The transcription factor masculinizer in sexual differentiation and achieved full functional sex reversal in prawn.

Some Zinc finger (ZnF) proteins are required for masculinization in silkworms. In the present study, a masculinizer gene (Mr-Masc) with multi-tissue expression is identified in the freshwater prawn Macrobrachium rosenbergii. The Mr-Masc is clustered into a separate branch with ZnF proteins from decapoda by phylogenetic tree analysis. Moreover, Mr-Masc silencing in male postlarvae prawn results in functional sex reversal females known as neo-females, which are applied to all-male monosex offspring breeding. This manipulation has been significant in sexually dimorphic cultured species. In addition, several significantly expressed transcripts are enriched and the effects of crucial signal pathways are focused through the comparative transcriptomic analysis in Mr-Masc gene knockdown. The significantly differentially expressed epidermal growth factor, upregulated low-density lipoprotein receptor, flotillin, and sex-lethal unigenes, downregulated heat shock proteins and forkhead box homologs are focused. The finding offers an innovative perspective on Masc proteins' evolution and physiological function.

Downregulation of a CT10 regulator of kinase (Crk) promotes the formation of diapause embryos in the brine shrimp Artemia.

To survive under harsh environments, embryonic development of Artemia was arrested at the gastrula stage and released as the diapause embryo. Cell cycle and metabolism were highly suppressed in this state of quiescence. However, cellular mechanisms underlying diapause remain largely unclear. In this study, we found that the expression level of a CT10 regulator of kinase-encoding gene (Ar-Crk) in diapause embryos was significantly lower than non-diapause embryos at the early embryogenetic stage of Artemia. Knockdown of Ar-Crk by RNA interference induced formation of diapause embryos, while the control group produced nauplii. Western blot analysis and metabolic assays revealed that the diapause embryos produced by Ar-Crk-knocked-down Artemia had similar characteristics of diapause markers, arrested cell cycle, and suppressed metabolism with those diapause embryos produced by natural oviparous Artemia. Transcriptomic analysis of Artemia embryos revealed knockdown of Ar-Crk induced downregulation of the aurora kinase A (AURKA) signaling pathway, as well as energetic and biomolecular metabolisms. Taken together, we proposed that Ar-Crk is a crucial factor in determining the process of diapause in Artemia. Our results provide insight into the functions of Crk in fundamental regulations such as cellular quiescence.

From ecology to oncology: To understand cancer stem cell dormancy, ask a Brine shrimp (Artemia).

The brine shrimp (Artemia), releases embryos that can remain dormant for up to a decade. Molecular and cellular level controlling factors of dormancy in Artemia are now being recognized or applied as active controllers of dormancy (quiescence) in cancers. Most notably, the epigenetic regulation by SET domain-containing protein 4 (SETD4), is revealed as highly conserved and the primary control factor governing the maintenance of cellular dormancy from Artemia embryonic cells to cancer stem cells (CSCs). Conversely, DEK, has recently emerged as the primary factor in the control of dormancy exit/reactivation, in both cases. The latter has been now successfully applied to the reactivation of quiescent CSCs, negating their resistance to therapy and leading to their subsequent destruction in mouse models of breast cancer, without recurrence or metastasis potential. In this review, we introduce the many mechanisms of dormancy from Artemia ecology that have been translated into cancer biology, and herald Artemia's arrival on the model organism stage. We show how Artemia studies have unlocked the mechanisms of the maintenance and termination of cellular dormancy. We then discuss how the antagonistic balance of SETD4 and DEK fundamentally controls chromatin structure and consequently governs CSCs function, chemo/radiotherapy resistance, and dormancy in cancers. Many key stages from transcription factors to small RNAs, tRNA trafficking, molecular chaperones, ion channels, and links with various pathways and aspects of signaling are also noted, all of which link studies in Artemia to those of cancer on a molecular and/or cellular level. We particularly emphasize that the application of such emerging factors as SETD4 and DEK may open new and clear avenues for the treatment for various human cancers.

