Bacterial c-di-GMP signaling gene affects mussel larval metamorphosis through outer membrane vesicles and lipopolysaccharides.

Biofilms serve as crucial cues for settlement and metamorphosis in marine invertebrates. Within bacterial systems, c-di-GMP functions as a pivotal signaling molecule regulating both biofilm formation and dispersion. However, the molecular mechanism of how c-di-GMP modulates biofilm-induced larval metamorphosis remains elusive. Our study reveals that the deletion of a c-di-GMP related gene in Pseudoalteromonas marina led to an increase in the level of bacterial c-di-GMP by knockout technique, and the mutant strain had an enhanced ability to produce more outer membrane vesicles (OMVs) and lipopolysaccharides (LPS). The mutant biofilms had higher induction activity for larval metamorphosis in mussels Mytilus coruscus, and OMVs play a major role in the induction activity. We further explored the function of LPS in OMVs. Extracted LPS induced high larval metamorphosis rate, and LPS content were subject to c-di-GMP and LPS-biosynthesis gene. Thus, we postulate that the impact of c-di-GMP on biofilm-induced metamorphosis is mediated through OMVs and LPS.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica.

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3' UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

Unraveling the transcriptomic landscape of eye migration and visual adaptations during flatfish metamorphosis.

Flatfish undergo a remarkable metamorphosis from symmetrical pelagic larvae to fully asymmetrical benthic juveniles. The most distinctive features of this transformation is the migration of one eye. The molecular role of thyroid hormone in the metamorphosis process in flatfishes is well established. However, the regulatory network that facilitates eye movement remains enigmatic. This paper presents a morphological investigation of the metamorphic process in turbot eyes, using advanced imaging techniques and a global view of gene expression. The study covers migrant and non-migrant eyes and aims to identify the genes that are active during ocular migration. Our transcriptomic analysis shows a significant up-regulation of immune-related genes. The analysis of eye-specific genes reveals distinct patterns during the metamorphic process. Myosin is highlighted in the non-migrant eye, while ependymin is highlighted in the migrant eye, possibly involved in optic nerve regeneration. Furthermore, a potential association between the alx3 gene and cranial restructuring has been identified. Additionally, it confirmed simultaneous adaptation to low light in both eyes, as described by changes in opsins expression during the metamorphic process. The study also revealed that ocular migration activates systems asynchronously in both eyes, providing insight into multifaceted reorganization processes during metamorphosis of flatfish.

The BTB transcription factor, Abrupt, acts cooperatively with Chronologically inappropriate morphogenesis (Chinmo) to repress metamorphosis and promotes leg regeneration.

Many insects undergo the process of metamorphosis when larval precursor cells begin to differentiate to create the adult body. The larval precursor cells retain stem cell-like properties and contribute to the regenerative ability of larval appendages. Here we demonstrate that two Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (BTB) domain transcription factors, Chronologically inappropriate morphogenesis (Chinmo) and Abrupt (Ab), act cooperatively to repress metamorphosis in the flour beetle, Tribolium castaneum. Knockdown of chinmo led to precocious development of pupal legs and antennae. We show that although topical application of juvenile hormone (JH) prevents the decrease in chinmo expression in the final instar, chinmo and JH act in distinct pathways. Another gene encoding the BTB domain transcription factor, Ab, was also necessary for the suppression of broad (br) expression in T. castaneum in a chinmo RNAi background, and simultaneous knockdown of ab and chinmo led to the precocious onset of metamorphosis. Furthermore, knockdown of ab led to the loss of regenerative potential of larval legs independently of br. In contrast, chinmo knockdown larvae exhibited pupal leg regeneration when a larval leg was ablated. Taken together, our results show that both ab and chinmo are necessary for the maintenance of the larval tissue identity and, apart from its role in repressing br, ab acts as a crucial regulator of larval leg regeneration. Our findings indicate that BTB domain proteins interact in a complex manner to regulate larval and pupal tissue homeostasis.

Intestinal remodeling during Xenopus metamorphosis as a model for studying thyroid hormone signaling and adult organogenesis.

