest has been induced through quiescence. Specifically: Stress response transcripts are prominent in the quiescent phase with a transcript like the mammalian autophagy gene Sequestosome-1/p62 (SQSTM) being upregulated. The present analysis provides a better understanding of the molecular mechanisms characterizing the quiescent embryonic state of A. tonsa.

Signaling Transduction Pathways and G-Protein-Coupled Receptors in Different Stages of the Embryonic Diapause Termination Process in Artemia.

Artemia is a widely distributed small aquatic crustacean, renowned for its ability to enter a state of embryonic diapause. The embryonic diapause termination (EDT) is closely linked to environmental cues, but the precise underlying mechanisms remain elusive. In this study, ATAC-seq and RNA-seq sequencing techniques were employed to explore the gene expression profiles in Artemia cysts 30 min after EDT. These profiles were compared with those during diapause and 5 h after EDT. The regulatory mechanisms governing the EDT process were analyzed through Gene Ontology (GO) enrichment analysis of differentially expressed genes. Furthermore, the active G-protein-coupled receptors (GPCRs) were identified through structural analysis. The results unveiled that the signaling transduction during EDT primarily hinges on GPCRs and the cell surface receptor signaling pathway, but distinct genes are involved across different stages. Hormone-mediated signaling pathways and the tachykinin receptor signaling pathway exhibited heightened activity in the '0-30 min' group, whereas the Wnt signaling pathway manifested its function solely in the '30 min-5 h' group. These results imply a complete divergence in the mechanisms of signal regulation during these two stages. Moreover, through structural analysis, five GPCRs operating at different stages of EDT were identified. These findings provide valuable insights into the signal regulation mechanisms governing Artemia diapause.

The small GTPase RAB-18 is involved in regulating development/diapause by recruiting the intestinal cholesterol transporter NCR-1 onto the apical side in Caenorhabditis elegans.

RAB family proteins, which are small GTPases, are integral to the process of eukaryotic membrane trafficking. In the nematode, Caenorhabditis elegans, 31 RAB proteins have been identified through genome sequencing. Using an RNAi screen specifically targeting C. elegans rab genes, we identified multiple genes that are involved in the regulation of larval development, in particular, the rab-18 gene. Our molecular genetic studies resulted in several findings. First, RAB-18 predominantly functions in the intestine to regulate larval development by modulating steroid hormone signaling. Second, the C. elegans cholesterol transporter NCR-1 is a target of RAB-18 in the intestine. Third, the membrane trafficking of NCR-1 to the apical side in intestinal cells is particularly influenced by RAB-18. Finally, RAB-18 and NCR-1 possibly co-localize on membrane vesicles. Our study is the first to demonstrate the relationship between a RAB protein and a cholesterol transporter, in which the RAB protein probably drives the transporter to the apical membrane in the intestine to regulate cholesterol uptake. This study provides insight into the molecular mechanisms underlying human disease stemming from a transport defect of cholesterol and its derivative.

Different photoperiodic responses in diapause induction can promote the maintenance of genetic diversity via the storage effect in Daphnia pulex.

Understanding mechanisms that promote the maintenance of biodiversity (genetic and species diversity) has been a central topic in evolution and ecology. Previous studies have revealed that diapause can contribute to coexistence of competing genotypes or species in fluctuating environments via the storage effect. However, they tended to focus on differences in reproductive success (e.g. seed yield) and diapause termination (e.g. germination) timing. Here we tested whether different photoperiodic responses in diapause induction can promote coexistence of two parthenogenetic (asexual) genotypes of Daphnia pulex in Lake Fukami-ike, Japan. Through laboratory experiments, we confirmed that short day length and low food availability induced the production of diapausing eggs. Furthermore, we found that one genotype tended to produce diapausing eggs in broader environmental conditions than the other. Terminating parthenogenetic reproduction earlier decreases total clonal production, but the early diapausing genotype becomes advantageous by assuring reproduction in 'short' years where winter arrival is earlier than usual. Empirically parameterized theoretical analyses suggested that different photoperiodic responses can promote coexistence via the storage effect with fluctuations of the growing season length. Therefore, timing of diapause induction may be as important as diapause termination timing for promoting the maintenance of genetic diversity in fluctuating environments.