A cytological revisit on parthenogenetic Artemia and the deficiency of a meiosis-specific recombinase DMC1 in the possible transition from bisexuality to parthenogenesis.

Although parthenogenesis is widespread in nature and known to have close relationships with bisexuality, the transitional mechanism is poorly understood. Artemia is an ideal model to address this issue because bisexuality and "contagious" obligate parthenogenesis independently exist in its congeneric members. In the present study, we first performed chromosome spreading and immunofluorescence to compare meiotic processes of Artemia adopting two distinct reproductive ways. The results showed that, unlike conventional meiosis in bisexual Artemia, meiosis II in parthenogenic Artemia is entirely absent and anaphase I is followed by a single mitosis-like equational division. Interspecific comparative transcriptomics showed that two central molecules in homologous recombination (HR), Dmc1 and Rad51, exhibited significantly higher expression in bisexual versus parthenogenetic Artemia. qRT-PCR indicated that the expression of both genes peaked at the early oogenesis and gradually decreased afterward. Knocking-down by RNAi of Dmc1 in unfertilized females of bisexual Artemia resulted in a severe deficiency of homologous chromosome pairing and produced univalents at the middle oogenesis stage, which was similar to that of parthenogenic Artemia, while in contrast, silencing Rad51 led to no significant chromosome morphological change. Our results indicated that Dmc1 is vital for HR in bisexual Artemia, and the deficiency of Dmc1 may be correlated with or even possibly one of core factors in the transition from bisexuality to parthenogenesis.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Embryogenic stem cell-derived intestinal crypt fission directs de novo crypt genesis.

Intestinal epithelial replenishment is fueled by continuously dividing intestinal stem cells (ISCs) resident at the crypt niche. However, the cell type(s) enabling replenishment upon damage and subsequent loss of whole crypts remain largely unclear. Using Set domain-containing protein 4 (Setd4), we identify a small population with reserve stem cell characteristics in the mouse intestine. Upon irradiation-induced injury, Setd4-expressing (Setd4+) cells survive radiation exposure and then activate to produce Sca-1-expressing cell types to restore the epithelial wall and regenerate crypts de novo via crypt fission. Setd4+ cells are confirmed to originate from the early fetal period, subsequently contributing to the development of embryonic gut and the establishment of postnatal crypts. Setd4+ cells are therefore represented as both originators and key regenerators of the intestine.

SETD4 cells contribute to brain development and maintain adult stem cell reservoir for neurogenesis.

Cellular quiescence facilitates maintenance of neural stem cells (NSCs) and their subsequent regenerative functions in response to brain injury and aging. However, the specification and maintenance of NSCs in quiescence from embryo to adulthood remain largely unclear. Here, using Set domain-containing protein 4 (SETD4), an epigenetic determinant of cellular quiescence, we mark a small but long-lived NSC population in deep quiescence in the subventricular zone of adult murine brain. Genetic lineage tracing shows that SETD4+ cells appear before neuroectoderm formation and contribute to brain development. In the adult, conditional knockout of Setd4 resulted in quiescence exit of NSCs, generating newborn neurons in the olfactory bulb and contributing to damage repair. However, long period deletion of SETD4 lead to exhaustion of NSC reservoir or SETD4 overexpression caused quiescence entry of NSCs, leading to suppressed neurogenesis. This study reveals the existence of long-lived deep quiescent NSCs and their neurogenetic capacities beyond activation.

Exosomal DEK removes chemoradiotherapy resistance by triggering quiescence exit of breast cancer stem cells.