Intestinal development takes places in two phases, the initial formation of neonatal (mammals)/larval (anurans) intestine and its subsequent maturation into the adult form. This maturation occurs during postembryonic development when plasma thyroid hormone (T3) level peaks. In anurans such as the highly related Xenopus laevis and Xenopus tropicalis, the larval/tadpole intestine is drastically remodeled from a simple tubular structure to a complex, multi-folded adult organ during T3-dependent metamorphosis. This involved complete degeneration of larval epithelium via programmed cell death and de novo formation of adult epithelium, with concurrent maturation of the muscles and connective tissue. Here, we will summarize our current understanding of the underlying molecular mechanisms, with a focus on more recent genetic and genome-wide studies.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles.

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a "molecular memory" of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3'-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Ontogeny of the Cytochrome P450 Superfamily in the Ornate Spiny Lobster (Panulirus ornatus).

Cytochrome P450s (CYP450s) are a versatile superfamily of enzymes known to undergo rapid evolution. They have important roles across growth and development pathways in crustaceans, although it is difficult to characterise orthologs between species due to their sequence diversity. Conserved CYP450s enzymes in crustaceans are those associated with ecdysteroidogenesis: synthesising and breaking down the active moult hormone, 20-hydroxyecdysone. The complex life cycle of the ornate spiny lobster, Panulirus ornatus, relies on moulting in order to grow and develop. Many of these diverse life stages have been analysed to establish a comprehensive transcriptomic database for this species. The transcripts putatively encoding for CYP450s were mapped using transcriptomic analysis and identified across growth and development stages. With the aid of phylogeny, 28 transcripts of 42 putative P. ornatus CYP450s were annotated, including the well conserved Halloween genes, which are involved in ecdysteroidogenesis. Expression patterns across the life stages determined that only a subset of the CYP450s can be detected in each life stage or tissue. Four Shed transcripts show overlapping expression between metamorphosis and adult tissues, suggesting pleotropic functions of the multiple Shed orthologs within P. ornatus.

Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm (Bombyx mori).

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Despite important progress in understanding various aspects of silkworm biology, the hormone signaling pathway in the silkworm remains poorly understood. Genome-wide screening using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)-based libraries has recently emerged as a novel method for analyzing genome function, enabling further research into essential genes, drug targets, and virus-host interaction. Previously, we constructed a genome-wide CRISPR/Cas9-based library of the silkworm (Bombyx mori) and successfully revealed the genes involved in biotic or abiotic stress factor responses. In this study, we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action. Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus. Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity, interfere with intracellular nutrition and energy metabolism, and eventually cause cell apoptosis. The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes, which had increased tolerance to 20E. Our findings provide a panoramic overview of signaling in response to 20E in the silkworm, underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

Juvenile hormone inhibits adult cuticle formation in Drosophila melanogaster through Kr-h1/Dnmt2-mediated DNA methylation of Acp65A promoter.

Differentiation of imaginal epidermal cells of Drosophila melanogaster to form adult cuticles occurs at approximately 40-93 h after puparium formation. Juvenile hormone (JH) given at pupariation results in formation of a second pupal cuticle in the abdomen instead of the adult cuticle. Although the adult cuticle gene Acp65A has been reported to be down-regulated following JH treatment, the regulatory mechanism remains unclear. Here, we found that the JH primary response gene Krüppel homologue 1 (Kr-h1) plays a vital role in the repression of adult cuticle formation through the mediation of JH action. Overexpression of Kr-h1 mimicked-while knocking down of Kr-h1 attenuated-the inhibitory action of JH on the formation of the adult abdominal cuticle. Further, we found that Kr-h1 inhibited the transcription of Acp65A by directly binding to the consensus Kr-h1 binding site (KBS) within the Acp65A promoter region. Moreover, the DNA methyltransferase Dnmt2 was shown to interact with Kr-h1, combined with the KBS to promote the DNA methylation of sequences around the KBS, in turn inhibiting the transcription of Acp65A. This study advances our understanding of the molecular basis of the "status quo" action of JH on the Drosophila adult metamorphosis.

Defining components of early thyroid hormone signalling through temperature-mediated activation of molecular memory in cultured Rana [lithobates] catesbeiana tadpole back skin.

Thyroid hormones (THs) are essential signalling molecules for the postembryonic development of all vertebrates. THs are necessary for the metamorphosis from tadpole to froglet and exogenous TH administration precociously induces metamorphosis. In American bullfrog (Rana [Lithobates] catesbeiana) tadpoles, the TH-induced metamorphosis observed at a warm temperature (24 °C) is arrested at a cold temperature (4 °C) even in the presence of exogenous THs. However, when TH-exposed tadpoles are shifted from cold to warm temperatures (4 â†’ 24 °C), they undergo TH-dependent metamorphosis at an accelerated rate even when the initial TH signal is no longer present. Thus, they possess a "molecular memory" of TH exposure that establishes the TH-induced response program at the cold temperature and prompts accelerated metamorphosis after a shift to a warmer temperature. The components of the molecular memory that allow the uncoupling of initiation from the execution of the metamorphic program are not understood. To investigate this, we used cultured tadpole back skin (C-Skin) in a repeated measures experiment under 24 °C only, 4 °C only, and 4 â†’ 24 °C temperature shifted regimes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA-sequencing (RNA-seq) analyses. RNA-seq identified 570, 44, and 890 transcripts, respectively, that were significantly changed by TH treatment. These included transcripts encoding transcription factors and proteins involved in mRNA structure and stability. Notably, transcripts associated with molecular memory do not overlap with those identified previously in cultured tail fin (C-fin) except for TH-induced basic leucine zipper-containing protein (thibz) suggesting that thibz may have a central role in molecular memory that works with tissue-specific factors to establish TH-induced gene expression programs.

Stem cell proliferation and differentiation during larval metamorphosis of the model tapeworm Hymenolepis microstoma.

Background: Tapeworm larvae cause important diseases in humans and domestic animals. During infection, the first larval stage undergoes a metamorphosis where tissues are formed de novo from a population of stem cells called germinative cells. This process is difficult to study for human pathogens, as these larvae are infectious and difficult to obtain in the laboratory. Methods: In this work, we analyzed cell proliferation and differentiation during larval metamorphosis in the model tapeworm Hymenolepis microstoma, by in vivo labelling of proliferating cells with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), tracing their differentiation with a suite of specific molecular markers for different cell types. Results: Proliferating cells are very abundant and fast-cycling during early metamorphosis: the total number of cells duplicates every ten hours, and the length of G2 is only 75 minutes. New tegumental, muscle and nerve cells differentiate from this pool of proliferating germinative cells, and these processes are very fast, as differentiation markers for neurons and muscle cells appear within 24 hours after exiting the cell cycle, and fusion of new cells to the tegumental syncytium can be detected after only 4 hours. Tegumental and muscle cells appear from early stages of metamorphosis (24 to 48 hours post-infection); in contrast, most markers for differentiating neurons appear later, and the detection of synapsin and neuropeptides correlates with scolex retraction. Finally, we identified populations of proliferating cells that express conserved genes associated with neuronal progenitors and precursors, suggesting the existence of tissue-specific lineages among germinative cells. Discussion: These results provide for the first time a comprehensive view of the development of new tissues during tapeworm larval metamorphosis, providing a framework for similar studies in human and veterinary pathogens.

Molecular identification and functional analysis of chitinase genes reveal their importance in the metamorphosis of Sarcophaga peregrina (Diptera: Sarcophagidae).

Chitinases play a crucial role in insect metamorphosis by facilitating chitin degradation. Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a typical holometabolous insect and an important hygiene pest that causes myiasis in humans and other mammals and acts as a vector for various parasitic agents, including bacteria, viruses, and parasites. Enhancing the understanding of the metamorphosis in this species has significance for vector control. In this study, we identified a total of 12 chitinase genes in S. peregrina using bioinformatic analysis methods. Based on transcriptome data, SpIDGF2 and SpCht10 were selected for further functional investigation. The down-regulation of these genes by RNA interference led to developmental delays, disruptions in molting, and differences in cuticle composition during the pupal stage. These findings underscore the pivotal role of chitinase genes in the metamorphic process and offer valuable insights for effective control strategies.