Tumor therapeutics often target the primary tumor bulk but fail to eradicate therapy-resistant cancer stem cells (CSCs) in quiescent state. These can then become activated to initiate recurrence and/or metastasis beyond therapy. Here, we identified and isolated chemoradiotherapy-resistant CSCs in quiescent state with high capacity of tumor-initiation and tumorsphere formation from three types of breast tumors in mice. Experiments of knockdown and rescue revealed DEK, a nuclear protein, as essential for CSC activation. Exogenous DEK was then used to trigger quiescence exit of CSCs. ChIP-seq and ATAC-seq showed that DEK directly binds to chromatin, facilitating its genome-wide accessibility. The resulting epigenetic events upregulate the expression of cellular activation-related genes including MYC targets, whereas cellular quiescence-related genes including the p53 signaling pathway are silenced. However, twinned with DEK-induced activation, formerly resistant CSCs were then destroyed by chemotherapy in vitro. In mice, traditional chemoradiotherapy concurrent with the injection of DEK-containing exosomes resulted in eradication of primary tumors together with formerly resistant CSCs without recurrence or metastasis. Our findings advance knowledge of the mechanism of quiescent CSC activation and may provide novel clinical opportunities for removal of quiescence-linked therapy resistance.

SETD4-expressing cells contribute to pancreatic development and response to cerulein induced pancreatitis injury.

In the adult pancreas, the presence of progenitor or stem cells and their potential involvement in homeostasis and regeneration remains unclear. Here, we identify that SET domain-containing protein 4 (SETD4), a histone lysine methyltransferase, is expressed in a small cell population in the adult mouse pancreas. Genetic lineage tracing shows that during pancreatic development, descendants of SETD4+ cells make up over 70% of pancreatic cells and then contribute to each pancreatic lineage during pancreatic homeostasis. SETD4+ cells generate newborn acinar cells in response to cerulein-induced pancreatitis in acinar compartments. Ablation of SETD4+ cells compromises regeneration of acinar cells, in contrast to controls. Our findings provide a new cellular narrative for pancreatic development, homeostasis and response to injury via a small SETD4+ cell population. Potential applications may act to preserve pancreatic function in case of pancreatic disease and/or damage.

Setd4 controlled quiescent c-Kit+ cells contribute to cardiac neovascularization of capillaries beyond activation.

Blood vessels in the adult mammal exist in a highly organized and stable state. In the ischemic heart, limited expansion capacity of the myocardial vascular bed cannot satisfy demands for oxygen supply and the myocardium eventually undergoes irreversible damage. The predominant contribution of endogenous c-Kit+ cells is understood to be in the development and homeostasis of cardiac endothelial cells, which suggests potential for their targeting in treatments for cardiac ischemic injury. Quiescent cells in other tissues are known to contribute to the long-term maintenance of a cell pool, preserve proliferation capacity and, upon activation, facilitate tissue homeostasis and regeneration in response to tissue injury. Here, we present evidence of a Setd4-expressing quiescent c-Kit+ cell population in the adult mouse heart originating from embryonic stages. Conditional knock-out of Setd4 in c-Kit-CreERT2;Setd4f/f;Rosa26TdTomato mice induced an increase in vascular endothelial cells of capillaries in both neonatal and adult mice. We show that Setd4 regulates quiescence of c-Kit+ cells by the PI3K-Akt-mTOR signaling pathway via H4K20me3 catalysis. In myocardial infarction injured mice, Setd4 knock-out resulted in attenuated cardiomyocyte apoptosis, decreased infarction size and improved cardiac function. Lineage tracing in Setd4-Cre;Rosa26mT/mG mice showed that Setd4+ cells contribute to each cardiac lineage. Overall, Setd4 epigenetically controls c-Kit+ cell quiescence in the adult heart by facilitating heterochromatin formation via H4K20me3. Beyond activation, endogenous quiescent c-Kit+ cells were able to improve cardiac function in myocardial infarction injured mice via the neovascularization of capillaries.

Full Functional Sex Reversal Achieved Through Silencing of MroDmrt11E Gene in Macrobrachium rosenbergii: Production of All-Male Monosex Freshwater Prawn.