Estrogen-related receptor functions via the 20-hydroxyecdysone and IIS/TOR signaling pathways to regulate the development and morphology changes of ant Polyrhachis vicina Roger (Hymenoptera, Formicidae).

Estrogen-related receptor (ERR) is a key regulator of insect growth, development, and metabolic processes in insects; however, the molecular mechanisms underlying its effects are not fully understood. We investigated roles of 20-hydroxyecdysone (20E) and insulin/insulin-like signaling/target of rapamycin (IIS/TOR) signaling pathways in the effects of PvERR on larval development, metamorphosis, and adult growth in ant Polyrhachis vicina Roger. PvFOXO expression levels depended on caste and developmental stage. PvERR RNAi significantly reduced the expression levels of IIS/TOR signaling pathway genes and 20E signaling pathway genes in fourth-instar larvae, pupae, females, and workers and significantly increased the expression levels of IIS/TOR signaling pathway genes PvFOXO and PvAkt in males. PvFOXO RNAi resulted in developmental defects and increased mortality. After PvFOXO RNAi, the expression of PvERR, 20E signaling pathway genes, and IIS/TOR signaling pathway genes decreased significantly in pupae, females, and workers and increased significantly in fourth-instar larvae. Exogenous 20E attenuated expression changes induced by PvFOXO RNAi in a sex- and stage-specific manner. These results indicate that ERR interacts with 20E and IIS/TOR signaling pathways to regulate caste determination, metamorphosis, and male fertility in P. vicina and that correlations between PvERR and PvFOXO are caste- and stage-specific.

Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.

Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate 'capstone' research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.

Regulation mechanisms underlying tail resorption in Bufo gargarizans metamorphosis.

Anurans have been excellent organisms for studying amphibian metamorphosis. Tail resorption is a remarkable event that occurs during amphibian metamorphosis. Although tail resorption has been previously studied in other anurans like Xenopus laevis and Rana chensinensis, there is no report on Bufo gargarizans. This paper thus explored the mechanism of tail resorption during metamorphosis in Bufo gargarizans tadpoles through some biological research methods. Histological results showed that the tail tissues of tadpoles gradually degraded as metamorphosis progressed. RNA sequencing analysis was performed to examine the expression level and functional enrichment of differentially expressed genes in the tail. In addition, we analyzed the mRNA expression levels of genes related to tail resorption by quantitative real-time polymerase chain reaction. We also speculated on three pathways that participate in the regulation of tail resorption based on the above results. The present study might provide a theoretical basis and novel insights for further research of complex molecular mechanisms of tail resorption in amphibians.

Metamorphic gene regulation programs in Xenopus tropicalis tadpole brain.

Amphibian metamorphosis is controlled by thyroid hormone (TH), which binds TH receptors (TRs) to regulate gene expression programs that underlie morphogenesis. Gene expression screens using tissues from premetamorphic tadpoles treated with TH identified some TH target genes, but few studies have analyzed genome-wide changes in gene regulation during spontaneous metamorphosis. We analyzed RNA sequencing data at four developmental stages from the beginning to the end of spontaneous metamorphosis, conducted on the neuroendocrine centers of Xenopus tropicalis tadpole brain. We also conducted chromatin immunoprecipitation sequencing (ChIP-seq) for TRs, and we compared gene expression changes during metamorphosis with those induced by exogenous TH. The mRNA levels of 26% of protein coding genes changed during metamorphosis; about half were upregulated and half downregulated. Twenty four percent of genes whose mRNA levels changed during metamorphosis had TR ChIP-seq peaks. Genes involved with neural cell differentiation, cell physiology, synaptogenesis and cell-cell signaling were upregulated, while genes involved with cell cycle, protein synthesis, and neural stem/progenitor cell homeostasis were downregulated. There is a shift from building neural structures early in the metamorphic process, to the differentiation and maturation of neural cells and neural signaling pathways characteristic of the adult frog brain. Only half of the genes modulated by treatment of premetamorphic tadpoles with TH for 16 h changed expression during metamorphosis; these represented 33% of the genes whose mRNA levels changed during metamorphosis. Taken together, our results provide a foundation for understanding the molecular basis for metamorphosis of tadpole brain, and they highlight potential caveats for interpreting gene regulation changes in premetamorphic tadpoles induced by exogenous TH.