The freshwater prawn Macrobrachium rosenbergii is one kind of important economic aquaculture species and displays remarkable sexual dimorphism. The molecular mechanism of sexual differentiation in M. rosenbergii has been primarily unraveled through the research efforts of the androgenic gland and its related genes. However, the understanding of conserved genes involved in the molecular mechanism underpinning sex determination and sexual differentiation of M. rosenbergii is still fragmentary. MroDmrt11E is a member of the doublesex and mab-3-related transcription factor (Dmrt) gene family and is prominently expressed in the testis. In the present study, in vivo knockdown of MroDmrt11E at the postlarva stage in male prawn induced a complete and functional sex reversal and achieved the production of an all-male monosex population. Furthermore, a great deal of new information of upregulated and downregulated transcriptions involved in sexual differentiation of MroDmrt11E knockdown was enriched by comparative transcriptomic analysis. The effects of RNAi-mediated gene knockdown of MroDmrt11E on the differentially expressed and sex-related candidate genes, such as transformer, fruitless, feminization, insulin-like androgenic gland gene, Dmrt gene family, were primarily focused on, and their possible molecular regulatory relationships in sexual differentiation were analyzed. Meanwhile, the response of primary Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathways was investigated to expound the potential roles of MroDmrt11E in male sexual differentiation, which provided a deeper understanding of the molecular regulatory network underlying sexual differentiation of M. rosenbergii. The finding provided a novel sexual manipulation technique through silencing of Dmrt gene family for achieving a complete and functional sex reversal and offered a new insight regarding the mechanism of the Dmrt gene family in the sexual differentiation of crustaceans.

SET Domain-Containing Protein 4 Epigenetically Controls Breast Cancer Stem Cell Quiescence.

Quiescent cancer stem cells (CSC) play important roles in tumorigenesis, relapse, and resistance to chemoradiotherapy. However, the determinants of CSC quiescence and how they sustain themselves to generate tumors and relapse beyond resistance to chemoradiotherapy remains unclear. Here, we found that SET domain-containing protein 4 (SETD4) epigenetically controls breast CSC (BCSC) quiescence by facilitating heterochromatin formation via H4K20me3 catalysis. H4K20me3 localized to the promoter regions and regulated the expression of a set of genes in quiescent BCSCs (qBCSC). SETD4-defined qBCSCs were resistant to chemoradiotherapy and promoted tumor relapse in a mouse model. Upon activation, a SETD4-defined qBCSC sustained itself in a quiescent state by asymmetric division and concurrently produced an active daughter cell that proliferated to produce a cancer cell population. Single-cell sequence analysis indicated that SETD4+ qBCSCs clustered together as a distinct cell type within the heterogeneous BCSC population. SETD4-defined quiescent CSCs were present in multiple cancer types including gastric, cervical, ovarian, liver, and lung cancers and were resistant to chemotherapy. SETD4-defined qBCSCs had a high tumorigenesis potential and correlated with malignancy and chemotherapy resistance in clinical breast cancer patients. Taken together, the results from our previous study and current study on six cancer types reveal an evolutionarily conserved mechanism of cellular quiescence epigenetically controlled by SETD4. Our findings provide insights into the mechanism of tumorigenesis and relapse promoted by SETD4-defined quiescent CSCs and have broad implications for clinical therapies. SIGNIFICANCE: These findings advance our knowledge on the epigenetic determinants of quiescence in cancer stem cell populations and pave the way for future pharmacologic developments aimed at targeting drug-resistant quiescent stem cells.

DEK terminates diapause by activation of quiescent cells in the crustacean Artemia.

To cope with harsh environments, the Artemia shrimp produces gastrula embryos in diapause, a state of obligate dormancy, having cellular quiescence and suppressed metabolism. The mechanism behind these cellular events remains largely unknown. Here, we study the regulation of cell quiescence using diapause embryos of Artemia We found that Artemia DEK (Ar-DEK), a nuclear factor protein, was down-regulated in the quiescent cells of diapause embryos and enriched in the activated cells of post-diapause embryos. Knockdown of Ar-DEK induced the production of diapause embryos whereas the control Artemia released free-swimming nuaplii. Our results indicate that Ar-DEK correlated with the termination of cellular quiescence via the increase in euchromatin and decrease in heterochromatin. The phenomena of quiescence have many implications beyond shrimp ecology. In cancer cells, for example, knockdown of DEK also induced a short period of cellular quiescence and increased resistance to environmental stress in MCF-7 and MKN45 cancer cell lines. Analysis of RNA sequences in Artemia and in MCF-7 revealed that the Wnt and AURKA signaling pathways were all down-regulated and the p53 signaling pathway was up-regulated upon inhibition of DEK expression. Our results provide insight into the functions of Ar-DEK in the activation of cellular quiescence during diapause formation in Artemia.