Steroid hormone regulation of innate immunity in Drosophila melanogaster.

Endocrine signaling networks control diverse biological processes and life history traits across metazoans. In both invertebrate and vertebrate taxa, steroid hormones regulate immune system function in response to intrinsic and environmental stimuli, such as microbial infection. The mechanisms of this endocrine-immune regulation are complex and constitute an ongoing research endeavor facilitated by genetically tractable animal models. The 20-hydroxyecdysone (20E) is the major steroid hormone in arthropods, primarily studied for its essential role in mediating developmental transitions and metamorphosis; 20E also modulates innate immunity in a variety of insect taxa. This review provides an overview of our current understanding of 20E-mediated innate immune responses. The prevalence of correlations between 20E-driven developmental transitions and innate immune activation are summarized across a range of holometabolous insects. Subsequent discussion focuses on studies conducted using the extensive genetic resources available in Drosophila that have begun to reveal the mechanisms underlying 20E regulation of immunity in the contexts of both development and bacterial infection. Lastly, I propose directions for future research into 20E regulation of immunity that will advance our knowledge of how interactive endocrine networks coordinate animals' physiological responses to environmental microbes.

[Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm].

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli.

Metamorphosis is one of the most important physiological processes in insects, which is coordinated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Ecdysone receptor (EcR) is a steroid receptor (SR), which usually presents in cytoplasm and transfers into nucleus after binding to 20E. Heat shock proteins (Hsps) are suggested to be important members of the SR complex. However, their role in nucleocytoplasmic shuttle of the EcR remains unclear. In the present study, we found that apoptozole (Hsp70 inhibitor) suppressed the larval molting by decreasing the expression of ecdysone signaling genes. Two cytoplasmic (Cy) Hsp70s (Hsp72 and Hsp73) interacted with both EcR and ultraspiracle (USP, the heterodimer partner of EcR). By immunohistochemistry experiments, we revealed that CyHsp70 co-localized with EcR in the cytoplasm, and that both apoptozole and interfering of CyHsp70 significantly inhibited the process of EcR entering the nucleus under 20E induction, while reducing the expression of ecdysone signaling genes. Interestingly, the nuclear localization of EcR was also promoted by two other stimuli, including JH and heat stress, and this promotion was inhibited by apoptozole. This implies that various stimuli can induce EcR entry into the nucleus, and that this process is mediated by CyHsp70. Curiously, neither JH nor heat stress activated the ecdysone signaling genes; instead, they have a significant inhibitory effect on them. Taken together, it seems that Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli, and that the biological effects of various stimuli passing through the EcR are different. Thus, our data provide a new viewpoint to understand the mechanism of nucleocytoplasmic shuttle of EcR.

Genome-wide chromatin accessibility and gene expression profiling during flatfish metamorphosis.

Metamorphosis is a widely studied post-embryonic process in which many tissues undergo dramatic modifications to adapt to the new adult lifestyle. Flatfishes represent a good example of metamorphosis in teleost fishes. During metamorphosis of flatfish, organ regression and neoformation occur, with one of the most notable changes being the migration of one of the eyes to the other side of the body. In order to create a useful and reliable tool to advance the molecular study of metamorphosis in flatfish, we generated a chromatin accessible atlas as well as gene expression profile during four developmental stages ranging from a phylotypic to a post-metamorphic stage. We identified 29,019 differentially accessible chromatin regions and 3,253 differentially expressed genes. We found stage-specific regulatory regions and gene expression profiles, supporting the quality of the results. Our work provides strongly reproducible data for further studies to elucidate the regulatory elements that ensure successful metamorphosis in flatfish species.