Identification and characterization of a symbiotic agglutination-related C-type lectin from the hydrothermal vent shrimp Rimicaris exoculata.

Rimicaris exoculata (Decapoda: Bresiliidae) is one of the dominant species of hydrothermal vent communities, which inside its gill chamber harbors ectosymbioses with taxonomic invariability while compositional flexibility. Several studies have revealed that the establishment of symbiosis can be initiated and selected by innate immunity-related pattern recognition receptors (PRRs), such as C-type lectins (CTLs). In this research, a CTL was identified in R. exoculata (termed RCTL), which showed high expression at both mRNA and protein levels in the scaphognathite, an organ where the ectosymbionts are attached outside its setae. Linear correlationships were observed between the relative quantities of two major symbionts and the expression of RCTL based on analyzing different shrimp individuals. The recombinant protein of RCTL could recognize and agglutinate the cultivable γ-proteobacterium of Escherichia coli in a Ca2+-dependent manner, obeying a dose-dependent and time-cumulative pattern. Unlike conventional crustacean CTLs, the involvement of RCTL could not affect the bacterial growth, which is a key issue for the successful establishment of symbiosis. These results implied that RCTL might play a critical role in symbiotic recognition and attachment to R. exoculata. It also provides insights to understand how R. exoculata adapted to such a chemosynthesis-based environment.

Optimization of ultrasound-microwave assisted acid extraction of pectin from potato pulp by response surface methodology and its characterization.

The ultrasound-microwave assisted HCl extraction of pectin from potato pulp was optimized using the response surface methodology. Effects of extraction temperature, pH, and time on the yield were evaluated, and structural characteristics of pectin extracted under optimal conditions were determined. The yield was 22.86 ±â€¯1.29% under optimal conditions of temperature 93 °C, pH 2.0, and time 50 min. The obtained pectin was rich in branched rhamnogalacturonan I (61.54 mol%). Furthermore, the pectin was a low-methoxyl (degree of methylation, 32.58%) but highly acetylated (degree of acetylation, 17.84%) pectin and the molecular weight was 1.537 × 105 g/mol. Fourier transform infrared spectroscopy and 1H nuclear magnetic resonance indicated that pectin had a linear region of α-1, 4-linked galacturonic acids which could be methyl and acetyl-esterified, and rhamnose linked with galacturonic acid to form rhamnogalacturonan which was branched with side chains. Scanning electron microscopy showed most of pectin had a lamellae structure.

An H-ferritin from the hydrothermal vent shrimp Rimicaris exoculata and its potential role in iron metabolism.

Rimicaris exoculata (Decapoda: Bresiliidae) is one of the dominant species among hydrothermal vent communities along the Mid-Atlantic Ridge. This shrimp can tolerate high concentrations of heavy metals such as iron, but the mechanisms used for detoxification and utilization of excess metals remain largely unknown. Ferritin is a major iron storage protein in most living organisms. The central heavy subunit of ferritin (H-ferritin) possesses ferroxidase activity and converts iron from Fe2+ to Fe3+, the non-toxic form used for storage. In the present study, the H-ferritin RexFrtH was identified in the hydrothermal vent shrimp R. exoculata, and found to be highly expressed in the gill, the main organ involved in bioaccumulation of metals, at both RNA and protein levels. Accumulation of RexFrtH decreased from efferent to afferent vessels, coinciding with the direction of water flow through the gills. Fe3+ was localized with RexFrtH, and in vitro iron-binding and ferroxidase assays using recombinant RexFrtH confirmed the high affinity for iron. Based on these results, we propose a model of iron metabolism in R. exoculata gills; ferrous iron from ambient hydrothermal water accumulates and is converted and stored in ferric form by RexFrtH as an iron reservoir when needed for metabolism, or excreted as an intermediate to prevent iron overload. The findings expand our understanding of the adaptation strategies used by shrimps inhabiting extreme hydrothermal vents to cope with extremely high heavy metal concentrations.

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia.

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We found that Vmem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas Vmem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high Vmem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and Vmem in the Artemia model.

TT(N)mGCCTC inhibits archaeal family B DNA polymerases.

The proofreading activity of the archaeal family B DNA polymerases enables PCR with high fidelity. However, thermostable proofreading DNA polymerases occasionally failed to amplify target fragment that could be amplified by Taq DNA polymerase. We have previously showed that G-rich sequences, which form G-quadruplex, can bind to and inhibit proofreading DNA polymerases. Here we showed that single-stranded oligonucleotides containing sequences of TT(N)mGCCTC can bind and inhibit archaeal family B DNA polymerases but not Taq DNA polymerase. It is very likely that TT(N)mGCCTC inhibits thermostable DNA polymerases during PCR in a single-stranded form. To the best of our knowledge, this is the first example of DNA sequence that could inhibit DNA polymerase in its single-stranded form.

Extraction, structure, and emulsifying properties of pectin from potato pulp.

Effects of HCl, H2SO4, HNO3, citric acid, and acetic acid on the yield, structure, and emulsifying properties of potato pectins were investigated. Results showed that the highest yield (14.34%) was obtained using citric acid, followed by HNO3 (9.83%), HCl (9.72%), H2SO4 (8.38%), and acetic acid (4.08%). The degrees of methylation (37.45%) and acetylation (15.38%), protein content (6.97%), and molecular weight (3.207 × 105 g/mol) were the highest for pectin extracted using acetic acid, and (galactose + arabinose)/rhamnose was 33.34, indicating that it had a highly branched rhamnogalacturonan I domain. Fourier transform infrared spectroscopy showed a specific absorbance peak at 1064 cm-1, which corresponds to the acetyl groups in potato pectins. SEM showed that all potato pectins are morphologically different. The emulsifying activity (EA, 44.97%-47.71%) and emulsion stability (ES, 36.54%-46.00%) of the pectins were influenced by acid types, and were higher than those of commercial citrus and apple pectin.

Identification and characterization of a Masculinizer (Masc) gene involved in sex differentiation in Artemia.

The sex of relatively primitive animals such as invertebrates is mostly determined by environmental factors and chromosome ploidy. Heteromorphic chromosomes may also play an important role, as in the ZW system in lepidopterans. However, the mechanisms of these various sex determination systems are still largely undefined. In the present study, a Masculinizer gene (Ar-Masc) was identified in the crustacean Artemia franciscana Kellogg 1906. Sequence analysis revealed that the 1140-bp full-length open reading frame of Ar-Masc encodes a 380-aa protein containing two CCCH-type zinc finger domains having a high degree of shared identities with the MASC protein characterized in the silkworm Bombyx mori, which has been determined to participate in the production of male-specific splice variants. Furthermore, although Ar-Masc could be detected in almost all stages in both sexual and parthenogenetic Artemia, there were significant variations in expression between these two reproductive modes. Firstly, qRT-PCR and Western blot analysis showed that levels of both Ar-Masc mRNA and protein in sexual nauplii were much higher than in parthenogenetic nauplii throughout the hatching process. Secondly, both sexual and parthenogenetic Artemia had decreased levels of Ar-Masc along with the embryonic developmental stages, while the sexual ones had a relatively higher and more stable expression than those of parthenogenetic ones. Thirdly, immunofluorescence analysis determined that sexual individuals had higher levels of Ar-MASC protein than parthenogenetic individuals during embryonic development. Lastly, RNA interference with dsRNA showed that gene silencing of Ar-Masc in sexual A. franciscana caused the female-male ratio of progeny to be 2.19:1. These data suggest that Ar-Masc participates in the process of sex determination in A. franciscana, and provide insight into the evolution of sex determination in sexual organisms.